Lysozyme and α-Lactalbumin: Structure, Function, and Interrelationships

LYSOZYME AND a-LACTALBUMIN: STRUCTURE. FUNCTION. AND INTERRELATIONSHIPS

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By HUGH A McKENZIE* and FREDERICK H WHITE. JR.t 'Department of Chemistry. University College. University of New South Wales. Australian Defence Force Academy. Canberra. ACT 2600. Australia t Department of Chemistry. Florida state Unhrersity. Tallahassee. Florida 32306

I . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I1. Early History . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Lysozyme and Its Function . . . . . . . . . . . . . . . . . . . . . B. a-Lactalbumin and Its Function . . . . . . . . . . . . . . . . . . . C. Sequence Homology between a-Lactalbumin and Lysozyme . . . . . . I11. Some Aspects of the Occurrence. Isolation, and Characterization of Lysozyme and a-Lactalbumin . . . . . . . . . . . . . . . . . . . . . A. Lysozyme: Occurrence, Isolation. and Kinetics of Cell Lysis . . . . . . B. a-Lactalbumin: Occurrence. Isolation. and Determination of Specifier Activity . . . . . . . . . . . . . . . . . . . . . . . . . IV. Three-Dimensional Structure of Lysozyme . . . . . . . . . . . . . . . A . X-Ray Crystal Structure of Domestic Hen Egg-White Lysozyme . . . . B. Mechanism of Cell Lytic Action . . . . . . . . . . . . . . . . . . . C. Structures of Other Lysozymes . . . . . . . . . . . . . . . . . . . D. Water in Lysozyme Crystals . . . . . . . . . . . . . . . . . . . . V. Three-Dimensional Structure of a-Lactalbumin . . . . . . . . . . . . . A . Models for the Three-Dimensional Structure of a-Lactalbumin (Based on Sequence Homology with Lysozyme) . . . . . . . . . . . . . . . . B. X-Ray Crystal Structure of Baboon Milk a-Lactalbumin . . . . . . . . C. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . VI . Comparative Binding of Metal Ions in Lysozyme and a-Lactalbumin . . . . A . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Metal Ion Binding to Lysozyme . . . . . . . . . . . . . . . . . . . C. Metal Ion Binding to a-Lactalbumin . . . . . . . . . . . . . . . . D. Structural Changes on Cation Binding by a-Lactalbumin and Their Implications in Lactose Synthase Activity . . . . . . . . . . . . E . Metal Ion Binding in a-Lactalbumin: Implications for Lysozyme . . . . VII . Amino Acid Composition and Sequence Homologies in Lysozyme and a-Lactalbumin . . . . . . . . . . . . . . . . . . . . . A . Amino Acid Compositions . . . . . . . . . . . . . . . . . . . . . B. Sequence Comparisons . . . . . . . . . . . . . . . . . . . . . . C. Summary of Important Features of Comparative Sequences . . . . . . VIII . Galactosyltransferase and the Lactose Synthase System . . . . . . . . . . A . Galactosyltransferases: Occurrence. Function. and Isolation . . . . . . B. Relationships of Structure to Function in Galactosyltransferase . . . . . C. Interactions of Galactosyltransferase and a-Lactalbumin in the Lactose Synthase System . . . . . . . . . . . . . . . . . . . . D. Structural Requirements of Substrate . . . . . . . . . . . . . . . . E. FinalRemarks . . . . . . . . . . . . . . . . . . . . . . . . . . 173 ADVANCES IN

PROTEIN CHEMISTRY. Vol. 41

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Copyright 0 1 9 9 1 by Academic Press Inc . All rights of reproduction in any form reserved.

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HUGH A. MCKENZIE AND FREDERICK H.

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IX. Some Additional Physical, Chemical, and Biological Comparisons between Lysozyme and a-Lactalbumin . . . . . . . . . . . . . . . A. Spectroscopic Studies . . . . . . . . . . . . . . . . . . . . . . . B. Small-Angle X-Ray Scattering . . . . . . . . . . . . . . . . . . . C. Electron Spin Resonance and Nuclear Magnetic Resonance . . . . . . D. Association and Aggregation . . . . . . . . . . . . . . . . . . . . E. Denaturation and Renaturation . . . . . . . . . . . . . . . . . . . F. Chemical Reactivities . . . . . . . . . . . . . . . . . . . . . . . G. Immunochemical Properties . . . . . . . . . . . . . . . . . . . . H. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . X. Evolutionary Origins of Lysozyme and a-Lactalbumin . . . . . . . . . . A. Introduction: Molecular Clocks and the Fossil Record . . . . . . . . . B. Evolution of Lysozyme and a-Lactalbumin: Divergenceand/orConvergence? . . . . . . . . . . . . . . . . . . C. Are the Functions of Lysozyme and a-Lactalbumin Mutudly Exclusive? . . . . . . . . . . . . . . . . . . . . . . . . XI. Conclusions and the Future . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Note Added in Proof . . . . . . . . . . . . . . . . . . . . . . . .

259 259 265 265 267 268 271 272 275 276 276 280 290 293 299 315

I. INTRODUCTION The initiation of a project to study a selected protein structure requires very careful consideration; it is rather like a decision as to which type of new aeroplane to build. The cost in manpower, time, and money is considerable, and if the structure proves to be obdurate, this expenditure shows little return. Lysozyme, which Dr. Poljak had already studied when he joined the Davy Faraday team in 1960, proved to be a fortunate choice. It is the third protein structure to be successfully analysed, and the first enzyme.

T h e above statement was made by the late Sir Lawrence Bragg (1967) at a Royal Society Discussion on the structure and function of lysozyme held at the Royal Institution on February 3, 1966. A 0.6-nm (6 A) resolution Fourier map of domestic hen egg-white lysozyme had been presented by Blake et al. (1962), and one at 0.2-nm (2 A) resolution was published in 1965 (Blake et al., 1965),just 5 years after Poljak initiated the original study. This work resulted not only in a structure for lysozyme itself, but also in specific information on the active site of the enzyme and the mode of catalysis (Johnson and Phillips, 1965; Blake et al., 196713). Important as this was in expanding our knowledge of enzymatic catalysis, it has had farther-reaching effects. At about the same time (1967), evidence began to accumulate that there was structural homology between lysozyme and the milk protein, a-lactalbumin. Furthermore, the functional role of a-lactalbumin in milk was elucidated for the first time. In the ensuing 20 years much more evidence has accumulated on the structural and functional relationships of these two proteins. During

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1965- 1974 there were some dramatic breakthroughs in our knowledge of the structure of these proteins, but the pace slowed later in that decade. This was followed by a period in which much information was accumulated on the evolution of primary structure and of antigenic sites. The resulting debate did not resolve the questions of convergent and divergent evolution. Despite predicted similarities in the structures of a-lactalbumin and lysozyme, technical and other difficulties resulted in little progress on the elucidation of the three-dimensional structure of a-lactalbumin. The discovery of the essential binding of calcium(I1) by a-lactalbumin in 1980 and ensuing studies resulted in a further attack on this obdurate problem. Success was finally achieved in 1986, and the results have far-reaching implications for the structural, evolutionary, and functional relationships of these two proteins. There have been previous reviews on a-lactalbumin and lysozyme, especially during 1970- 1975. The reviews on lysozyme include the conference proceedings edited by Osserman et al. (1974) and the reviews by Imoto et al. (1972), Jolles and Jollb (1984), and Proctor and Cunningham (1988). Work on a-lactalbumin and lactose synthase has been reviewed by Brew (1970), Hill and Brew (1975), Brew and Hill (1975), and Hall and Campbell (1986). In this article we have a somewhat different purpose: to consider both the structure and function of a-lactalbumin and lysozyme in relation to each other, especially in light of the work done in the past few years. We consider also the potential significance of the studies in health and the pathology of disease, such as cancer. We open with a brief report of the early discovery of the occurrence and isolation of these proteins and the elucidation of their function and homology, followed by a brief discussion of some problems in their isolation and the determination of their activity. We then consider various aspects of their three-dimensional structures and their significance. We summarize studies on the implications of their sequence similarities, and also on the binding of metal ions, especially calcium(II), and consider their implications. Then follows a brief discussion of lactose synthase, an enzyme of which a-lactalbumin and galactosyltransferase are essential components. We then examine the evidence concerning the evolution of the two proteins, about which there are conflicting views (see Section X,B). Some conclusions and predictions of future directions are made. The subjects chosen and the emphasis placed on certain aspects reflect the experience and interest of the authors. We make no attempt to be encyclopedic, and the omission of any work does not imply that it is unimportant. However, we do hope that those not working in this field

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will appreciate the progress that has been made and that we also give some inspiration and challenge to others working in this area. 11. EARLYHISTORY A. Lysozyme and Its Function It has been reported that the early Romans used egg white in the treatment of eye infections, and some mothers are reputed to have used human milk for the same purpose; both media contain appreciable amounts of lysozyme. Later, antibacterial properties of leukocytes (Metschnikoff, 1883),cow milk (Fokker, 1890), Bacillussubtilis (Nicolle, 1907), domestic hen egg-white (Laschtschenko, 1909), and nasal secretions (Bloomfield, 1919, 1920) were described from 1880 to 1920. It has been stated (e.g., Fleming and Allison, 1922) that none of the workers prior to 1920 considered “bacteriolysis” in their publications. Nevertheless, Nicolle ( 1907) does discuss “taction bacttkiolysente” in his paper. While Laschtschenko (1909) does not consider bacteriolysis, he does attribute the spore-destroying properties of egg white to “proteolytic enzymes.” However, it was Fleming who first clearly showed that an enzymic substance present in a wide variety of secretions is capable of rapidly lysing (i.e., dissolving)certain bacteria, particularly a yellow “coccus”that he studied. Fleming’s chief, Almroth Wright (quoted by Maurois, 1959), who delighted in constructing words from Greek roots, suggested that the substance be called lysozyme and that the microbe be called Micrococcus lysodeikticus. In December 1921 Fleming presented a paper on his work to the Medical Research Club in London, but it was coldly received. However, the icy reception did not deter Fleming (1922) from submitting his classical paper, “On a Remarkable Bacteriolytic Element Found in Tissues and Secretions,” to the Royal Society in February 1922. Despite the lack of interest, Fleming continued his work on lysozyme for several years and remained convinced of its importance. In 1936 Meyer et al. showed that lysozyme is a protein, and in the following year Abraham and Robinson (1937) first reported its crystallization. Various biochemical and chemical investigations were made of lysozyme over the next 30 years, but it was not until the 1960s that Fleming’s great faith in lysozyme was vindicated. Although Fleming showed the presence of lysozyme in an amazing variety of secretions, he does not appear to have been the first to show its presence in milk. Bordet and Bordet (1924) showed that it is present in human colostrum and milk, but failed to detect it in cow milk. Later, Fleming (1932) concluded that it was present in cow milk, but at a much

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lower level than in human milk. Indeed, the level of lysozyme in cow milk became controversial, and some workers even disputed its presence at all. This controversy has only recently been resolved (see Section 111,A). The nature of the role of lysozyme in the lysis of bacteria was finally elucidated by Salton and co-workers nearly 40 years after Fleming’s original work (Salton, 1964). It had been evident from the earlier studies by Meyer et aZ. (1936) and Epstein and Chain (1940) that the characterization of the substrate for lysozyme and the products formed as a result of its action should throw light on the nature of the structure of the bacterial cell wall. Salton (1952) subsequently showed that the isolated cell wall of M. Zysodeikticus could be used as the substrate for lysozyme and that this could be exploited to determine the nature of the digestion products. A series of studies showed that the lysis of bacteria by lysozyme involved a specific cleavage of cell wall mucopolysaccharides. Of the saccharides liberated, the simplest found was a disaccharide; its structure was investigated by Perkins (1960) and by Salton and Ghuysen (1959, 1960). Since their original work there has been some refinement in our knowledge of the nature of the linkage cleaved between N-acetylglucosamine and N-acetylmuramic acid [/3( 1 4 4 ) linkage, not /3( 1-6) as originally proposed]. A schematic diagram of the cleavage is shown in Fig. 1.

---

o-(NAM)--CH3CHCOOH

I I

(NAM)

(NAG)

Lysozyme

(NAM)

(NAG)

FIG. 1. Catalysis by lysozyme (1,4-p-N-acetylmuramidase)of the cleavage of the glycosidic linkage between the C-1 of N-acetylmuramic acid (NAM) and the C-4 of Nacetylglucosamine (NAG) in a polymer of NAM and NAG. The vertical broken line shows the point of cleavage.

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HUGH A. MCKENZIE AND FREDERICK H . WHITE, JR.

In this period it was also shown by Berger and Weiser (1957) that lysozyme can also degrade highly purified chitin, and it was proposed that lysozyme and related enzymes be called muramidases (Salton, 1964). Lysozymes are now defined as 1,4-P-N-acetylmuramidases cleaving the glycosidic linkage between the C- 1 of N-acetylmuramic acid and the C-4 of N-acetylglucosamine in the bacterial peptidoglycan. B . a-Luctulbumin and Its Function

From 1885 to 1965 work on another protein, a-lactalbumin, was proceeding independently of, and without apparent relevance to, that on lysozyme. It is difficult to assess some of the early work because of confusion in nomenclature, especially with respect to the terms “lactalbumin” and “lactoglobulin” (McKenzie, 1967). The first extensively reported study of lactalbumin in bovine milk and colostrum seems to be that by Sebelien (1885), who also referred to some earlier analytical studies of lactalbumin. However, the first reported crystallization of a lactalbumin is that by Wichmann (1899), who appears to have established that his preparation is different from serum albumin (see also Mann, 1906). Following Svedberg’s development of the ultracentrifuge, it was possible in the 1930s to perform ultracentrifugal studies of skim milk and proteins isolated by this method. The noncasein fraction was found to exhibit three peaks, designated as a, P, and 7 , in sedimentation velocity patterns (Sjogren and Svedberg, 1930).The sedimentation coefficient of the /3 peak was shown by both Phillipi and Pedersen in 1935-1936 to be identical to that of the lactoglobulin isolated by Palmer (see Pedersen, 1936a). In 1935 Kekwick isolated a lactalbumin from milk, and soon afterward Pedersen (1936b) concluded from ultracentrifugal studies that it was the protein responsible for the a peak. [It was later shown to be similar to the “crystalline insoluble substance” of Sorensen and Sorensen (1939).] Thus, the two proteins were called P-lactoglobulin and a-lactalbumin. Although it was soon established that these two proteins were the dominant “whey” proteins of cow milk, their functions proved to be elusive. Indeed, the biological function of the former is still uncertain, and it was only 20 years ago that the function of the latter was established. In the early 1960s Hassid and collaborators demonstrated that the enzyme lactose synthetase (now synthase) exists as a microsomal enzyme in the mammary glands of lactating cows and guinea pigs (Watkins and Hassid, 1962) and in a soluble form in cow milk (Babad and Hassid, 1964, 1966). They confirmed an earlier suggestion by Wood and co-

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workers that lactose is enzymatically synthesized in the mammary gland from UDP-galactose and glucose (see Brew and Hill, 1975). Soon afterward, Brodbeck and Ebner (1966) showed that the milk enzyme (or the microsomal enzyme when solubilized by sonic oscillation) could be resolved into two protein fractions, A and B. Neither A protein nor B protein alone exhibited lactose synthase activity, but recombination of the two fractions restored catalytic activity. Subsequently, Ebner et al. (1966; see also Brodbeck et al., 1967) showed that the B protein is a-lactalbumin. The substrate specificity of the lactose synthase system was studied further by Brew et al. (1968). They confirmed that neither A protein nor B protein alone was active for the synthesis of lactose, but the A protein catalyzed the following reaction (see also Fig. 2): UDP-galactose

+ N-acetylglucosamine + N-acetyllactosamine

+ UDP

(1)

Thus, protein A is a galactosyltransferase (UDP-galactose :N-acetylglucosamine-P-4-galactosyltransferase;EC 2.4.1.38). Its normal function

UDP-Gal

NAG

NAL (a)

UDP-Gal

Glucose

Lactose

(b) FIG.2. Reactions catalyzed by galactosyltransferase (GT). (a) The incorporation of galactose (Gal) into a p(1-4) linkage with N-acetylglucosamine (NAG) to form N-acetyllactosamine (NAL). UDP, Uridine diphosphate. (b) Modification of the activity of GT by a-lactalbumin (a-LA) to convert it to a lactose synthase catalyzing the formation of lactose from UDP-Gal and glucose.

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is to catalyze the incorporation of galactose (Gal) into p( 1+4) linkage with N-acetylglucosamine (N-AcGlc) during the synthesis of the oligosaccharide prosthetic groups of certain glycoproteins (Hill et al., 1969): UDP-Gal

+ N-AcGlc + Gal-N-AcGlc + UDP

1 I Protein

Protein

(2)

With the onset of lactation, a-lactalbumin is formed in the mammary gland and alters the substrate specificity of the transferase from N acetylglucosamine to glucose, enabling lactose synthesis to be effected: UDP-Gal

+ glucose + lactose + UDP

(3)

[Note that the protein glycosylation reaction (2) is not inhibited when a-lactalbumin is formed.] Hereafter we will not use the terms “A protein” and “B protein,” but will refer to them as galactosyltransferase and a-lactalbumin, respectively. Because of its unique ability to alter the specificity of the galactosyltransferase to a lactose synthase (UDP-D-galactose:D-glucose P-~-Dgalactosyltransferase; EC 2.4.1.22), a-lactalbumin has been designated a “specifier” protein. A unique biological role (other than a minor nutritional one) finally had been demonstrated for a-lactalbumin. Although there are several enzymes that consist of two proteins, the unique feature of the involvement of a-lactalbumin is that it is able to change the acceptor specificity of the galactosyltransferase (Ebner, 1970).

C. Sequence Homology between a-Lactalbumin and Lysozyme In 1958 Yasunobu and Wilcox drew attention to certain similarities between a-lactalbumin and lysozyme (see Gordon, 1971). A few years later Brew and Campbell (1967) also drew attention to their marked similarity in molecular weights, amino acid composition, and the aminoand carboxy-terminal amino acid residues. They stated, “To the extent that the properties mentioned reflect similar primary structures, the a-lactalbumins may have evolved by gradual modification from lysozyme, which is found in the milk of many species” (p. 263). This proposal prompted Brew et al. (1967, 1970) to determine the amino acid sequence of bovine a-lactalbumin, which proved to have a high level of sequence identity with domestic hen egg-white lysozyme. Thus, these studies were in accordance with the proposal that the two proteins had diverged from a common ancestor (see also Hill et al., 1969, 1974). They stated that “although lysozyme does not participate in lactose synthesis and a-lactalbu-

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min does not act on lysozyme substrates, the integral role of a-lactalbumin in lactose synthesis implies a functional as well as a structural similarity between a-lactalbumin and lysozyme. One enzyme is involved in the cleavage and the other in the synthesis of a p( 1+4)-glucopyranosyl linkage.’’ We will discuss in Section X,C how far these statements need modification in light of subsequent work. 111. SOMEASPECTS OF THE OCCURRENCE, ISOLATION, AND CHARACTERIZATION OF LYSOZYME AND ~-LACTALBUMIN A. Lysozyme: Occurrence, Isolation, and Kinetics of Cell LyszS

Lysozyme occurs in domestic hen egg-white to the extent of -30 mg g- ’. It is the most extensively studied lysozyme and is representative of a class of lysozymes, designated chicken- or chick-type lysozymes, now usually abbreviated c-type lysozymes. Although the majority of amino acid sequences of egg-white lysozymes determined have been for the c type, this type has been found at high concentration in only two orders of birds: the Galliformes and the Anseriformes. The c-type lysozymes consist of a single amino acid chain of 129 residues and a molecular weight of -14,500. A different type of lysozyme was found by Dianoux and Jollks (1967) and by Canfield and McMurry (1967) in Embden goose egg white. It was shown by Prager et al. (1974) to be present in at least nine orders of birds. This lysozyme has been designated to be of the goose type, usually abbreviated g type. In the egg white of the black swan (Cygnus atratus) both c and g types occur. Despite their ubiquity, few sequences of the g type have ever been determined. Each contains 185 residues, with an M , of -20,500 (Canfield et al., 1971; Simpson et al., 1980; Simpson and Morgan, 1983). All mammalian lysozymes thus far examined have proved to be of the c type. Bacteriophage lysozymes contain about 164 amino acid residues with M , 18,700. Fungus and bacterial lysozymes show considerable differences from the c type (Fouche and Hash, 1978). Powning and Davidson (1976) have characterized a c-type lysozyme from the wax moth (Galleria mellonella). Jolles et al. (1979b) found the c type among some members of the insect order Lepidoptera, but found a different type in eggs of Ceratitis capitata. By DNA sequencing, Engstrom et al. (1985) unequivocally demonstrated that the Hyalophoru (moth) lysozyme is c type. Other invertebrate lysozymes have been discussed by Jollks and Jollks ( 1984). T h e first plant lysozyme appears to have been isolated from papaya latex by Smith et al. (1955).

-

-

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HUGH A. MCKENZIE A N D FREDERICK H . WHITE, JR.

Because of the high isoelectric point and limited molecular size, it is not surprising that isolation of the lysozymes has proceeded generally along fairly similar paths. In the isolation from milk and egg white from a variety of species, the lysozyme has been adsorbed on an ion exchanger, such as Amberlite IRC-50 (Rohm and Haas, Philadelphia, PA), CM-cellulose (Whatman Inc., Clifton, NJ), CM-Sephadex (Pharmacia, Uppsala, Sweden), and BioGel-CM 30 (Bio-Rad, Richmond, CA), from an ammonium sulfate fraction of the original egg white, or skim milk. After elution it is often purified further by gel filtration on Sephadex G50 or another suitable medium. Whitaker (1963) noted abnormal retention on Sephadex gel under certain conditions. He attributed this to interaction with the dextran, possibly related to the enzymatic function. In more recent work selective removal from immunoadsorbents (e.g., MacKay et al., 1982), heparin-Sepharose (e.g., Boesman-Finkelstein and Finkelstein, 1982; Teahan et al., 1991b), and chitin (e.g., Grinde et al., 1988) was effected. Isoelectric focusing (e.g., Lie and Syed, 1986) has also been used. There are several problems requiring careful attention. Lysozyme has a tendency to form complexes with many substances [e.g., alkyl sulfates, fatty acids, aliphatic alcohols (Smith and Stocker, 1949), cephalins (Brusca and Patrono, 1960), and other proteins]. Of particular importance is its tendency to form complexes with transferrins [e.g., ovotransferrin (Ehrenpreis and Warner, 1956)l. These interactions lead to difficulties in the isolation of lysozyme. Some recent workers have used fast protein liquid chromatography (FPLC) and high-performance liquid chromatography (HPLC) (e.g., Ekstrand and Bjorck, 1986). The resolution in these procedures may not always be satisfactory, and in HPLC pressure and solvent effects must be monitored carefully if the product is to be suitable for conformation and activity studies. There have been special problems in the isolation of some lysozymes, particularly those from cow milk and the milk of monotremes. Isolation from colostrum has proved difficult because of the previously noted tendency of lysozyme to complex with other proteins (e.g., immunoglobulins). Some proteins, particularly lactoferrin and transferrin, may elute from ion exchangers similarly to lysozyme, and it is frequently necessary to take the precaution of using multiple passages of the lysozyme-containing product thereafter, through an appropriate gel filtration column. The isolation of lysozyme from cow milk proved especially difficult. There were three problems: (1) Lysozyme is present in low concentration (-100 pg liter1), necessitating a large volume of starting milk. (2) The enzyme is unstable in raw milk, requiring the immediate start of the processing procedure (White et al., 1988), after which the enzyme is

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more stable. The instability in fresh milk may be due to proteolytic action on the lysozyme. (3) The enzyme was found to be closely associated with another protein in the final stage of purification, from which lysozyme could be separated only partially, but sufficiently to permit partial amino acid sequence determination (White et al., 1988). This problem of separation had not been realized earlier, when Chandan et al. (1965) reported isolation of cow milk lysozyme, and the subsequent characterization of their product was unknowingly done on a mixture of lysozyme and the contaminating protein. This combination of proteins which were difficult to separate was first observed in attempts to undertake sequencing of the milk lysozyme that had been purified by White et al. (1988). The results showed the existence of two molecular species, one minor, amounting to -30% by weight. This species, however, was identifiable as a c-type lysozyme by its partial sequence. The partial sequence of the remaining component was not identifiable with any known protein sequence. A partial separation could be obtained with HPLC, eluting with dilute acetic acid. T h e trailing edge of the more slowly moving component was confirmed as lysozyme by activity determination and partial sequencing to demonstrate the presence of lysozyme. The question can now be raised as to whether other lysozymes “purified” from sources of low lysozyme levels might be similarly contaminated. Most of the methods by which lysozyme activity has been detected and studied are variations on the principle first discovered by Fleming, that is, the lysis of bacterial cell walls. Typically, a turbid cell suspension (Mzcrococcus luteus) is observed to clear in the presence of lysozyme, and the rate of change of turbidity is equated with lysozyme activity. Some recent workers (e.g., Hammer and Wilson, 1990) detect lysozyme in electrophoretic gels by overlaying the gel with another gel containing M. luteus cells [see also Osserman and Lawlor (1966) and the modification by Lie et al. (1986)l. Also noteworthy as a means for lysozyme determination is radioimmunoassay (Canfield et al., 1974). Another means involves use of the synthetic substrate 3,4-dinitrophenyltetra-N-acetyl-P-chitotetraoside with a spectrophotometric determination (Ballardie and Capon, 1972). This substrate, however, did not lend itself to high sensitivity determinations and was not found to be satisfactory for the determination of lysozyme at low concentration levels (our unpublished observations). A highly sensitive, yet practical, means of lysozyme determination is that of McKenzie and White (1986), which uses the turbidimetric principle. An important feature of their method is the prolonged incubation of the reaction mixture so as to magnify traces of enzymatic activity.

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HUGH A. MCKENZIE AND FREDERICK H. WHITE, JR.

Although extended reaction time was also used by Selsted and Martinez (1980) in their turbidimetric method, they did not stress the importance of the kinetic order of cell lysis. McKenzie and White (1986) showed that, while the reaction is biphasic, simple kinetics are obeyed over a sufficiently long period that this property can be exploited in the determination. The kinetic order must be considered in the quantitative treatment of the results. The limit of detection was found to be 100 pg ml-' of reaction mixture, equivalent in the case of bovine milk lysozyme to 6 ng ml-' of skim milk, for a 50-1.11 sample. The method of McKenzie and White (1986) readily lends itself to the study of reaction kinetics. A number of lysozymes have been assessed according to kinetic order in the primary phase. Thus, Table I shows that second order predominates in either Tris or imidazole buffer, except that zero order is observed for the platypus milk and tammar wallaby stomach mucosal enzymes. A range of kinetic orders is observed in phosphate buffer, with equine and domestic hen egg-white lysozymes being first order, and others being either second or zero order. Particularly difficult to understand is the exhibition of second-order kinetics for a reaction that basically involves a single reactant, that is, the TABLE I Kinetic Ordersfor Lysis of Cells of Micrococcus l&us with Lysozymes from Various Sources"

Kinetic order Enzyme source Domestic hen egg white Black swan egg white, c and g Human milk Horse milk Cat milk' Echidna milk Platypus milk Cow milk Bovine stomach mucosa Tammar wallaby stomach mucosa

Imidazole or Trisb Phosphateb 2 2 2 2 2 2

0

2 2

2

0

0

2 2

1

2 2

0

1 0

"Different buffer systems for reaction. All results are from McKenzie and White (1986, 1987), except where indicated. Reaction mixture buffer. 'Unpublished results of J. Halliday, H. A. McKenzie, and F. H. White, Jr.

LYSOZYME AND Q-LACTALBUMIN

185

bacterial cell wall. Various workers have reported kinetics of cell lysis with lysozyme as being zero order (Shugar, 1952; Smolelis and Hartsell, 1949), first order (Dickman and Proctor, 1952; Kerby and Eadie, 1953), or second order (Smith et al., 1955; Howard and Glazer, 1969; Prasad and Litwack, 1963). The latter group speculated that the phenomenon of second order in the main phase might be caused by two points of attack on the cell surface. They suggested that lysozyme may not release readily from the cellular debris after cell wall cleavage and that the ensuing slow stage involves cleavage of one linkage at a time. Howard and Glazer (1969) later presented a detailed sequential mechanism for the mode of action of papaya lysozyme. Another possibility for explaining second-order kinetics is that each lysozyme molecule, being trapped in the cellular debris, is, in effect, eliminated from the reaction mixture after it participates in a single lysis. The lysozyme so involved then behaves kinetically as if it were a reactant rather than a catalyst, since it cannot separate from the product in order to participate in another reaction. However, the extent to which lysozyme behaves in this way, as a second reagent, to account for secondorder kinetics, remains to be assessed. M. F. Hammer and A. C. Wilson developed a method by which lysozyme can be detected after the electrophoresis of biological fluids (Dobson et al., 1984). In this method the unstained electrophoretic gel was overlaid with a polyacrylamide gel containing a suspension of M. luteus cells. The lysozyme was allowed to diffuse into the overlay gel, where it catalyzed lysis of the suspended cells. The resulting change in opacity of the overlay could then be quantified by densitometric scanning. In a recent modification of this method (Cortopassiand Wilson, 1989), the sensitivity of lysozyme detection was increased 25-fold, being in the nanogram range. An additional means of quantifying the lysozyme after electrophoresis, again involving an overlay containing a suspension of M. luteus cells, exploited a different, previously untried, principle. Advantage was taken of the fact that lysis of the bacteria cells releases a number of dehydrogenases, principal among which is isocitrate dehydrogenase. The staining method used is suitable for all enzymes that regenerate NADPH or NADH and involves the formation of an insoluble tetrazolium dye (Giblett, 1969). Thus, staining of the overlay reveals the position and intensity of the lysozyme, which can be quantified by densitometric scanning. Moser et al. (1988) described a fluorometric method of detection of lysozyme down to levels of 0.1 pg. They used highly purified cell walls to prepare a peptidoglycan which is labeled with fluorescamine. After

186

HUGH A. MCKENZIE AND FREDERICK H . WHITE, JR.

hydrolysis of a suspension of the fluorescamine-labeled proteoglycan for a fixed time, followed by filtration, the fluorescence of the filtrate is determined and the lysozyme level is estimated. Morsky (1988) has described a method for detecting human lysozyme (with a sensitivity of 5 5 ng) in body fluids, by a procedure involving immunoblotting.

B . a-Lactalbumin: Occurrence, Isolation, and Determination of Specifier Activity Although lysozymes have been isolated from milk, tears, egg white, plant and insect materials, etc., a-lactalbumin has been found only in milk and colostrum (Table 11). However, there is now some evidence for a-lactalbumin-like proteins occurring elsewhere (e.g., in the male reproductive tract). Until the mid-l970s, there was little investigation of the proteins of milk in species other than bovine. A solid body of knowledge of the cow milk proteins has been built up gradually, so that they have become a valuable point of reference for the proteins of other mammals. During the past 10 years sufficient evidence has accumulated to indicate that there are both significant qualitative and quantitative differences in the protein composition of the other species. For example, the casein group of proteins is the dominant protein group in cow milk, but in human milk the whey proteins (i.e., noncasein proteins) quantitatively exceed the caseins. The major whey protein in cow milk is p-lactoglobulin, present in mature phase milk of Western dairy breeds, to the extent of 2.6-4.0 g 1- l , followed by a-lactalbumin at 1 g I-' (McLean et al., 1984). In contrast, human milk contains, at most, traces of plactoglobulin, possibly none of which is synthesized de nova in the human mammary gland. a-Lactalbumin is the dominant whey protein. The occurrence of any a-lactalbumin in the milk of the monotreme Tachyglossus aculeatw multiaculeatw is controversial. Thus, the level of a-lactalbumin in the milk of different species can vary from trace levels (or even zero) to concentrations on the order of 1 g I-'. Because of this wide range in concentration, the complex protein composition of milk, and the even greater complexity of colostrum, considerable care must be exercised in the isolation of a-lactalbumin (indeed, the same may be said of the isolation of lysozyme from these secretions). Furthermore, if the study of the isolated protein is to include conformation and/or studies of enzymatic (and immunological) activity, considerable care must be taken that the method of isolation does not alter the conformation or interfere with the activity. This is particularly important in determining whether or not the isolated protein has a-lactalbu-

-

LYSOZYME AND a-LACTALBUMIN

187

min-like or lysozyme-like activity, or a major activity of one type and a minor activity of the other. In dealing with the milk of rare monotremes and marsupials, samples of which are limited in volume, fractionation by HPLC methods might appear very attractive. However, because of the pressures involved and the nature of the solvents frequently used, both conformation changes and loss of activity may occur. Thus, considerable care should be exercised in the use of HPLC. While known sequencing methods may enable the sequence o r partial sequence to be established for an impure protein [e.g., cow milk lysozyme (White et al., 1988)], protein of high purity is required for other purposes (e.g., determination of trace lytic activity in an isolated alactalbumin or trace lactose synthase specifier activity in a lysozyme). Otherwise, erroneous conclusions may be drawn with respect to structure and function and their evolutionary relationships. Even for the well-characterized cow milk, considerable care must be exercised in the isolation of a-lactalbumin. Armstrong et al. (1967, 1970) made a careful study of the salt-pH solubility relationships of cow “whey” obtained by fractional ammonium sulfate precipitation from whole milk. If the pH is too low (i.e., 5 3 ) , irreversible changes may occur in the state of association and of conformation of both a-lactalbumin and P-lactoglobulin. Although there is evidence (Kronman et al., 1965) that a-lactalbumin, for example, undergoes reversible changes at low pH, it is the experience of one of the authors (H. McK.) that the behavior of the pure individual proteins is not necessarily always the same as that of the respective protein of the a-lactalbumin-P-lactoglobulin mixture in the “whey” medium. On the other hand, there are constraints on the higher pH side as well: The pH must be sufficiently high to maintain a reasonable solubility of the a-lactalbumin, but not so high that it undergoes aggregation (P-lactoglobulin is also sensitive to pH >7.0). It is evident from recent studies of metal ion binding to a-lactalbumin that spurious results will be obtained if aggregation occurs and if the order of mixing is varied. Diuturnal effects have been observed. Metal ion binding studies are critical to our future understanding of the mechanism of action of a-lactalbumin. Thus, it is critical that reaction only be performed with freshly prepared a-lactalbumin that has not been subjected to conditions that may cause irreversible or quasiirreversible changes. Thus, it is best, if possible, to work in a narrow pH region during the fractionation. It is generally possible in the column chromatography of whey proteins to achieve satisfactory fractionation between pH 6.3 and 7.5. Lindahl and Vogel (1984) studied the purification of bovine, human, caprine, ovine, and equine a-lactalbumins, exploiting the property of

TABLE I1 a-Lactalbumin in Milk of Various Specks Species Bovine (Bos)

w

00 00

Sheep (OvW aries)

Variant

B: Most common variant, occurs in Bos taurus, Bos indicur, Bos (Poephagw g r u n n i m ) ; A: Occurs in Bos indicw, Bos taurus, and crosses; C: Occurs in Bos (bzbos) javanicus

Goat (Capra hircus) Camel (Camelus dromedarius) Human (Homo sapienr) Pig (Sw scrofa) Horse (Equus caballus)

Guinea pig (Cavia porcellus) Rabbit (Oryctolagw cuniculw) Rat (Rattwnoruegzcus)

B: Most common variant; A: Less common A: Common variant

Note

Refs."

B and A variants, homozygotes shown to contain 1 major, 3 minor components

1

Major and possible minor components Major and possible minor components Heterogeneity observed in camel Similar to, but not identical with, baboon (Papio cynocephalw) and chimpanzee (Pan troglodytes) Minor component also in each homozygote B and C variants isolated from colostrum of Arabian horse (Equus caballus caballus perissodactyla)

2

Glycoprotein 140 residues (chain extension and is glycoprotein)

3 4

5

6

7

8 9 10

I

02 rc)

Mouse ( M u musculus) Dog (Canisfamiliaris) Cat (FelW catus) Marsupials Red kangaroo (Macropus rufus) Grey kangaroo (Macropus giganteus) Tammar wallaby (Macropus eugenii) Red-necked wallaby (Macropzcs rufognseus) Ring-tailed possum (Pseudocheirtu peregnnus) Monotremes Echidna (Tachyglossus aculeatus) Platypus (Ornithorhynchw anatinus)

11 12 13 14 Two variants

One variant present throughout lactation

15 16 17 18

? ?

Occurrence controversial

19

20

“References: (1) Aschaffenburg and Drewry (1957), Bell et al. (1970, 1981a), Blumberg and Tombs (1958), Gordon (1971), Grosclaude et al. (1976), Hopper and McKenzie (1973), Proctor et al. (1974); (2) Bell and McKenzie (1964), Schmidt and Ebner (1972); (3) Jenness (1982), Schmidt and Ebner (1972); (4) Beg et al. (1985), Conti et al. (1985); (5) Findlay and Brew (1972), Hanson (1960),Jenness (1982), Nagasawa et al. (1973), Phillips and Jenness (1971), R. Greenberg, unpublished observations (see Stuart et al., 1986); (6) Bell et al. (1981~); (7) Bell et al. (1981b), GodovacZimmermann et al. (1987); (8)Brew and Campbell (1967), Brew (1972); (9) Hopp and Woods (1979),Quarfoth and Jenness (1975); (10) Brown et al. (1977), Nicholas etal. (1981), Prasad et al. (1982), Qasba and Chakrabartty (1978); (11) Nagamatsu and Oka (1980), Bhattacharjee and Vonderhaar (1983); (12) Quarfoth and Jenness (1975); (13) Halliday etal. (1990); (14) Bell et al. (1980), McKenzie etal. (1983); (15) Bell et al. (1980), Brew et al. (1973); (16) Nicholas etal. (1987); (17) Shewale etal. (1984); (18) Nicholas etal. (1989); (19) Hopper and McKenzie (1974), Teahan (1986), Teahan etal. (1991a); (20) Teahan et al. (1991b).

190

HUGH A. MCKENZIE AND FREDERICK H. WHITE, JR.

their binding to phenyl-Sepharose in the presence of EDTA and their elution in the presence of Ca(I1). Nevertheless, they caution against using this procedure to determine the quantitative binding of metal ions to a-lactalbumin, because the hydrophobic support stabilizes one conformation, and the binding constants are then not comparable to those determined in free solution. It is now well known that a-lactalbumin may exhibit apparent as well as true heterogeneity. This apparent heterogeneity exhibited in electrophoresis and column chromatography probably involves interactions with buffer ions, but appears to be complex in nature (see, e.g., Gordon, 1971; Hopper, 1973; Prieels and Schlusselberg, 1977). In view of the recent demonstration that a-lactalbumin is a metalloprotein (containing calcium), and the finding by Fenna (1982a) that a-lactalbumin only gave apparent heterogeneity on columns when not fully saturated with Ca(II), it may be that the previous observations on apparent heterogeneity are related, at least in part, to this phenomenon. The occurrence of genetic variants of bovine a-lactalbumin, reflecting autosomal alleles without dominance, is now well established (Table 11). Porcine a-lactalbumin appears to be the only other a-lactalbumin for which similar genetic variation has been established (for references, see Table 11, footnote a). Hopper and McKenzie (1973) observed that each homozygote consists of a major component and three minor ones: one moving faster than the main component and two moving more slowly in electrophoresis at alkaline pH. The fast-moving minor component (F) possibly differs from the main component (M) in an amide residue. The two slower components (S, and S,) have the same amino acid composition as M, but contain a carbohydrate moiety. That of S, differs from that of S, by one sialic acid residue. These observations, although controversial at the time, were later confirmed by Proctor et al. (1974). The heterogeneity reported for other ruminant a-lactalbumins (Table 11) probably involves, at least in part, glycosylation. The rabbit and rat proteins are unusual in that the main protein is a glycoprotein in each case. The rat protein is unique, furthermore, in having an extension of the peptide chain at the carboxy-terminal end, giving a total of 140 residues versus the usual 123 residues. A necessary, but insufficient, property for a protein to be an alactalbumin is its ability to act as a specifier in the lactose synthase system. There is, at present, controversy as to whether a true a-lactalbumin occurs in the milk of monotremes. Thus, it is essential to have good methods for the determination of galactosyltransferase and lactose synthase activities. They must enable the detection of low levels of activity. This is

LYSOZYME AND a-LACTALBUMIN

191

especially important in considering the possibility of some lysozymes exhibiting weak specifier activity. It is beyond the scope of this article to make a detailed critical evaluation of available methods. Nevertheless, it is important to make several comments. In general, the determination of lactose synthase and galactosyltransferase activities will be necessary on milk samples of the species being studied during the course of fractionation, and on the isolated alactalbumin (or lysozyme). Regardless of the method used, a high-quality galactosyltransferase and a reference a-lactalbumin (usually bovine) will be necessary. It is our experience that some, although not all, commercial preparations of galactosyltransferase and a-lactalbumin are not satisfactory. The latter is frequently impure. It may contain substantial amounts of lactoferrin; indeed, one group described the isolation of lactoferrin from commercial a-lactalbumin as a convenient method of preparation of the former (Castellino et al., 1970). Thus, in general, it is preferable to use high-quality laboratory preparations. Until recently, methods for determination of both activities were essentially of two types: the determination of UDP formation enzymatically by a spectrophotometric method and the determination of the incorporation of UDP[U-14C] galactose into ["C] lactose. The first type is limited to purified systems, since crude systems catalyze the endogenous oxidation of NADH (Brodbeck and Ebner, 1966). The effects of varying conditions of pH and concentrations of substrate (glucose or N-acetyllactosamine), UDP-galactose, and MnC1, on the I4C incorporation determination of lactose synthase and galactosyltransferase in both crude systems and purified proteins have been studied, for example, by Fitzgerald et al. (1970b). The use of UTP in the inhibition of interfering hydrolase activity was discussed by McGuire (1969). In more recent procedures, products (including degradation products) and unused UDP-galactose are separated by high-voltage electrophoresis, and the substrate may be 3H-or 14C-labeled(Ram and Munjal, 1985). Hopper and McKenzie (1974), in their study of the possible ability of echidna lysozyme to act as specifier protein for the production of weak lactose synthase activity, found that the conditions of the determination needed modification for these purposes. A preliminary study was made by H. A. McKenzie and V. Muller (unpublished observations) of optimum conditions for such determinations. However, it was evident that much further work was necessary. The effect of lipids on galactosyltransferase activity has been studied by Mitranic and Moscarello (1980). More recently, Hymes and Mullinax (1984) introduced the use of HPLC in the determination of galactosyltransferase activity. This method

192

HUGH A. MCKENZIE AND FREDERICK H.WHITE, JR.

does not require radioactive substrates. Further, it permits the use of saturating levels of UDP-galactose and the monitoring of side reactions. Thus, its further development looks promising for studies of systems exhibiting weak activity.

IV. THREE-DIMENSIONAL STRUCTURE OF LYSOZYME A . X-Ray Crystal Structure of Domestic Hen Egg-White Lysozyme

As indicated in Section I, the determination of the three-dimensional structure of domestic hen egg-white lysozyme was the first elucidation of the X-ray crystal structure of an enzyme (Blake et al., 1962, 1965, 1967a; Phillips, 1966, 1967). One reviewer stated, “up until that time very little was known about its catalytic properties” (Creighton, 1984). Actually, prior to this work a good deal of important information on the nature of the linkages attacked by lysozyme had accumulated due to the important work by Ghuysen, Salton, and others. The X-ray studies indicated the nature of the active site of the enzyme and the mode of binding to inhibitors and substrate. The studies were also important in that they demonstrated the first example in a globular protein of the P-sheet structure and differences from the protein structures previously determined: myoglobin and hemoglobin. The elucidation of the structure was considerably facilitated by the determinations made in two laboratories (those of Canfield and Jolles) of the amino acid sequence, emphasizing the critical importance of adequate sequence information for X-ray studies (the electron density map did not show the individual atoms separately resolved). In summary, the main structural features of the domestic hen eggwhite lysozyme molecule (see Fig. 3) are: 1. The molecule has approximately the shape of a prolate ellipsoid,

4.5 x 3.0 x 3.0 nm (no allowance being made for bound water). It has

a deep cleft on one side. The cleft divides the molecule roughly into two lobes. The first consists of the two ends of the chain (residues 1-39 and 85- 129), while the second (comprising residues 40-84) is rather sheetlike and consists of residues either in the outer surface or lining the cleft. 2. In contrast to myoglobin and hemoglobin, lysozyme has a fairly small proportion of helix and reasonably long stretches of chain with essentially irregular conformation. Several parts of the chain have, as already mentioned, an extended conformation closely similar to the p sheet seen in fibrous proteins.

LYSOZYME AND a-LACTALBUMIN

193

FIG.3. Perspective drawing of the main-chain conformation of domestic hen egg-white lysozyme. The view is an elevation from the active-site side of the molecule (Imoto et al., 1972). Only the positions of the a-carbon atoms are shown. An instructive colored painting by I. Geis of the original three-dimensional model of lysozyme is reproduced in the early review by Phillips (1966). Of historic interest is the drawing of the model by the late Sir Lawrence Bragg (reproduced by Blake et al., 1965, and Phillips et al., 1987). It is to be noted that Bragg’s diagram is a free-hand drawing and not an accurate computergenerated representation of the molecule. (Reproduced with permission from Imoto et al., 1972.)

3. In detail: The first lobe (residues 1-39 and 85- 129)contains four helices that are close to the Pauling-Corey a-helix type, and one singleturn 310-typehelix. There are short stretches (each five to nine residues) of backbone loops and turns connecting the helices. Three a helices (helix A, residues 4- 15; helix C, residues 88-99; helix D, residues 108-115) are on the protein surface and are partially exposed to solvent. The a helix (B) consisting of residues 24-36 is totally buried. The 310helix (residues 119- 124) is partially exposed to solvent. The second lobe (residues 40-84) contains a three-stranded antiparallel P-pleated

194

H U G H A. MCKENZIE AND FREDERICK H. WHITE, JR.

sheet (residues 42-60), a small p sheet (residues 1-2 and 39-40), and a single-turn 3,, helix (residues 79-84). There is a long coiled-loop region, residues 61-78, between the large p sheet and the 3,, helix. Residues that line the cleft include p-sheet residues 43, 44, 46, 52, and 56-59, helix B residue 35, the loop connecting helices C and D (residues 98 and 101- 103), helix D residues 107- 110 and 112, and residues 62, 63, and 73. 4. Cystine bridges occur between residues 6 and 127, 30 and 115, 64 and 80, and 76 and 94. The first two pairs have negative torsion angles, but the last two pairs have positive angles. All are-in the range 100” ? 10”. 5. Following the original work by Kauzmann on hydrophobic interactions and the determinations of the structures of myoglobin and hemoglobin, it was stated, and is still stated frequently (despite evidence to the contrary), that hydrophobic residues are buried in the interior of proteins and hydrophilic residues are exposed to solvent water. It was first shown by Klotz (1970; see also Lee and Richards, 1971) that a substantial proportion of the exposed solvent-accessiblesurface area of proteins is composed of nonpolar groups. This matter has been stressed in lectures for many years by one of the authors (H. McK.) (for a discussion of various approaches to this problem, see Edsall and McKenzie, 1983). In the case of lysozyme, a substantial proportion of the hydrophobic residues Leu, Val, Ile, Ala, Gly, Phe, Tyr, Trp, Met, and Pro are either fully exposed to solvent or at least have some atoms that are solvent accessible. Examples of “hydrophobic” residues that are “surface” exposed are Val-2, Phe-3, Leu-17, Phe-34, Leu-75, Trp-123, Pro-’70, and Pro-79, with Trp-62, Trp-63, Ile-98, Trp-108, and Val-109 being on the surface of the cleft. Examples of the least-exposed ionizable side chains are Asp-66, Asp-52, Tyr-53, His-15, and Glu-35. The above summary is based on the structure of the tetragonal crystalline form of domestic hen egg white lysozyme determined at 0.2-nm (2 A) resolution. A refined high-resolution (0.15 nm, 1.5 A) study has been made by Handoll et al. (unpublished observations, quoted by Post et al., 1986). This study includes refinement of the positions for interior and surface water molecules. As shown in Table 111, other crystalline forms have been isolated and studied by X-ray crystallography. Joynson et al. (1970) studied the triclinic and tetragonal forms of hen egg-white lysozyme; Moult et al. (1976) studied the triclinic form; Hogle et al. (1981) compared monoclinic, triclinic, and tetragonal forms; and Artymiuk et al. (1982) studied the monoclinic and orthorhombic forms (see also Table 111). The results from these studies have shown essen-

LYSOZYME AND LY-LACTALBUMIN

195

tially the same conformational structure for all of these crystalline forms. However, it is important to realize that the lysozyme molecules are more closely packed in the triclinic crystals than in the tetragonal crystals. This may account for the fact that the apparent thermal factor ( B ) ' is lower in the triclinic form (B = 8) than in the tetragonal form (B = 15). In several instances long flexible side chains have very different conformations in the two structures (e.g., Arg-14, Lys-33, Phe-38, Arg-61, Arg-73, Arg-114, and Arg-128). There are also some differences in main-chain conformation, especially in the p-loop region between residues 44 and 50. Jolles and Berthou (1972) observed that tetragonal crystals of lysozyme were unstable above 25"C, especially at physiological temperatures, and transformed into orthorhombic crystals which are stable u p to 55°C (see also Berthou and Jollts, 1974). Berthou et al. (1983) found that, although the conformations obtained from orthorhombic and tetragonal forms are similar, there are differences caused by crystal contact. Thus, Trp-63 and Pro-71 are much better ordered than in the tetragonal form, where they are exposed to solvent. These differences may account for the observed difficulty of inhibitor binding in the hightemperature crystalline form, but do not seem to reflect the behavior of lysozyme in solution at the same temperature. B . Mechanism of Cell Lytic Action

We have already seen that lysozyme is a glycosidase hydrolyzing the glycosidic bond between C-1 of N-acetylmuramic acid (NAM) and C-4 of N-acetylglucosamine (NAG) of bacterial cell wall polysaccharide (Section 11,A).The polysaccharide chitin, found in crustacean shells, consists only of NAG residues joined by p( 1+4)-glycosidic links. It is also a substrate for lysozyme. The identification of the substrate binding site and the mechanism of the catalysis were not immediately evident from the original X-ray studies of lysozyme. One approach would be to apply the difference Fourier method (see Blundell and Johnson, 1976) to elucidate the structure of the enzyme-substrate complex during catalysis. Such an approach is impractical at room temperature because of the slow rate of X-Ray results provide important information regarding molecular and atomic motions, through determination of the thermal factor ( B ) ,which gives a measure of the mean square (harmonic) displacement (3) of an atom or group from its equilibrium position. The two are related by the Debye-Waller equation: B = 8+$. A highly mobile protein side chain may have a B value as high as 40 A2,corresponding to a mean square displacement of 0.5 A2 (see also the discussion in Section IV,D).

TABLE 111 Crystallografhic Data for Lysozyme and a-Lactalbumin Cell dimensions

Source Lysozyme Domestic hen egg white

Crystal form Tetragonal

Orthorhombic Monoclinic

Triclinic Tortoise (Tnmyx gangetuus) Cuvier egg white Human urine (leukemic)

Growth conditions (medium, pH, temperature)

-0.9 M NaCI, pH 4.5-4.7, 18°C 0.3-1.5 M NaClb, pH 4.3, 18°C 0.5-1.1 M KClb, pH 4.3, 18°C 0.5-1.1 M NH4Clb, pH 4.3, 18°C 0.4-1.1 M MgClz', pH 4.1, 18°C 0.5-1.2 M ammonium citrateb, pH 4.7, 18°C 0.9-1.5 M NH40Acb, pH 4.5, 18°C 1.1-1.2 M NaHzP04*, pH 4.5, 18°C 0.9 M NaCI, pH 10, RTc 0.36 M N a N Q , H N Q , pH 4.5, RT 0.77 M Na2SO4, 0.5 M NaOAc + HzS04, pH 4.5, RT 0.2 M Nal, pH 4.5, RT 0.075-0.20 M KSCNb, pH 4.5,

Space

group

P43212

P212p21 p2 I

18°C

Moll asym. unit Refs."

a (")

(i) (A) (i)

p

y

(")

(")

79.1 79.2 79.2 79.2 79.2

79.1 79.2 79.2 79.2 79.2

37.9 38.0 38.0 38.1 37.9

90 90 90 90 90

90 90 90 90 90

90 90 90 90 90

1 1 1 1

78.8 79.2 79.0 56.3 27.9

78.8 79.2 79.0 65.2 63.1

38.3 37.9 38.1 30.6 60.6

90 90 90 90 90

90 90 90 90 90.5

90 90 90 90 90

1 1 1 1 2

28.6 28.1

63.0 63.1

61.6 60.4

90 90

93.5 91.0

90 90

2 2

28.1

63.0

60.4

90

90.4

90

2

4

108.8 90

111.5 90

1 1

6

90

90

1

7,s

b

Orthorhombic

0.24 M N a N Q , 0.025 M NaOAc, pH 4.5, RT NaZHFQ4, KHzP04, pH 6.6, RT

P1 P212121

27.5 58.0

32.0 58.9

34.4 43.1

88.5 90

Orthorhombic

7 M NH4N03, pH 4.5, RT

P212121

57.1

61.0

32.9

90

1-3 4

1

2 3

3,5

a-Lactalbumin Baboon (Papio qynocephaluc) milk Human milk

Goat (Capra hircw) milk

Orthorhombic (spheroidal) Orthorhombic Orthorhombic Monoclinic

Cow milk

Triclinic Tetragonal Monoclinic Trigonal I Hexagonal Trigonal I1

-1 u satd. (NH4)2SO4 + 1 u 0.2 M PO;, pH 6.8, RT 1.8 M (NH4)2S04, 01. M PIPES, -0.006 M CaCb, pH 6.5, 35°C -1 u satd. (NH4)2SO4 + 1 u 0.2 M PO4 (or 0.1 M Tris-HCI), pH 6.6, RT, “high salt crystals” Water + minimum satd. NaCI, p H 5.3, RT, “low salt crystals” 0.5 satd. (NH4)2S04. 0.1 M Pod, p H 6.6, 25°C 0.5 satd. (NH4)2S04. 0.1 M PO4, p H 6.6, 4°C 0.5 satd. (NH&S04, 0.2 M PO,, p H 6.6, 4°C 1.9 M (NH4)2S04, 0.2 M PO,, p H 6.5, 4°C 1.8 M (NH&S04. 0.2 M PO,, p H 6.5, 35°C 1.9 M (NH4)2S04, 0.1 M PIPES, 0.01 M CaCI2, pH 6.5, 35°C

33.6 35.5* 33.6

69.6 69.1d 69.9

47.0 46.1d 47.3

90 90 90

90 90 90

90 90 90

1 1 1

67.6

109.7

68.9

90

90

90

2

45.0

89.0

32.1

90

92.6

90

2

94.7

122.9

117.9

90

116

91

32

119.6

119.6

153.2

90

90

90

8

140.7

196.7

63.2

90

111

90

24

57.4

57.4

75.0

90

90

90

I

94.0

94.0

67. I

90

90

90

1

93.7

93.7

66.9

90

90

90

2

“References: (1) Alderton and Fevold (1946),(2) Palmer et al. (1948), (3) Steinrauf (1959), (4) Ries-Kautt and Ducruix (1989). (5) Moult et al. (1976), (6)Aschaffenburg al. (1980). (7) Osserman and Lawlor (1966), (8) Banyard et al. (1974), (9) Aschaffenburg et al. (1979), (10) Fenna (1982b), (11) Aschaffenburg el al. (1972h), (12) Aschaffenburg et al. (1972a), (13) Fenna (1982a). b0.005 M in NaOAc.
198

HUGH A. MCKENZIE AND FREDERICK H. WHITE, JR.

diffusion of substrate into the enzyme crystal compared to the rate of conversion of bound substrate into product. Another approach is to cool the crystal (e.g., - 20 to - 50°C) to slow the catalytic process and to examine the enzyme-substrate complex under the low-temperature conditions. The feasibility of this cryoenzymological approach has been discussed by Douzou (1977). As far as we are aware, the only attempt to determine the feasibility of this method for lysozyme is that by Douzou et al. (1974). Blake et al. (1967b; see also Johnson and Phillips, 1965) used another approach: a difference electron density study of the complex of lysozyme with an “unreactive” analog of the substrate, compared with lysozyme. Oligomers of N-acetylglucosamine consisting of fewer than five residues either are not hydrolyzed or are hydrolyzed only very slowly, but they do bind to the enzyme. Tri-N-acetylglucosamine (tri-NAG) is a potent competitive inhibitor of lysozyme. Thus, the &-ray study (2 resolution) of the tri-NAG-lysozyme complex revealed the active site and the interactions involved. The binding of the tri-NAG was shown unequivocally to be in the enzyme cleft. The three residues (at positions designated A, B, and C) filled only one-half of the cleft. There was room for three additional residues (at positions D, E, and F); this is an important point because hexa-NAG is rapidly hydrolyzed by lysozyme. The positions (D, E, and F) of the three additional rings were deduced from model building. This is shown in Fig. 4, and a schematic diagram is given in Fig. 5. Important evidence from chemical studies considerably assisted the crystallographic work. Rupley (1967) showed that the relative rates of hydrolysis of n-mers of NAG (trimer taken as unity at M substrate concentration) are 1 (trimer), 8 (tetramer), 4 x 103 (pentamer), 3 x 104 (hexamer), 3 x lo4(octamer); that is, there is a dramatic increase in rate from (NAG)4to (NAG)5,with a further increase for (NAG)6,but a negligible change thereafter. Since (NAG)s is stable, the A-B and B-C bonds cannot be the site of cleavage for (NAG)6.Furthermore, Rupley found that lysozyme cleaves the hexamer into a tetramer and a dimer, the latter two residues being at the reducing end of the oligomer; that is, the cleavage is between residues D and E (see also Shrake and Rupley, 1980). If we consider the cleavage of the cell p( 1+-4)-glycosidiclinkage, it will be recalled that this involves the linkage between NAM and NAG residues and not that between NAG and NAM. In their model-building experiments Blake, Johnson, Phillips, and co-workers found that residue C could not be NAM because there was no room for the lactyl side chain. T h e same was true for residue E, so that only residues B, D, and F could be NAM. Since cleavage of the linkage between residues B and C has

FIG. 4. The active site of hen egg-white lysozyme, showing the mode of binding of a hexasaccharide (hexa-N-acetylchitohexaose),as proposed and developed by Blake, Johnson, Phillips, and co-workers. The sugars in sites A-C are based on the crystallographic determination of the binding of tri-N-acetylchitotriose, and those in sites D-F are based on model building. Cleavage of the hexasaccharide is considered to take place between D and E (see text). The monosaccharide N-acetylglucosamine was found to bind to site C in two slightly different orientations, one of which corresponds to the anomer (as here), the other corresponding to the binding of the a anomer (see Fig. 1B of Perkins et al., 1981). (Reproduced with permission from Perkins et al., 1981.)

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HUGH A. MCKENZIE AND FREDERICK H . WHITE, JR.

Trp-62 Trp-63

Asn-103

Asn-59

Ala-107

Asn-46

Tr p-108 Val-1 09

0

Asn-37

0

Phe-34

Arg-114

FIG. 5. The binding sites for the hexasaccharide to lysozyme, indicating residues implicated in the catalytic cleavage.

already been excluded, it was evident that the linkage between residues D and E was highly likely to be the one cleaved, and this is supported from the above chemical studies on the cleavage of (NAG)G. Further information was obtained from chemical studies in which the enzyme-catalyzed hydrolysis was performed in the presence of water enriched with l 8 0 . It was concluded that the bond split is that between C-1 of residue D and the oxygen of the glycosidic linkage to residue E. It is evident that any detailed mechanism of the catalytic action of lysozyme in the hydrolysis of glycosides would involve the role for proton donors and/or acceptors. The most plausible residues near the cleavage site are Asp-52 and Glu-35. The Asp residue lies in a polar environment, where it is a hydrogen bond acceptor in a complex network of hydrogen bonds. Because of its location, it has virtually a normal pK, of 3.5 ? 0.2. At pH 5.0, which is near the optimum pH value of hydrolysis of chitin by lysozyme, Asp-52 is in the ionized form. The Glu residue lies in a nonpolar region, has an increased pK, of 6.3 k 0.2, and would be largely un-ionized at pH 5.0. The mechanism involving these residues, as developed by Blake,Johnson, Phillips, and co-workers over the period 1965- 1972 (see also Vernon, 1967)is as follows: A hydrogen ion is donated from the carboxyl of Glu-35 to the glycosidic oxygen between the rings in sites D and E, resulting in the cleavage of the bond between C-1 of the ring at D and the glycosidic oxygen, creating a positive charge on C-1. This transient species

20 1

LYSOZYME AND OI-LACTALBUMIN

is designated as an oxocarbonium ion, which may be stabilized by the negative charge of Asp-52. With the removal of the NAG dimer from sites E and F, the carbonium ion intermediate reacts with an OH- of surrounding water. Also, Glu-35 becomes protonated, tetra-NAG moves from sites A, B, C, and D, and another round of catalysis may then occur. An essential feature of this proposed mechanism (Fig. 6, Scheme 11) is the distortion of the NAG residue at site D from its normal chain conformation. The resultant twist boat conformation enables stereoelectronic assistance to be obtained from ring oxygen 0, in the transition state, leading to cleavage of the exocyclic C,-0, bond. The overall mechanism has received support from a good deal of experimental work (see, e.g., Imoto et al., 1972; Ford et al., 1974; Banerjee et al., 1975; Rosenberg and Kirsch, 1981). When tetra-NAG is treated with lysozyme, some hexa-NAG and diNAG are formed. This reaction, transglycosylation, was first proposed by Maksimov et al. (1965) to account for hydrolysis of tri- and tetrasaccharides, during which an insoluble chitinlike carbohydrate was formed. Thus, short-chain oligosaccharides are cleaved by lysozyme, with the products transferred to longer-chain oligosaccharides, which are subsequently broken down. Much work has focused on this phenomenon,

0 Glu-35

Scheme I

\

H

\

Glu-35

Scheme 11

0

5\

Glu-35

-0

FIG. 6. Proposed reaction mechanisms by which lysozyme catalyzes the cleavage of a polysaccharide substrate. Scheme I is that proposed by Post and Karplus (1986) and indicates the possibility of cleavage with no prior assumption of distortion of the sugar ring at site D. Scheme I1 is that originally developed by Blake, Johnson, Phillips, and co-workers and involves ring distortion as a critical step. (Reproduced with permission from Post and Karplus, 1986.)

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HUGH A. MCKENZIE AND FREDERICK H . WHITE, JR.

which is well treated in the review by Imoto et al. (1972). While transglycosylation complicates kinetic studies of lysozyme catalysis, it has also indicated general support for the above mechanism. However, a good deal of recent experimental and theoretical work does not lend support for the distortion of the sugar ring at site D. In an X-ray study [0.25-nm (2.5 hi) resolution], Kelly et al. (1979) found no direct evidence for distortion of ring D in the complex between lysozyme and the trisaccharide NAM-NAG-NAM, bound in subsites B, C, and D. They concluded that it was more likely that they were looking at a Michaelis complex. In a series of papers, Pincus, Scheraga, and co-workers (see, e.g., Pincus and Scheraga, 1981) studied the conformational energies of complexes of lysozyme with oligomers of NAG and copolymers of NAG and NAM. T h e search of the conformational space at the active site of lysozyme and the minimization of the conformational energies of the complexes indicated: the hexasaccharide (NAG), binds preferentially on the “left” side of the lysozyme cleft. The fourth residue from the nonreducing end is bound in the chair form at a D site close to the surface of the cleft, somewhat removed from Glu-35 and Asp-52. The binding of the fifth and sixth residues of (NAG), at sites E and F involved residues such as Arg-45, Asn-46, and Thr-47. This D-site binding mode is in accord with the solution studies by Schindler et al. (1977) and the X-ray studies by Kelly et al. (1979). There were two other structures, but of somewhat higher energy. One of them had a distorted conformation, but the best contacts, at sites E and F, were on the “right” side of the cleft. There were contacts at site F with residues Asn-113 and Arg-114, the structure being similar to the model discussed by Imoto et al. (1972). T h e hexasaccharide (NAG-NAM),-(NAG), was also found to bind on the “left” side of the cleft. In contrast, the alternating copolymer (NAG-NAM)3 was bound with its F-site residue on the “right” side, residues such as Phe-34 and Arg-114 being involved (the lacteal side chain of NAM prevents F-site binding on the “left” side). The calculations indicated that the highest affinity of the disaccharide NAG-NAM is for sites C and D and the “right”-side sites E and F, in agreement with the experimental study by Sarma and Bott (1977). T h e conformational energy calculations received support subsequently from two types of experimental study by Smith-Gill et al. (1984). T h e affinity of ring-necked pheasant lysozyme, in which Asn and Arg at positions 113 and 114 are replaced by Lys and His, respectively, is the same for (NAG), as that of domestic hen egg-white lysozyme (i.e., the right side is not involved). They also showed that a monoclonal antibody bind-

LYSOZYME AND (Y-LACTALBUMIN

203

ing specifically to an epitope including residues Arg-45, Asn-46, Thr-47, Asp-48, and Arg-68 on the “left” side of hen egg-white lysozyme, is competitively displaced by (NAG), and (NAG),, but not by NAG, (NAG),, o r (NAG), [i.e., the terminal residues of (NAG)s and (NAG), bind to the “left” side]. In a molecular dynamics study of native and substrate-bound hen eggwhite lysozyme, Post et al. (1986) found the structural features analyzed agreed well with the results of X-ray studies at 0.15-nm ( I .5 h;) resolution, except for some surface residues. Appreciable differences were found in residue mobilities between the simulations of the native and substrate-bound states in the region of the enzyme that is in direct contact with the substrate and in a region that is distant from the active site cleft. This study enabled Post and Karplus (1986) to develop a case for an undistorted ring at site D in their proposal of an alternative pathway for lysis of the oxygen bridge between rings at sites D and E. An essential feature of their mechanism is that no twist-boat (“half-chair”) conformation for ring D is necessary, assuming certain minor rearrangements among residue side chains are made in the structure of lysozyme. Another essential feature is that, with this undistorted substrate in place within the binding site cleft, the endocyclic bond between the oxygen and C, of the ring at site D (Fig. 6, Scheme I), rather than the exocyclic bond originally proposed by Blake et al. (1967b), breaks first. T h e initial step in the reaction is protonation of ring 0, by Glu-35, followed by cleavage of the endocyclic bond C- 1-0-5 with formation of the oxocarbonium ion intermediate, stabilized by Asp-52. Hydrolysis, cleavage of the C,-OS bond, and ring closure give rise to the reaction products. T h e importance of entropic, rather than enthalpic, contributions, as in the “classic”mechanism, has been discussed by Post and Karplus (1986), who carefully stress that, although their mechanism is in accord with experimental results, it is only suggestive at this stage. Genetic engineering of hen egg-white lysozyme has been used by Kirsch et al. (1989) as an approach to studying the structure-function relationships of lysozyme. Thus, they offer evidence from site-directed mutagenesis of cloned lysozyme (expressed in yeast), that Asp-52 and Glu-35 are vital for the expression of lysozyme. However, it is curious that conversion of Asp-52 to the amide resulted in a form of the enzyme that still had 5% of the normal activity. Conversion of Glu-35 to the amide, on the other hand, resulted in a lysozyme that was devoid of all activity. It was demonstrated by mutagenesis of Asp-101 to Gly that the ionization of this residue contributes thermodynamically to the association of lysozyme with the inhibitor chitotriose.

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HUGH A. MCKENZIE AND FREDERICK

H. WHITE, JR.

Dao-pin et al. (1989) stressed that the enzymatically catalyzed hydrolysis of polysaccharides proceeds at more than five orders of magnitude faster than that for model compounds mimicking the substrate in the active site of the lysozyme. Although many workers have stressed that electrostatic interactions of specific residues with the substrate are an important feature of the mechanism, Dao-pin et al. suggest, rather, on the basis of results obtained by classical electrodynamics, that the charge distribution of the enzyme as a whole is the important feature. C . Structures of Other Lysozymes

In addition to the studies of various crystalline forms of domestic hen egg-white lysozyme, the structures of human and tortoise egg-white lysozymes have been determined (for crystal data see Table 111). Artymiuk and Blake (1981) refined the structure of the human enzyme to 1.5 8, resolution. The main objectives of this study were to determine the extent of differences in structure from that of the hen egg-white protein, to discover the location of water molecules, and to test the validity of the method of restrained refinement. The particular restrained leastsquares approach to refinement described in their paper appears to have been validated. The two proteins were found to be closely homologous, but there were small differences (e.g., in a helices), details of which can be obtained from consulting their paper. The X-ray refinement of the tortoise enzyme has been used by Blake et al. (1983) primarily to study the location of water molecules, as discussed in Section IV,D. The egg white of the Australian black swan, Cypus atratus, contains two forms of lysozyme c and g types. The X-ray structure of the g type has been determined by Isaacs et al. (1985)at 0.28-nm (2.8 A) resolution. A comparison of the structures of chicken egg white, goose egg white (g type), and bacteriophage T4 types of lysozyme has been made by Weaver et al. (1985), and the evolutionary relationships have been discussed in light of this study. This work is discussed in Section X.

D . Water in Lysozyme Crystals The total volume of protein crystals is 25-65% water, and problems that are not yet completely resolved are the nature and location of the water molecules. However, much light is being shed on the problem by high-resolution (0.12-0.18 nm; 1.2- 1.8 A) X-ray diffraction studies, supplemented more recently by neutron diffraction studies (Kossiakoff, 1985). Edsall and McKenzie (1983) have made a tentative classification

LYSOZYME A N D (Y-LACTALBUMIN

205

of the major categories of water associated with proteins, summarized as follows: 1. Outside the immediate neighborhood of the protein surface, the

water is essentially bulk water (modified only by the presence of salt ions and small organic molecules). 2. Water at the protein surface consists of three subcategories: (a) highly mobile water molecules adjacent to nonpolar surface atoms, (b) somewhat less mobile water molecules hydrogen bonded to polar groups of Ser and Thr, C=O and NH groups of the peptide chain, and (c) water molecules that are probably more retarded around groups carrying a formal charge (Lys, Arg, Asp, Glu, etc.). 3. Internal water molecules within the folded peptide chain. 4. Water molecules at the interface of subunits, for those proteins consisting of subunits. T h e results of the study by Blake et al. (1983) of human and tortoise egg-white lysozymes at high resolution is broadly consistent with the above classification. (In all such studies certain assumptions are made regarding the B factors and occupancy and displacement.) Blake et al. found that, although the tortoise lysozyme crystals had -650 water molecules per protein molecule versus 350 for the human protein molecule, the numbers of ordered water molecules were similar for both (i.e., 128 versus 140) (cf. also triclinic and tetragonal forms of domestic hen egg-white lysozyme; 110 and 140, respectively). Their results are summarized in Table IV. On average, protein oxygen atoms interact with twice as many water molecules as protein nitrogen. In conformity with results with other proteins, twice as many water molecules are hydrogenbonded to peptide C=O groups as to peptide NH. It is evident from the results of Blake et al. that, as a group, the charged side chains-Asp, Glu, Lys, and Arg-have the highest solvation. (Other results are given in detail in the original paper.) Neutron diffraction crystallographic studies of the dynamics and hydration of lysozyme are discussed in Section XI. In an investigation of the role of water in enzymic catalysis, Brooks and Karplus (1989) chose lysozyme for their study. Stochastic boundary molecular dynamics methodology was applied, with which it was possible to focus on a small part of the overall system (i.e., the active site, substrate, and surrounding solvent). It was shown that both structure and dynamics are affected by solvent. These effects are mediated through solvation of polar residues, as well as stabilization of like-charged ion pairs. Conversely, the effects of the protein on solvent dynamics and

206

HUGH A. MCKENZIE AND FREDERICK H. WHITE, JR.

TABLE IV Protein- Water Interactions in Human and Tortoise Lysozymes a Property No. of ordered water moleculesb No. making hydrogen bonds to bound water6 No. making at least two hydrogen bonds with protein* (b) No. making one hydrogen bond with protein* (c) Total no. bound to protein* [(b) + (c)] % Bound to peptide CO % Bound to peptide NH % Bound to side chains % Bound to protein oxygens % Bound to protein nitrogens Mean H20-0 distance (A) Mean H2O-N distance (8) Mean H 2 0 - H 2 0 distance (8)

Human

Tortoise

140 35 35 70 105 42 18 40 68 32 2.83 +- 0.15 2.96 t 0.12 2.82 2.0.23

128 19 33 76 109 44 18 38 64 36 2.81 2 0.15 2.96 t 0.17 2.84 t 0.23

“From work by Blake el al. (1983). Per molecule of protein.

structure were also observed to be significant. In particular, the water surrounding apolar groups is less mobile than bulk water, or the water solvating polar groups.

V. THREE-DIMENSIONAL STRUCTURE OF a-LACTALBUMIN

A. Mo&h for the Three-Dimnsional Structure of a-Lactalbumin (Based on Sequence Homology with Lysozyme) Because of the high level of identity in amino acid sequence between lysozyme and a-lactalbumin (see Fig. lo), it was inevitable that interest turned to the three-dimensional structure of a-lactalbumin when the structure of lysozyme was determined in 1965 by the group at the Royal Institution. However, there were unforeseen difficulties in the direct experimental determination, as discussed below. Hence, attention was directed to models for the a-lactalbumin structure based on the coordinates for lysozyme and on energy minimization programs. I . Model of B r o w et al. ( I 969) A wire skeletal model of lysozyme was constructed and then modified to accommodate the a-lactalbumin sequence, by changing side chains that differ between them and by rearranging the main chain to accommodate the various deletions.

LYSOZYME AND a-LACTALBUMIN

207

Browne et al. (1969) concluded that the differences between the two sequences were compatible with their having similar conformations; that is, hydrophobic side chains are usually replaced by other hydrophobic side chains, and a change in one residue is often accompanied by a compensating change in a neighboring residue. All of these changes were so readily accommodated, without producing major rearrangement of the main-chain conformation, that the proposed model for a-lactalbumin seemed likely to be substantially correct. The effect of the changes in the upper part of the substrate cleft appears to be that sites A and B are blocked off, largely a consequence of the replacement of Ala-107 (see Section VI) in lysozyme with Tyr (or His in the rabbit). These changes make it unlikely that sites A and B would remain attractive saccharide binding sites in a-lactalbumin. However, as discussed in Section VI, 107 is not always Ala in lysozyme. The lower part of the cleft, where residues E and F bind in lysozyme, is changed both in topology and in the nature of surface groups. While it appears from this model that this part of the cleft could bind saccharides, the precise mode of binding would be expected to differ from that in lysozyme. Residue 52, Asp, is invariant in lysozyme, the equivalent residue in a-lactalbumin being Glu-49. Residue 35 is Glu in lysozyme, but the equivalent residue in a-lactalbumin is variable. However, if residues 32-36 are rearranged to give maximum sequence identity: Hen egg-white lysozyme

32 33 34 35 35a 36

Ala LYS Phe Glu

J.

Ser

Bovine a-lactalbumin Thr

30

Phe His Thr Ser

31 32 33 34

I

then the equivalent bovine a-lactalbumin residue for hen egg-white lysozyme residue 35 (Glu) is His-32. This residue could assume the function of Glu-35. On the basis of this model, it might be anticipated that a-lactalbumin and lysozyme have similar biological functions, as well as similar conformations. 2. Calculations of Lewis and Scheraga (1971) The probability that particular residues start or end in helix was calculated for bovine a-lactalbumin and hen egg-white lysozyme. There is a one-to-one correspondence of location of helical regions, predicted and found for lysozyme and predicted for a-lactalbumin. Thus, the

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HUGH A. MCKENZIE AND FREDERICK H.

WHITE, JR.

overall impression is one of great similarity between the two proteins. The helical content in the carboxy-terminal region, however, is predicted to be greater for a-lactalbumin.

3. Model of W a r n et al. (1974) When Warme et al. (1974) used their method for minimizing the total potential energy of a protein, in the examination of hen egg-white lysozyme, they found a low conformational energy that still maintained a close resemblance to the original X-ray structure. Then they adapted their procedures to compute a low-energy conformation for a-lactalbumin, based on the known structure of lysozyme. The following points especially stand out. In the cleft region the His-32 side chain points down and away from the cleft region and does not occupy the position corresponding to Glu35 of lysozyme. This differs from the conclusion of Browne et al. (1969), who, as we have seen, suggested that His-32 might substitute for Glu-35 in the active site. In the Warme et al. (1974) model residue 33, T h r in bovine a-lactalbumin, but not invariant in other species, appears to correspond more closely in position to Glu-35 in lysozyme. In the upper part of the cleft region, Tyr-103 tends to block Trp-60 and Trp-104 from contact with substrates. This agrees with the finding by Browne et al. (1969), that is, that sugar binding sites A and B of lysozyme are blocked in a-lactalbumin. Reactivities of Tyr residues were found to be somewhat difficult to interpret in terms of past experimental observations, although the model and the experimental results were in “reasonable” agreement. Warme et al. (1974) concluded, for example, that Tyr-18 and Tyr-103 would be those most easily acetylated with acetylimidazole (Kronman et al., 1972a), while Tyr-36 and Tyr-50 would react more slowly. Castellino and Hill (1970) reported that the Met is readily accessible to reagents, in agreement with the model. Also in agreement were the reactivities of His residues. Thus, with iodoacetate, carboxymethylation proceeds in the order His-68 > His-32 > His-107. Helical contents are much the same as those reported for lysozyme (Robbins and Holmes, 1970; Bare1 et al., 1972). Carboxyls are, in general, more exposed in a-lactalbumin than in lysozyme (Lin, 1970), in agreement with the model. Immunochemical differences observed experimentally (i.e., no crossreactivity) are not incompatible with the model, which shows many surface differences with lysozyme. The greatest difficulty with this model, as with that of Browne et al. (1969), lay in predicting the structure of the carboxy-terminal end of

LYSOZYME AND a-LACTALBUMIN

209

a-lactalbumin: neither of the two groups was able to suggest a unique structure for this part of the molecule. Indeed, the elucidation of this structure had to await the solution of the X-ray structure.

B . X-Ray Crystal Structure of Baboon Milk a-Lactalbumin Attempts to produce crystals of bovine a-lactalbumin from the milk of Western dairy breeds of cattle for X-ray crystallographic studies met with appreciable difficulties. While crystallization from concentrated ammonium sulfate was not difficult, the crystals were very small. Thus, Aschaffenburg et'al. (1972a) were led to the study of crystallization of the goat milk protein. Freeze-dried caprine a-lactalbumin was dispersed in water and dissolved by the addition of a minimum volume of saturated NaCl solution, giving a final protein concentration of 10 g dl-I. T h e pH was adjusted to 5.3 and the solution was dialyzed against water at 4°C for several days. The mixture was then warmed to -17"C, resulting in the formation of lozenge-shaped crystals. Attempts to produce heavy metal derivatives of these crystals were not satisfactory. Accordingly, Aschaffenburg et al. (1972b) turned their attention to crystallization from concentrated ammonium sulfate solution. This resulted in crystals that gave a complex diffraction pattern, the additional reflections of which could be eliminated by soaking the crystals in 0.001 M K,PtCl,. Although the caprine crystals looked promising, they proved difficult to analyze. Hence, attention was then directed toward a-lactalbumins of other species, especially the baboon (Papio cynocephalus). Aschaffenburg et al. (1979) found the crystals to be relatively easy to prepare and suitable for structural analysis at high resolution. The turning point in the structural studies came after the work by Hiraoka et al. (1980) revealed that a-lactalbumin is a metalloprotein in which calcium is strongly bound (see also Sections VI and VII). Soon afterward three new crystal forms of bovine a-lactalbumin were isolated by Fenna (1982a), particularly trigonal Form 11. Fenna (1982b) also isolated calcium-containing crystals of human a-lactalbumin suitable for X-ray structural analyses. T h e various crystalline forms of a-lactalbumin are summarized in Table 111. In any event it was the analysis of baboon a-lactalbumin crystals for which the first X-ray crystal structure was produced, initially at 0.6 nm (6 A) and 0.45 nm (4.5 A) (Phillips et al., 1987; Smith et al., 1987). More recently, the structure has been refined at 0.17-nm (1.7-A) resolution, enabling comparison with the high-resolution c-type lysozyme structure (Acharya et al., 1989) (see Fig. 7). As already indicated, difficulties were experienced in the preparation

210

HUGH A. MCKENZIE AND FREDERICK H . WHITE, JR.

C

FIG. 7. The tertiary structure of (a) baboon a-lactalbumin and (b) domestic hen eggwhite lysozyme. (Reproduced with permission from Acharya et al., 1989; based on a program of J. P. Priestle.)

of heavy-atom derivatives of a-lactalbumin. In particular, the attempts to prepare chloroplatinite and bromoplatinite derivatives by the crystalsoaking method were disappointing. Recourse was had to the insertion of a mercury atom in the disulfide bridge linking residues 6 and 120. This involved reduction of cystine residues in the a-lactalbumin crystals soaked in another liquor containing dithiothreitol, and then in another liquid containing mercury(I1) acetate. Although the resultant electron density maps (Smith et al., 1987) did not enable a high resolution of structure, the overall features of the models of Browne et al. (1969) and Warme et al. (1974) were confirmed. Also, the identification of helix B (residues 24-36 in hen egg white lysozyme) in a-lactalbumin enabled resolution of a problem found in the

LYSOZYME AND a-LACTALBUMIN

21 1

earlier work. Although maximum identity could be achieved by deletion at residue 33 (as indicated above) with consequent loss of helix, it had been decided to take a conservative approach and retain the helix. It now appeared that the latter decision was correct. Subsequently, the baboon a-lactalbumin structure was refined at 1.7-A resolution by Acharya et al. (1989). Using the structure of domestic hen egg white lysozyme as the starting model, preliminary refinement was made using heavily constrained least-squares minimization in reciprocal space. Further refinement was made using stereochemical restraints at 1.7-A resolution to a conventional crystallographic residual of 0.22 for 1141 protein atoms. Some features of the refined structure are: 1. The human a-lactalbumin amino acid sequence was used in the refinement since the baboon sequence has not been determined, although it was known from the unpublished work by R. Greenberg to be close to the human sequence. However, it became evident in the course of the X-ray work that there were eight sequence changes in baboon a-lactalbumins (see Section VII,B). 2. The disulfide bridges are similar to those of lysozymes, with the exception of one bridge in echidna lysozymes I and 11, discussed in Section VII,B. 3. There are similarities in the helices and /3 sheets between baboon a-lactalbumin and hen egg-white lysozyme, as summarized in Table V. However, there are important differences, for example, in hen egg-white lysozyme residues 41-60 form an irregular antiparallel @pleated sheet; in this protein a residue is deleted at position 48 (human lysozyme numbering), but two residues are deleted in a-lactalbumin at positions 47 and 48 (human lysozyme numbering). Residue 47 is the most exposed to solvent in the hen egg-white lysozyme and forms part of the irregular p turn. These residues occur in a P-pleated sheet and the deletions are accommodated with minimal disruption to the pleated sheet (see the comparison in Acharya et al., 1989). 4. There are differences in the carboxy-terminal region of a-lactalbumin from lysozyme (see Acharya et al., 1989). This work resolves the inconclusive nature of the earlier models that could not resolve the structure of a-lactalbumin in this region. Also, changes occur in the loop region. 5. Of the 150 water molecules in the a-lactalbumin structure, four have been shown to be internal. Of the two cavities in a-lactalbumin, one small cavity around residues Leu-12, Phe-53, Met-90, and Ser-56 is fairly devoid of water. The second channel starts at

TABLE V Comparison of Structural E l m & for Domestic Hen Egg-White Lysorym and Baboon a-Lactalbumin" a Helix

DHEL (A) 4-15

(B) 24-36

(C) 88-99 (D) 108-115

3 10 Helix

a-LA 5- 11 (5- 16) 23-34 (25-36) 86-99 (89-103) 105- 109 (109- 1 13)

DHEL

79-84 119-124

/3 Sheet

a-LA

DHEL

a-LA

12-16 (12-18) 17-21 (19-23) 76-82 (79-85) 101-104 (105-112) 115-1 19 ( 1 19- 126)

42-60

40-43 (42-45) 47-50 (50-53)

1-2 39-40

O(A), (B), (C), and (D), a Helix A, B, C, and D. DHEL Domestic hen egg-white lysozyme; a-LA, baboon a-lactalbumin. Numbers in parentheses signify equivalent residues in domestic hen egg-white lysozyme. Based on results by Acharya et al. (1989) and by Blake et al. (1967a).

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Ile-27, runs to Asp-88, and is partially occupied by water molecules. The channel is “blocked” by Tyr-103 (which is in the cleft region). There are corresponding cavities in hen egg-white lysozyme. T h e second cavity in the vicinity of Ser-91 is occupied by internal water molecules in egg-white lysozyme. This residue becomes Asp (residue 88) in a-lactalbumin. Due to calcium binding properties in a-lactalbumin, the locations of internal water molecules are somewhat different from those in lysozymes that do not bind calcium. 6. T h e location of the bound calcium(I1) ion was unequivocally identified (see Fig. 8). This is probably the most important feature of this work and is further discussed in Sections VI and VII (see also Table IX). It should be emphasized here that the calcium-binding fold in a-lactalbumin resembles only superficially the “EF-hand” of those calcium-binding proteins that exhibit this feature (Friedberg, 1988; see also Stuart et al., 1986). 7. In the course of their nuclear Overhauser effect (NOE) studies of a-lactalbumin, Poulsen et al. (1980), and later Koga and Berliner (1985), reported that the “hydrophobic box” region of hen eggwhite lysozyme, first noted by Blake et al. (1967a), is conserved in a-lactalbumin. This is confirmed in the X-ray crystal structure: residues Ile-95, Tyr-103, Trp-104, and Trp-60 form the box. In contrast for hen egg-white lysozyme the box is composed of residues Tyr-20( 18), Tyr-23(21), Trp-28(26), Trp-108( 104), Trp-

FIG. 8. Stereo view of the Ca(I1) binding site in baboon a-lactalbumin. (Reproduced with permission from Phillips et al., 1987.)

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111(107),Leu-17(15), Ile-98(95),and Met-105(101). (The equivalent a-lactalbumin numbering is shown in parentheses.) It should be noted that Ala-107 in lysozyme is replaced by Tyr-103 in alactalbumin, with the potential of blocking saccharide binding, to which we have alluded above.

C . Conclusions In conclusion, attention is drawn to several puzzling features: the differences found in the cleft region suffice to predict that a-lactalbumin would have no cell lytic activity. It remains an anomaly, however, that weak activity has been demonstrated for a-lactalbumin from various sources by McKenzie and White (1987) (Section X), and it is an unresolved problem as to how such activity could be explained, except by the possible involvement of His-32 in a-lactalbumin as an active site residue, in place of Glu-35, which appears in lysozyme (for further discussion see Section X). In addition, there are numerous discrepancies between the reactivities of a-lactalbumin and lysozyme. The former is generally a more reactive protein (Section IX), and these differences could not have been predicted by consideration of the above models, nor from the X-ray structural analysis. BINDINGOF METALIONS IN LYSOZYME VI. COMPARATIVE AND a-LACTALBUMIN

A. Introduction We have already seen in Section V that the determination of the highresolution structure of a-lactalbumin was frustrated by a variety of problems. Eventually, the evidence for the binding of calcium in the protein crystals led the Phillips group to a structure in which the binding site is part of a specific structural feature. Proposals for the binding of calcium were heavily dependent on previous studies of calcium binding by a-lactalbumin in solution, as well as on amino acid sequence studies. It is about 10 years since the binding of calcium by a-lactalbumin was first noted, and since that time the binding has been intensively studied, resulting in a voluminous and sometimes conflicting literature. Metal ion binding by lysozyme has been studied over a somewhat longer period. A brief review of the studies of both proteins is important for comparative purposes and for elucidation of the evolutionary rela-

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tionships of the two proteins. This comparison takes on a particular significance, since it constitutes an area of contrast between the two proteins, against a background of structural similarity.

B . Metal Ion Binding to Lysozyme The first systematic investigation of the binding of a metal ion by lysozyme is probably that by Fiess and Klotz (1952), who found the affinity of five proteins for Cu(I1) at pH 6.5 to be in the order bovine a-casein > &casein > serum albumin > P-lactoglobulin > hen egg-white lysozyme. Soon afterward Carr (1953), using membrane electrodes, found that -0.7 mol of Ca(I1) was bound per mole of hen egg-white lysozyme at pH 7.4, compared with 6.7 mol of Ca(I1) per mole of bovine serum albumin. In commenting on the lack of correlation of Ca(I1) binding for different proteins with their isoelectric points, Carr ( 1953) made the perceptive statement: there is a heterogeneity in available binding spots which has not been fully explained. It is most likely that the explanation lies in the structural relationships between the various active groups as they occur in a particular protein molecule. Thus a further understanding of these interactions will await further information about protein structure such as the effect of hydroxyl and other functional groups, amino acid sequences, and the three dimensional nature of the polypeptide chains.

Some years later, McDonald and Phillips (1969) studied a shift in the nuclear magnetic resonance (NMR) spectrum of hen egg-white lysozyme induced by Co(I1) and concluded that this cation participates in coordinative binding to a single site. Gallo et al. (1971), using electron paramagnetic resonance (EPR), studied the binding of Mn(II), as well as Co(II), to lysozyme. The binding of each involved Asp-52 and Glu-35. Both metal ions are inhibitors of lysozyme activity, but Mn(I1) binds more strongly than Co(I1).Jori et al. (197 1) coordinated Zn(1I) as well as Co(I1) to lysozyme and again found Glu-35 and Asp-52 to be involved. Ikeda and Hamaguchi (1973) studied the binding of Mn(II), Co(II),and Ni(I1) to lysozyme by circular dichroism (CD) and determined their binding constants. Teichberg et al. (1974) studied the binding of Cu(I1) to lysozyme by spectrofluorometry and X-ray crystallography. With spectrofluorometry, they determined that Cu(I1) was located in the neighborhood of Trp-108, the association constant being 1.8 x lo2M - I . This observation was confirmed and extended by their use of X-ray analysis, whereby Cu(I1) was placed at 0.7 nm (7 A) from Trp-108. In addition, this cation was found to be 0.2-0.3 nm (2-3 A) from the car-

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boxyl side chain of Asp-52 and 0.5 nm (5 hi) from that of Glu-35. Secemski and Lienhard (1974), measuring proton release and ultraviolet (UV) difference spectra, found that Gd(1II) is also bound between these residues, being attached to their carboxyls at the junction of binding sites D and E, in accordance with X-ray crystallographic findings. Kurachi et al. (1975) made a crystallographic study of the positions of Mn(II), Co(II), and Gd(II1) in triclinic egg-white lysozyme. The first two of these were 0.25 nm (2.5 hi) from one of the oxygen atoms in the Glu-35 side chain. There were two Gd(II1) binding sites. The one of highest affinity was 0.32 nm (3.2 A) from an oxygen in the Glu-35 side chain, and the other was 0.32 nm (3.2 h;) from an oxygen in the Asp-52 side chain. Jones et al. (1974) determined the water-proton relaxation times of the Gd(II1)lysozyme complex in aqueous solution. Perkins et al. (1979), using X-ray analysis, also found that Gd(1II) binds at two sites, one close to Glu-35 and the other close to Asp-52 [cf. the lanthanide complexes (Dobson and Williams, 1977)l. The two sites are 0.036 nm (0.36 hi) apart. There were numerous small conformational changes on the binding of Gd(III), as well as NAG, which had been complexed with Gd(II1). Some 13 years after Carr’s original observations, Kretsinger (1976), in his review of calcium-binding proteins, assumed that lysozyme can attach Ca(II), as well as other cations. It was not until 1981 that binding of Ca(I1) to lysozyme was further studied. Imoto et al. (1981) determined the stability (association) constant (40 M - I ) and found that lysozyme is inhibited in the presence of Ca(II), showing only 26% of the activity of the free enzyme toward hexa-N-acetylglucosamine. Because of this inhibition, they predicted that Ca(I1) binds near the catalytic carboxyls. Furthermore, Ca(I1) shifts the native-denatured transition in lysozyme toward the native state, and thus has some preservative effect on the protein. We will see in Sections VII and X that the recent elucidation by X-ray crystallography of the binding sites for Ca(I1) in baboon a-lactalbumin has led to a flurry of studies of potential binding by variants of lysozyme in a wide range of species.

C . Metal Ion Binding to a-lactulbumin

The first substantive report of the binding of Ca(I1) by cw-lactalbumin appears to be that by Hiraoka et al. (1980), who found that there is one site to which Ca(I1) is strongly bound in this protein, and some evidence of other weak binding sites. They concluded that a-lactalbumin is a cal-

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cium metalloprotein, and that calcium stabilizes the protein against unfolding by heat and by guanidine hydrochloride. This work led to an investigation by Permyakov et al. (1981), who studied a low-pH conformational shift involved in binding of one Ca(I1) to a-lactalbumin, which caused a change in the Trp fluorescence quantum yield and a spectral shift toward shorter wavelengths. They concluded that the shift at low pH resulted from competitive replacement of the bound Ca(I1) by hydrogen ions. On the basis of fluorescence changes during EGTA {[ethylenebis(oxyethylenenitri1o)ltetra-aceticacid}titration of Ca(I1)-a-lactalbumin and in pH titrations, Permyakov et al. (1981)found that the first association (stability) constant (&) for Ca(I1) binding by bovine a-lactalbumin is 4.5 ( & 1.5) x lo* M - I . Furthermore, Van-Ceunebroeck et al. (1985) found Ks,lfor bovine a-lactalbumin to be greater than lo7M - I . Herein lies a major difference between the 1: 1 binding of Ca(I1)by bovine a-lactalbumin and domestic hen egg white, the ratio of the two association constants being on the order of lo7:1. (The values of Ks,l determined by Kronman’s group would give a ratio of lo5: 1, for which see below.) During the past 10 years Berliner and associates have made an extensive study of the binding of metal ions by a-lactalbumin and their role in the action of lactose synthase (for review see Berliner and Johnson, 1988).This work includes a study by Murakami et al. (1982) of the binding strength of Ca(I1) by bovine, caprine, human, and guinea pig alactalbumins. They found that & for Ca(I1) is of the order of 1010-1012 M-1, and that for Mn(I1) is -lo6 M-’. They also concluded, on the basis of hypsochromic wavelength shift and quenching of Trp fluorescence, that the metal ion induced a conformational shift. As well as the strong binding site, they found evidence of three weaker binding sites. Finally, they stressed the need for determination of the equilibrium constants by a method such as ESR, in addition to the fluorescence method, in order to avoid potential errors. Soon afterward, Kronman, who has made a long study of a-lactalbumin reactions, considered that there was an experimental artifact in the use of chelating metal ion buffers (e.g., EGTA and EDTA) in the determination of association constants for metal ions with proteins by fluorescence titration. Kronman and Bratcher ( 1983)concluded that their observations explained the discrepancy between K 1for Ca(I1)and bovine a-lactalbumin reported by Kronman et al. (1981) (2.7 X lo6 M - l ) , Permyakov et al. (1981) (6.3 x lo8 M - l ) , and Murakami et al. (1982) (4 x lo9 M - I ) . Some years later a strong rebuttal was made by these groups to the criticism by Kronman and Bratcher (1983). Permyakov et al. (1987) stated that there is no valid evidence of artifact in their determinations and

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HUGH A. MCKENZIE AND FREDERICK H. WHITE, JR.

reiterated that the value of K S , ] for , Ca(I1) and bovine a-lactalbumin, is in the range of 0.25- 1.0 X lo9 M-I. Kronman (1989) has reiterated his criticism in his recent review of metal ion binding by a-lactalbumin. In the meantime Japanese and Dutch groups also made determinations of Ks,,. Segawa and Sugai (1983) concluded that bovine, human, and caprine a-lactalbumins “prepared by ordinary methods” contain 1.1-1.3 Ca(I1) ions per protein molecule and that the removal of the calcium “destabilizes the tertiary structures in these proteins.” They concluded, on the basis of changes in CD ellipticity, that KS,] values for these proteins are, respectively, 2.5 X lo8 M - I , 3.0 x lo8 M - l , and 2.8 X lo8 M-I. Later, Hamano et al. (1986), using a calcium-sensitive electrode, determined K s s 1in , 0.06 M Tris buffer (pH 7.8-8.5) in the presence of varying concentrations of NaC1. They found Ks,, for Ca(I1) and Na(1) to be 2.2 (+0.5) x lo7 M-I and 99 (&33) M - l , respectively, at pH 8.0 and 37°C. More nearly in agreement with Bratcher and Kronman (1984) are the results of Schaer et al. (1985), who found Ks,l for Ca(I1) binding to be from 1.2 X lo6 M-I to 2.5 X lo6 M - I , depending on the means of separation of the metal ion from the protein. The wide range of values for K , , is considered again in Section XI. D. Structural Changes on Cation Binding ly a-Lactalbumin and Their Implications in Lactose S y n t h e Activity

As indicated in Section III,B, Kronman and collaborators, in their early spectroscopic and sedimentation studies of a-lactalbumin, noted changes in a-lactalbumin as the pH was lowered below -4.0. Later, Kronman et al. (1972a,b) found that the low pH form (currently called an A form) differs from the native form (called the N form) in being somewhat less compactly folded and in a number of other properties; for example, there are changes in the environment of the tryptophan residues, but with no changes in their average extent of exposure to solvent. The nature of these changes and the origin of the terms N and A are considered in Section IX. It suffices to mention at this point that the N 4 A transition can be produced by a variety of conditions. It is now believed that the transition usually involves the dissociation of Ca(I1) from the a-lactalbumin. More recently, Kronman and Bratcher (1984) found that Tb(II1) displaces Ca(1I) in a-lactalbumin. With increasing concentration, Tb(II1) binds to a second site with a concomitant decrease in affinity for metal ion binding to the first site, resulting in a decreased stability of the native conformation (or N) conformer, and thus renders more favorable the

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conversion of a-lactalbumin to an “A-like” state, as determined by fluorescence measurements. The term “A state” was used first by Kuwajima (1977) and appears to be synonymous with “U state,” which had been used by Kronman and co-workers (e.g., Kronman et al., 1972a,b, 1981) to denote not only the conformational state that results from acid denaturation, but also that which results from Ca(I1) removal. Much work has focused on partially folded conformers of this protein (for more discussion see Section IX,A, E, F, and H). Kronman and Bratcher (1984) found additionally that a third, weaker, binding site exists for Tb(II1) in a-lactalbumin, and concomitant with this binding was a further conformational change, as judged by fluorescence properties, which they termed the “expanded” A-like state. Kronman and Bratcher (1984) found two binding sites for Zn(I1) in bovine a-lactalbumin. At the site of lower affinity, Zn(I1) caused conversion to the expanded A-like state [presumably the same as that seen also (above) with Tb(II1) binding]. There appear to be three binding sites in a-lactalbumin for Mn(I1). Of particular interest in the study of effects of metal ion binding on the conformation of a-lactalbumin are the contributions of Berliner and co-workers. Murakami et al. (1982), in a study of bovine, caprine, human, and guinea pig a-lactalbumin, observed metal ion-induced conformational change resulting in a unique hypsochromic shift and quenching of tryptophan fluorescence. They found that Ca(I1) and the lanthanides Tb(III), Eu(III), Gd(III), Yb(III), Pr(III), and Dy(II1) could be bound extremely strongly to a specific site. They also found that Mn(II), Ca(II), and Mg(I1) could be weakly bound to the same site. Murakami and Berliner (1983) later reported the existence of a zinc binding site in bovine, human, guinea pig, and rabbit a-lactalbumins, in which the zinc site is physically distinct from the site for binding calcium. This proposal was supported by the fact that when a cation binds to one site, the ensuing conformational shift excludes binding to the other site. All metal ions that were bound to apo-a-lactalbumin at the calcium site caused the same fluorescence shift. Titration of Ca(I1) or Mn(I1) protein with Zn(I1) or Al(II1) caused a complete return to apo-a-lactalbumin fluorescence parameters. I n contrast, titration of apo-a-lactalbumin with Zn(I1) caused no change in fluorescence parameters. Berliner et al. (1983) sabstituted l13Cd(II) or Mn(I1) for Ca(1I) in bovine and caprine a-lactalbumins. On the basis of NMR and ESR studies, respectively, of the 113Cd(II)and Mn(I1) proteins, they concluded that coordination to the metal ions was through oxygen. They considered the relationship of the binding site in a-lactalbumin to the “EF-hand domain” in calcium binding sites, as discussed in Section V.

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By studying the binding of the fluorescent probe, 4,4’-bis[l-(phenylamino)-8-naphthalene sulfonate] (bis-ANS), Musci and Berliner (1985a) were able to differentiate between a new apo-like conformation, which was locked in by binding with either Zn(I1) or AI(III), the true apo form with no metal ion attached, and that induced by the binding of Ca(I1). They concluded that their experimental evidence enabled a distinction to be made between site I, the calcium binding site, and site 11, the site that binds Zn(I1). T h e results also were suggestive of a-lactalbumin possessing an hydrophobic surface that becomes somewhat less accessible on 1: 1 calcium binding in the absence of metal ions that bind to site I1 [see also Desmet et al. (1987) in Section IX,A for further use of bis-ANS in the study of a-lactalbumin]. Musci and Berliner (1986), using Forster energy transfer measurements between donor Eu(1I) or Tb(II1) at site I and acceptor [Co(II)] at site 11, estimated the distance between these sites to be 11.5 _t 1.5 A. They also measured the distance between the locus of bis-ANS and Co(I1) at site I1 to be 13.6 +- 1.0 h;. Also determined was the distance between bis-ANS and a fluorescein moiety covalently bound to Met-90, which was 33.5 & 3.1 A, and between Met-90 and Co(I1) at site 11, which was 16.7 & l . O h ; . Further determinations of intramolecular distances have been made by Musci et al. (1987). Met-90 in a-lactalbumin was spin-labeled. Paramagnetic line broadening of the spin-labeled ESR lines by Gd(III), substituted at the high-affinity site, yielded a distance of 8 f 1 h; between the spin label and the metal binding site. Distances between the Met and several resolvable protons were also determined from paramagnetic line broadening, with the use of NMR. Musci and Berliner (1985b) concluded that apo-a-lactalbumin is more efficient as the modifier protein in the lactose synthase system than is the Ca(I1)-bound form. They found that V, for the apo form shows a 3.5-fold increase over that for the Ca(1I)-bound form, but there is no difference in K, (app.) between the two forms. They also confirmed that calcium stabilizes the protein against thermal denaturation (see Section IX,E), but that zinc is crucial in shifting the protein toward the apo-like form that is optimally active in lactose synthase. Their model is summarized schematically in Fig. 9. The question as to possible differences in conformation between the apo and Ca(I1)-bound forms of a-lactalbumin was also addressed by Kuwajima et al. (1986), who found that the Ca(I1)-bound and free forms can assume essentially the same folded conformation, as evidenced by similarity in their CD and proton NMR spectra. However, on the basis of CD studies of aromatic side-chain effects, they concluded that the stability of the folded state is markedly enhanced by Ca(I1).

22 1

LYSOZYME AND a-LACTALBUMIN

11

11

FIG. 9. Conformational states of a-lactalbumin in solution, as suggested by Musci and Berliner (1985b). (Reproduced with permission from Musci and Berliner, 1985b.)

In contrast to these findings are those by Van Ceunebroeck et al. (1986), who used a 1251-labeledhydrophobic dye in the study of the apo and Ca(I1)-bound forms of bovine a-lactalbumin. The former protein was more heavily labeled with the dye than the latter, and a larger hydrophobic surface was therefore concluded to be exposed in the absence of Ca(I1). Some other recent studies have been concerned with the effects of monovalent cations. Hiraoka and Sugai (1984) showed that one Na(1) ion binds to a specific site in a-lactalbumin, presumably the Ca(I1) binding site. The bound Na(1) stabilizes the native form of the protein. Hiraoka and Sugai (1985) reported that both Na(1) and K(I) stabilize the nativelike state of a-lactalbumin. However, the conformational change induced by these ions, from the partially unfolded apo form to the native form, is slow compared to that brought about by Ca(I1). Permyakov et al. (1985) studied the binding of Na(1) and K(I), as well as of Ca(I1) and Mg(II), to bovine a-lactalbumin by intrinsic protein fluorescence. Urea- and alkali-induced unfolding transitions involve stable partially unfolded intermediates for the ion-bound forms of this protein (see also Section IX,E).

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E . Metal Ion Binding in a-Lactulbumin: Implications for Lysozyme The studies in solution, by several schools, of the binding of calcium to a-lactalbumin, as well as the realization that crystals of a-lactalbumin (as ordinarily prepared) contain calcium, were of great importance in the elucidation of the three-dimensional structure of baboon alactalbumin by Phillips and co-workers, as already indicated. At the same time the precise delineation of the calcium binding site, discussed in Section VII (see also Table IX and Fig. 8), naturally led to the consideration of the minimum number of these residues that must be present and the relevant conformation of the peptide chain to enable calcium binding to occur. Questions that arise immediately are: Can any Iysozyme bind Ca(I1) in a comparable manner? To what extent can lysozymes exhibit weak lactose synthase activity and a-lactalbumins exhibit weak lytic activity? A crucial issue in the binding of Ca(I1) and other metal ions to a-lactalbumin is the number and nature of the binding sites. D. C. Phillips (personal communication, 1989) only found evidence for one binding site in crystalline a-lactalbumin. In a comprehensive review of the binding of metal ions to a-lactalbumin, Kronman (1989) postulates up to six ion binding sites. This is considered in Section XI. Many years ago Hopper and McKenzie (1974) noted structural similarities between equine and echidna lysozymes. They also obtained some evidence, albeit controversial, of a weak ability of echidna lysozyme to act as a modifier in the lactose synthase system. More recently, McKenzie and White (1987) noted very weak lytic activity in a variety of a-lactalbumin preparations. Also, Teahan et al. (1986, 1990) confirmed certain essential structural features for Ca(I1) binding in echidna lysozymes I and 11 and noted the potential binding of Ca(I1) by equine and pigeon lysozymes. D. C. Shaw and R. Tellam (quoted by Godovac-Zimmermann et al., 1987) made preliminary fluorometric observations that indicated binding of Ca(I1) by echidna and equine lysozymes. Subsequently, Nitta et al. (1987) concluded that equine lysozyme was a metalloprotein, containing one Ca(I1) ion per molecule. They recently determined K , , for binding of Ca(I1) by equine and pigeon lysozymes to be 2 X lo6 M-I and 1.6 X lo7 M - l , respectively (Nitta et al., 1988). More recently Desmet et al. (1989) found that removal of Ca(I1) from equine lysozyme induces a small but significant change in CD behavior, indicating a slightly unfolded apo conformation, apparently similar to that of the apo form of a-lactalbumin. Sugai et al. (1988), Nitta and Sugai (1989), and Acharya et al. (1991) have discussed the evolution of metal binding sites in proteins. We leave

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an assessment of the evolutionary aspects for discussion in Section X. In the interim we make a few other comments. Some additional comparative inference may lie in the nature of the research thus far conducted on these proteins. It is seen that, for lysozyme, emphasis has been mostly in the direction of determining the cation-binding loci for the various metal ions studied. With a-lactalbumin, on the other hand, studies of conformational changes occurring upon addition or removal of cations have been heavily emphasized in the literature, probably because such changes occur readily for alactalbumin and thus are more in evidence than for lysozyme. Hence, alactalbumin may possess greater conformational flexibility, with greater adaptability to complex formation, as is evident, for example, in its ability to combine with galactosyltransferase to form lactose synthase. That a-lactalbumin is inherently more susceptible to denaturative influences and other reactions is well established (see Section IX,E). What purpose metal ion binding may serve for lysozyme and alactalbumin in nature is largely unclear, despite proposals by some authors that cations may serve to stabilize a given conformational structure, as well as exerting control over their activities by inhibitory effects. According to Lonnerdahl and Glazier (1985), only 1% of the calcium content of human milk and 0.15% of the calcium content of cow milk are bound to a-lactalbumin. Hence, this protein is quantitatively unimportant for calcium nutrition of the infant. They point out, rather, that the primary role of calcium may be to regulate lactose synthesis and possibly to aid in the secretion of a-lactalbumin. On the other hand, Rao and Brew (1989) have found that Ca(1I) is essential for the formation of correct disulfide bonds and the development of native conformation. They suggest that Ca(I1) may function to guide the folding of the nascent protein. Musci and Berliner (1985b) have suggested that a balance between Ca(I1) and Zn(I1) may serve to “fine-tune” the protein conformation, affecting the release of this protein from binding with the membrane of the endoplasmic reticulum, as well as its modifier activity. AND SEQUENCE HOMOLOGIES IN VII. AMINOACIDCOMPOSITION LYSOZYME AND a-LACTALBUMIN

A . Amino Acid Compositions

In early comparative studies of proteins, both those of the same protein from different species and of genetic variants of a protein within a

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given species, it was necessary to compare their amino acid compositions, because sequence information was not available. Such comparisons had limitations: At best, they enabled workers to gain some idea of the lower limit to the number of differences to be expected in an amino acid sequence (see, e.g., Cornish-Bowden, 1979). Later, the experimental determination of sequences became easier, with respect both to speed of sequencing in automated sequencers and to the improvement in sensitivity, enabling much lower amounts of protein to be sequenced. Hence, attention was naturally directed to sequence determination, but to some extent relationships of particular groups of residues in evolution may be lost from sight in the emphasis on identity of residues in individual positions. Hence, we have made comparisons of amino acid compositions of alactalbumins (Table VI), mammalian c-type lysozymes (Table VII), and egg-white lysozymes (a variety of c type and one g type) (Table VIII). Where the sequence information is available, the compositions have been deduced from these results; otherwise, the amino acid compositions are obtained from amino acid analysis of the protein. The residues have been listed in the tables in the following order: the acidic amino acids (Asp and Glu) and their amides (Asn and Gln) are listed first, followed by His and then the basic amino acids (Lys and Arg). They are followed by the remaining amino acids in, broadly, their order of increasing hydrophobicity. This order is a crude “consensus” order based on the several hydrophobicity scales discussed by Edsall and McKenzie (1983). T h e comparison of the amino acid compositions may be summarized as follows: 1. On the basis of monomer molecular weights (from sedimentation-equilibrium and sedimentation-diffusion studies, amino acid sequences and compositions), the a-lactalbumins, with one exception (rat a-lactalbumin, 140 residues, see Section VII,B), have a single chain of 123 residues and M , values of 14,000. The mammalian lysozymes have 128- 130 residues and M , values of -14,400, except echidna lysozyme, which has -125 residues. The c-type hen egg-white lysozymes have -127-131 residues, in contrast to the g type, which has -185 residues. 2. All a-lactalbumins and c-type lysozymes have eight half-cystine residues (four disulfide bridges). There have been no reports of the presence of cysteine. There has been one report of a bovine a-lactalbumin having six half-cystines (Barman, 1973). As far as we know, no further work has been done on this variant. 3. In the a-lactalbumins (with the exception of the rat) the sum of

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the number of Asp and Glu residues (Asp + Glu) exceeds the sum of the Lys and Arg residues (Lys Arg) by five to nine residues (mean, seven residues). This difference is reflected in their low isoelectric points of pH -4.5. In contrast, for c-type hen egg-white lysozymes Asp Glu is substantially less than Lys + Arg ( - 6 to - 11; mean, - 8), leading to high isoelectric points. The position for mammalian c-type lysozymes is more complex. Human milk, rat urine, pig stomach mucosa, and echidna milk lysozymes have differences ranging from -6 to -8 residues, with a mean of -7. These lysozymes all have high isoelectric points (pH 11). In contrast, lysozymes from bovine stomach c z , baboon milk, equine milk, and deer stomach have differences ranging from - 1 to + 3 residues, with a mean of +2. This difference is reflected, for example, in the estimated isoelectric point for bovine c p , pH 7.6 k 0.2 (experimental value pH, 7.5 k 0.1) and the low pH for optimum catalytic activity. Another feature of the lysozymes is the marked variation in LysIArg ratios. These unusual differences and their significance are discussed in Section X. 4. In the 13 a-lactalbumins listed in Table Vl, seven have His contents of three residues per molecule, four have four residues, and two have two residues. Four of the mammalian lysozymes have two His residues, the remainder varying from one to five residues. Four of the hen egg-white proteins have no His residues, the remainder varying from one to five residues. 5. The Pro content of five a-lactalbumins is two residues per molecule; the remainder (with the exception of rat) are also low in Pro (one to three residues). Most of the hen egg-white lysozymes have two Pro residues, the remaining egg white and mammalian lysozymes ranging from one to five residues (except canine spleen). 6. Two variants of a-lactalbumin, caprine and ovine, have no Met residues, indicating that this residue plays no direct role in the lactose synthase system. Of the lysozymes only baboon milk and pigeon eggwhite lysozymes have no Met residues. 7. The numbers of Tyr, Trp, and Phe show small variation in alactalbumins, but appreciably greater variation in lysozymes. The greatest of the latter variations is the absence of phenylalanine in chachalaca lysozyme (see also Section VI1,B).

+

+

-

B . Sequence Comparisons The sequence comparisons of a-lactalbumins and of mammalian and avian c-type lysozymes that have been made in this article are summarized schematically in Fig. 10. In general, as far as practicable, only those sequences that have been

TABLE VI Amino Acid Compositions of a-Lactalbuminsfrom Vanow Mammals Number of Residues per Monomer Amino acid residue Asp

+ Asn

Glu

+ Gln

His LYS

'4% Pro M cys Met Ser Thr GlY Ala

Bovine B

Caprine

Ovine

13+8 = 21 8+ 5 = 13 3 12 1 2 8 1 7 7 6 3

1 4 + 8 = 22 6+ 7 = 13 3 13 1 2 8 0 6 6 5 5

1 4 + 8 = 22 6+ 7 = 13 3 13 1 2 8 0 5 5 5 6

Porcine B 21 11

3 11 1 1 8 4 6 7 7 3

Camel

Human

1 3 + 9 = 22 10+ 4 = 14 3 13 3 1 8 3 6 5 7 3

1 2 + 4 = 16 8+ 7 = 15 2 12 1 2 8 2 8 7 6 5

Equine A

Guinea Pig

1 1 + 6 = 17 8+ 6 = 14 2 12 2 3

1 6 + 4 = 20 6+ 6 = 12 4 11 2 2

8

8

3 8

7 7 2

1 8 6 4 5

Red Red-necked kanWallaby garoo

Rabbit

Rat

1 0 + 9 = 19 9+ 5 = 14 3 12 2 3 8 2 8 10 5 2

1 2 + 5 = 17 15+ 3 = 18 3 10

2

7 8 2 9 7 8 9

1 1 + 5 = 16 9+ 8 = 17 4 9 2 3 8 2 7 4 7 6

Grey kangaroo

16

16

19

19

4 10 3 3 8 2 7 5 7 6

4 10 3 4" 8 3 7 5 7 6

Leu Val lle Tyr Trp Phe Total Methodb, Refc Other variants

.I

13 6 8 4

4

4

13 6 8 4 4 4

13 5 7 4 4 4

12 2 10 4 4 4

11 2 10 3 5 4

14

2

12 4 3 4

13 4 9 4 4 4 123

s (7)

14 3 12 5 3 3

13 4 8 2 4 3

9 10 8 4 4 5

11 5 10 3 3 4 121

I1 5 9 3 3 5

-126

11 5 9 3 3 5 -127

S (11) A (12) A ( 1 2 )

'Very approximate value. bMethods for deriving composition: S, T h e composition has been determined from the complete amino acid sequence; A, the composition has been obtained from amino acid analysis. (References: ( I ) Brew et al. (1970), Vanaman el al. (1970, Shewale et al. (1984); (2) MacGillivray et al. (1979), Shewale et al. (1984); (3)Gaye et al. (1987); (4) Bell et al. (1981~); (5) Beget al., (1985); (6) Findlay and Brew (1972), Hall etal. (1982); (7) Kaminogawa et al. (1982, 1984);(8) Brew (1972), Hall etal. (1982); (9) Hopp and Woods (1979); (10) Prasad etal. (1982); (11) Shewale etal. (1984); (12) McKenzie elal. (1983). dThree genetic variants: A, B, and C. A differs from B by substitution of Gln for Arg at position 10 (Bell et al., 1970); substitution in C is not known (Bell et al., 1981a). Minor components: see Section lI1,B. 'Minor components have been identified by Schmidt and Ebner (1972); see Section Il1,B. The amino acid analysis values of 3 for Pro in caprine and ovine [given by Schmidt and Ebner (1971)] are too high. fTwo genetic variants: A and B. A differs from B in having as its amino-terminal residue Arg instead of Lys (Bell et al., 1981c). The value for Pro in the B variant is given correctly in Table 4 of Bell et al. (1981c), but it is printed incorrectly as 1.9, instead of 1.0, in Table 3. The value for Pro in the A variant has not been determined precisely, but it is assumed to be the same as for B. ZGodovac-Zimmermann et al. (1987) have examined two variants, B and C, from equine colostrum, having three and four differences from A, respectively. hHopp and Woods (1979) showed that rabbit a-lactalhumin is a glycoprotein. 'Brown el al. (1977) found that rat a-lactalbumin contains 13.4% (w/w) carbohydrate.

TABLE VII Amino Acid Compositions of Mammalian c-Type Lysozymes N N

00

Number of Residues per Monomer Amino acid residue Asp+Asn Glu

+ Gln

His LYS '4% Pro VZ cys Met Ser Thr GlY Ala

Human milk, leukemic Baboon urine milk 8+10 = 18 3 6

+

= 9

1 5 14

2 8

9+11 = 20 3 + 8 = 11 3 5 8 3 8

Horse milk

Rat urine

10+13 = 23 6+ 2 = 8 2 15

9 + 9 = 18 3+ 9

4

1

8

2 6

0

7

13

11

10

7

5

14

6

12

4

1

11

=

12

2

6

12 4

8 1

7

6

10 11

Bovine stomach mucosa c2

Deer stomach mucosa

Langur stomach mucosa

Pig stomach mucosa 3

7 + 8

7 + 8 = 15

7+11 = 18 5 + 5

1 0 + 9 = 19 3+ 6 = 9 2 13

= 15

8+ 2

= 10

4

11 3 2 8 1

13 8 8 10

9+ 3 =

2 8

10 2 9 6 3 8

I

0

12

3

10 4

10

9 10 9

=

8 5

I1

13

Rabbit spleen

9 + 9 = 18 3+ 4

20

18

17

11

10

13

1

5

= 7

3 8

5 15 3 3 8

1 10 4 8 10

9 9 9 8

7

Grey kangaroo

Echidna milk 1

1

1

6 6 5 8 2 9 7

12 12

Canine spleen

8 8

5 9

8 2 9

8

8

7

10 15

1

1

7 6

10 10

Leu Val Ile TYr TrP Phe Total Method," Ref Other variants

r c

(0

8 9 5 6 5 2

8 9 7 6 5

10 5 3

2

5 5

7 8 4 2

130

130

129

s (1)

s (2)

s (3)

4

C

6 6

9 9 5 5

9 9 5 5

2

6 9 7 6 5 3

10 3 7 4 4 2

8

10 6 6

2

6 2

130

129

129

130

130

125

s (4)

s (5)

S (6)

s (7)

S (6)

S (8)

e

f

6

d

5

10 6 7 3 2 3 -130 A(9)

9 9 5

3 6 3

8 8 4 4 5 3

-139

-124

A(10)

A(11)

"Methods for deriving composition: S, The composition has been determined from the complete amino acid sequence; A, the composition has been obtained from amino acid analysis. 'References: Jo1lt.s and Jolles (1971, 1972), Canfield etal. (1971), Thomsen etal. (1972); (2) Hermann etal. (1973); (3) McKenzie and Shaw (1982, 1985); (4) White etal. (1977); (5) Jollts et aE. (1984), A. C. Wilson (personal communication, 1983); (6)Jolles etal. (1989); (7) Stewart etal. (1987), includescorrection given by Jolles etal. (1989); (8) Teahan et d.(199lb); (9) Jolles and Fromageot (1954); (1O)Jolles and Ledieu (1959); McKenzie et al. (1983). [ A similar, but not identical, lysozyme has been isolated from donkey milk by Godovac-Zimmermann el al. (1988). d T ~ other o variants, c, and q ,have been identified by Dobson rt al. (1984) and by Jolles et al. (1984).
TABLE VIII Amino Acid Comflositiom of Avian Egg-White Lysozymes ~

~~~~

~~~~~~~~~~~

Number of Residues per Monomer

10 03

0

Amino acid residue Asp Glu

+ Asn

+ Gln

His LYS

A% Pro Y2 cys Met Ser Thr GlY Ala

Black swan Domestic hen 74-14 = 21 2+ 3

= 5 1

6 11 2 8 2 10 7 12 12

C

17 8 0 11 10 2 8 2 8 8 11 11

California Bobwhite quail quail

g 1 2 + 8 = 20 6 + 8 =14

5 18 11 5 4 3 9 14 21 15

7+14 = 21 2 + 2 = 4 2 6 11 2 8 2 10 7 12 12

8+13 = 21a 2+ 3 = 50

1 7 10 2 8

Turkey 7+13 =

20"

2+ 1 =

3a 2 7 10

2 8

2

2

10 7 12 12

10 7 13 13

Ringnecked pheasant 8+12 = 20 2+ 1 = 3 2 8 9 2 8 3 10 7 14 11

Guinea hen

Kaki duck 11

Peking duck 1

8+12 = 20 2 + 3

8+11

8+11 =19 3 + 2 = 5

=

5 2 8 10 2 8 2 10 7 12 12

=I9

4 + 1 = 5 0

6

13

2 8

0 6

13 2 8

2

2

11 7 12 11

11 7 12 11

Chachalaca 8+11 = 19

2 + 2 =

Japanese quail 9+13 = 22" 2+ 3

4

= 5 a

2

0 5 12

9 10 2 8 3 10

7

11 12

2 8

2

10

7

13 12

Pigeon 8+11 = 19 4+ 3 = 7 2 13 10 3 8 0 7 5 11 8

7 11 13 9 3 3

8 7 5 3 6 3

8 7 5 3 6 3

9 5 6 4 6 2

8 7 6 4 6 2

7 7 5 3 6 3

8 7 6 5 6 1

8 7 6 5 6 1

7 5 7 7 6 0

8 5 8 2 6 4

129

185

129

129

129

130

129

129

129

129

131

A (2)

S (3)

S (4)

S, P (5)

S, P ( 6 )

S (7)

S, P (8)

Leu Val Ile T Yr TrP Phe Total Method? Ref' Other variants

s (9) s, P ( 1 0 ) d

127

s (11) s, P(12)

d

'Exact distributions of Asp-Asn and of Glu-Gln are uncertain. "Methods for deriving composition: S, the composition has been determined from complete amino acid sequences; A, the composition has been determined from amino acid analysis; P, some of the residues have been obtained by peptide compositions and/or assumed identities. 'References: (1) Canfield (1963), Jolles et al. (1963), Canfield and Liu (1965), Imoto et al. (1972), Rees and Offord (1972), Phillips (1974), Ibrahimi et al. (1979; (2) Arnheim et al. (1973); (3) Simpson et al. (1980); (4) Ibrahimi et al. (1979); (5) Prager et al. (1972); (6) La Rue and Speck (1970); (7) Jollks et al. (1979a); (8)Jollks et al. (1972); (9) Hermann and Jolles (1970); Hermann etal. (1971); (10) Kondo etal. (1982); (11)Jolles etal. (1976); (12) Kaneda etal. (1969); (13) Rodriguez etal. (1985). dTwovariants of Kaki duck-namely, I1 and 111-and three variants of Peking duck-namely, 1, 2, and 3-have been studied. For their proposed relationships see Section VI.

232

HUGH A. MCKENZIE AND FREDERICK H . WHITE, JR.

completely determined are included in the comparisons. It has become evident, as substantial numbers of these proteins have been sequenced, that there is sufficient variation in sequence that it is unsatisfactory to designate residues (especially critical residues) on the basis of assumed identity or amino acid composition of peptides. However, in some lysozymes, as indicated below, we have had to make certain assumptions, especially in the designation of some Asp, Asn, Glu, and Gln residues. With improved methodology for sequence determination, important corrections have been made to some original sequences (as shown below). The basis of the individual sequences presented is summarized as follows (the notes are numbered to refer to the numbers in parentheses on Fig. 10, indicating the variant of the protein for which the sequence appears in Fig. 10). 1 . Bovine [Bos (Bos}] a-Lactalbumin

The sequence of 123 residues shown is that of the common B variant of Bos (Bos)primigenius f.d. taurus (f.d. = forma domestica) determined by Brew et al. (1970) and Vanaman et al. (1970). It includes the corrections made subsequently by Shewale et al. (1984): Residue Now Was

43 Gln Glu

46 Asp Gln

49 Glu Asp

82 ASP Asn

83 Asp Asn

87 Asp Asn

88 ASP Asn

The amino acid sequence derived by Vilotte et al. (1987; see also Hurley and Schuler, 1987) from the nucleotide sequence differs from the corrected chemical sequence presented here as follows: Residue Nucleotide Chemical

39 Gln Glu

63 Asp Asn

66 Asn Asp

Note (1): The A variant from Bos (Bos) namadicus f.d. indicus differs from the B variant at residue 10: Gln in A substituted for Arg in B (Bell et al., 1970). The sequence difference for the C variant from Bos (Bibos)javanicus (Table 11)is unknown.

2 . Caprine (Capra hircus} and Ovine (OvzS aries} a-Lactalbumin The sequence of 123 residues shown for caprine a-lactalbumin is that determined by MacGillivray et al. (1979) and corrected by Shewale et al. (1984): Residue Now Was

43 Gln Glu

46 Asp

49 Glu Asp

82 Asp Asn

83 Asp Asn

Kumagai et al. (1987) confirmed this sequence, except for residue 66,

233

LYSOZYME AND a-LACTALBUMIN

which they deduce from the nucleotide sequence to be Asn, not Asp. There could be an error in the chemical sequencing, it could be a different variant, or there could be an error in the reverse transcription process. Note (2): The amino acid sequence of ovine a-lactalbumin was derived by Gaye et al. (1987) from their determination of the complete nucleotide sequence of a-lactalbumin mRNA. This sequence (not shown in Fig. 10) is identical with the amino acid sequence of caprine a-lactalbumin except for residue 8 (assuming residue 66 in caprine is Asn): Caprine Ovine

Val Ala

3 . Guinea Pig (Cavia porcellus) a-Lactalbumin The original sequence of 123 residues was due to Brew (1972),but the subsequent DNA studies by Hall et al. (1982) resulted in the following important corrections [some of these residues are important in the binding of Ca(II), see Section V]: Residue Now Was

46 Asp Asn

57 Asp Asn

84 Asp Asn

82 Asp Asn

71 Asn Asp

87 Asp Asn

88 Asp Asn

102 Asp Asn

4 . Human (Homo sapiens) a-Lactalbumin

The sequence of 123 residues is that of Findlay and Brew (1972), with corrections from the DNA studies by Hall et al. (1982): Residue Now Was

45 Asn Asp

46 Glu Gln

82 Asp Asn

84 Asp Asn

87 Asp Asn

88 Asp Asn

102 Asp Asn

The X-ray crystallographic determination of the structure of alactalbumin has been made on baboon (Papio cynocephalus) a-lactalbumin. Its amino acid sequence has not been determined. However, on the basis of preliminary work by R. Greenberg (personal communication), it is evident, as would be expected, that its sequence is similar to that of human a-lactalbumin. On the basis of their X-ray studies, Acharya et al. (1989) concluded that the following are possible sequence changes from that of the human protein: Residue Human Baboon

11 Leu Asn

13 Lys Tyr

20 Gly Arg

58 Lys Ala

66 Val Ser

76 Ser Thr

105 Leu Ile

123 Leu Glu

5. Rabbit (0ryctoIagu.s cuniculus) a-Lactalbumin This sequence of 122 residues is that of Hopp and Woods (1979). It is predominantly a glycoprotein with the carbohydrate attached at residue 45 (Asn).

Key to Protein Abbreviations

Mammalian Proteins a-Lactalbumin Bovine B variant Caprine (goat) Guinea pig Human Rabbit Rat Equine (horse) N w Camel rp Red-necked wallaby Milk, urine, and stomach mucosa c-type lysozymes Human milk (and human leukemia urine) Baboon milk Equine milk Rat urine Bovine stomach mucosa, c type 2 (6) Deer stomach mucosa 1 Langur stomach mucosa Pig stomach mucosa 3 Echidna milk Tachyglossus aculeatus multiaculeatus, type I ( 8 )

Avian Proteins (BB a-la) ( 1 ) (C a-la) (2) (GP a-la) (H a-la) (Rb a-la) (Ra a-la) (E a-la) (3) (Ca a-la) (RW a-la) (HM lz) (BaM lz) (EM 12) (4) (RU 12) ( 5 ) (BSs Iz) (DS lz) (LS 12) (Piss lz) (7) (TMI lz)

c-Type lysozymes Domestic hen California quail Bobwhite quail Turkey Ring-necked pheasant Guinea hen Kaki duck I1 Peking duck 1 Chachalaca Pigeon

(DH lz)

(CQ 14 (BWQ 12)

(T 12)

(RNP lz) (GH lz) (KDII lz) (9) (PDl Iz) (9) (Chac lz) (P 12)

Key to notes (1)-(9) (see text). ( 1 ) Bovine a-la A (2) Ovine a-la (3) Equine a-la B,C (4)Donkey lz (5) Mouse M lz (6) Bovine stomach lz 1,3; Ovine stomach lz 1,2,3; Caprine stomach lz 1,2; Camel stomach Iz 1 (7) Pig stomach lz 1,2 (8) Echidna lz I1 (9) Kaki duck lz I; Peking duck lz 2,3

07.

T

FIG. 10. Comparison OF sequences of a-lactalbumins and lysozymes, including a key to the abbreviations. l’he highest numbers of-residues showing homology in a given position are boxed with continuous lines, the next highest are boxed in broken lines, and the third highest are boxed in dotted lines. a-la,a-Lactalbumin; lz, lysozyme. For further details see text. (Reproduced from H. A. McKenzie. Copyright 0 1983- 1989 by H. A. McKenzie.)

236

60

BB

c

o-la(1)

GP H Rb Pa

*-la121 a-la o-la o-la *-la

,z

(I-1a111

ca *-la

Ru

HM Bau EM RU BL DS LS Pis)

TUI

o-la

1Z

1r 1IaI 17.1II

lz I 0 11

1r 1~17)

17. 181

DH

lr

CQ

17.

B W T

1E

RNP GH KDII

lZ

lz lz

PD1

lzlrl 1z 191

Chac P

1z 17.

FIG. 10. See legend on p. 235.

70

80

BB

11-11(11

GP

.-la (21 0-1s

c

n a-la --la Pa a-la E *-la (:l ca e-1a Rb

m

.-la

Hn

17.

Bald

1Z

En

11111 lZ(11

Ru BSs 0s

1r.16) 1z

IS Pis)

1r

M I

lE(O1

DH

1Z

CQ BWQ

17.

lZ(7,

17.

T RWP

1z

GH

17.

KDII PD1 Chac

lZ(91 lz(9)

P

1r

1r

lr

90

100

239

240

HUGH A. MCKENZIE A N D FREDERICK H. WHITE, JR.

6 . Rat (Rattus nomegtcus) a-Lactalbumin Rat a-lactalbumin is unique in having a sequence of 140 residues. The sequence shown is that of Prasad et al. (1982). There are appreciable discrepancies between it and the nucleotide sequence of Dandeker and Qasba (1981). K. E. Ebner (personal communication, 1986) was unable to explain the difference between the work of his laboratory (Prasad et al., 1982) and the work at the National Institutes of Health (Dandeker and Qasba, 1981), for example: Residue Petal. DQ

38 Thr Ser

39 Glu Gln

41 Ser Ile

44 Asp Asn

59 Asp Asn

63 Glu Ser

64 Asn Ser

65 Gln Glu

67 Val Pro

102 Asn Asp

105 Leu Lys

Residues 28-58 are stated by Dandeker and Qasba (DQ) to differ only from Prasad (P) et al. at residues 39 and 44. This does not appear to be correct. Their comparison is actually made with Prasad and Ebner (1980).

7. Equine (Equus caba1lusf.d. caballus) (Perissodactyla) a-Lactalbumin The 123-residue sequence shown is that of the A variant (isolated from the milk of an Australian thoroughbred horse), as determined by Kaminogawa et al. (1982, 1984). A misprint in the 1984 paper has been corrected: Residue 10 should have read Gln, not Glu. Note (3): T h e B and C variants (isolated from the colostrum of a Persian Arab horse) differ from the A variant as follows (Godovac-Zimmermann et al., 1987): Residue A B

C

7

Glu Gln Gln

33 Ser Asn Asn

78 Asp Asn Asn

95 Ile Asp Ile

In this paper the residue at position 54 (A variant) is given incorrectly as Glu: it should read “Gln.”

8. Camel (Camelus dromedarius) a-Lactalbumin The 123-residue sequence is due to Beg et al. (1985).

9. Red-Necked Wallaby (Macropus rufogresius) a-Lactalbumin The sequence has only 121 residues and was determined by Shewale et al. (1984). 10. Human (Homo sapiens) Lysoqme The sequence of lysozyme isolated from the urine of human leukemic patients was determined by Canfield et al. (1971), and that of human milk Iysozyme was determined by Jollks and Jollks (1971). Canfield et al.

24 1

LYSOZYME AND CY-LACTALBUMIN

(1971), in their original sequence, showed a deletion after residue 100. The work by Thomsen et al. (1972) for the leukemic urine protein and that by Jolles et al. (1972) for the milk protein show that there is a ValVal sequence for residues 99-100 instead of a single Val residue. The revised sequences of both lysozymes are the same and are 130 residues long. The nucleotide sequences determined by Castafion et al. (1988), Chung et al. (1988), and Peters et al. (1989) are in accordance with the revised chemical sequence. 11. Baboon (Papio cynocephalw) Milk Lysozyme This sequence of 130 residues was determined by Hermann et al. (1973).

12. Equine (Equus caba1lusf.d. caballus) (Perissodactyla) Lysozyme The sequence (2) (Fig. 10) of 130 residues is that of McKenzie and Shaw (1982, 1985). Note (4): Recently, the sequence of donkey (Equus asinus) milk lysozyme was determined by Godovac-Zimmermann et al. (1988). They found the following differences: Residue Horse Donkey

61 Asn Ser

52 Ser Tyr

87 Glu ASP

13. Rodent Lysozymes: Rat (Rattus norvegicus) Urine Lysozyme and Mouse (Mw domesticus) Lysozyme M The 130-residue sequence of rat lysozyme, from the urine of rats bearing a transplantable chloroleukemic tumor, was determined by White et al. (1977). Note (5): A. B. White (personal communication from A. C. Wilson, 1986) compared the rat sequence with the results of a partial sequence determined by R. J. Riblet for mouse lysozyme. After making some realignments, White concluded that there were 14 changes in the 106 residues compared (residues 78-101 were not determined). Later, Cross et al. (1988) isolated and characterized both cDNA and genomic DNA of mouse (spleen) lysozyme M gene. They deduced the amino acid sequence from the nucleotide sequences, but found only 13 differences in sequence between the mouse and the rat. The differences found by both groups of workers are shown below, W and C signifying White and Cross, respectively: Rat

Mouse Worker

2 Thr Val W,C

18 Ser

Ah W.C

41

43

Gln Arg W,C

Arg Thr W,C

46 Asp Asn

W(?).C

47

Pro Arg W,C

74 Lys Val C

120

80 Pro

91

113

114

117

Gln

Asn W,C

W,C

Gln Arg W,C

Arg Ala W,C

Lys Asp Gln Val W(?),C W

Ah

122 Ser Arg

W

123 125 cly Ile Gln Val W(?),C W

126

Arg Glx W

242

HUGH A. MCKENZIE AND FREDERICK H. WHITE, JR.

“W(?)” signifies that the residue was identified by White only as Asx or Glx. Residue 74 was found by White to be the same as for the rat. 14. Bovine [Bos (Bos) taurus]Stomach Mucosa Lysoqme cp

Jolles et al. (1984) determined the 129-residue sequence of one of the three variants (variant 2) of the three c-type lysozymes isolated by Dobson et al. (1984) from bovine stomach mucosa. The sequence is given here. However, residue 98 has been altered from the original designation of Lys to His in accordance with the revision by P. Jolles and J. Jollks (quoted by Prager and Wilson, 1988). Note (6): Work by Joll&s et al. (1990) on the other bovine variants and two variants of caprine stomach mucosa lysozyme, and by Irwin and Wilson (1990) on three ovine variants, was in press at the time of writing and the following information was made available courtesy of Ellen Prager and Allan Wilson. Bovine stomach mucosal lysozyme c1 and c3 differ from c, as follows: Residue Bovine cq c, c3

c:,

48 Ser Gly

98 His Gln

125 Glu

Gln

The caprine and ovine stomach lysozyme variants differ from bovine as follows:

Residue Bovine cp Caprine 1 Caprine 2 Ovine 1 Ovine 2 Ovine 3

14 Lys Glu Glu Glu

19 Gly Asp

Asp

37 Ser Gly

48 Ser Gly Gly

63 Trp Phe Phe

72 Asn Asp

Gly

83 Glu

88 Asp

Ala Ala

Asn Asn Asn Asn

90 Ala Glu

98 His Gln

Glu Glu

128 Thr Ser

The 130-residue sequence of camel stomach lysozyme 1 differs from that of bovine stomach c2 in 36 positions: Residue Bovinecp Camel 1

3 Phe Trp

7 Glu Ala

11 Thr Lys

14 Lys Glu

17 Leu Met

21 Lys Arg

29 Leu Met

37 Ser Asp

63 Trp Tyr

67 Asp Asn

72 Asn His

75 Asp Asn

78 His Gly

79 Val Ser

80 Ala Asn

82 Ser Asn

87 Asn Asp

90 Ala Thr

94 Ala Gln

98 His Arg

99 Ile Val

101 Ser Arg

102 Glu Asp

-

114 Ser Asn

117 Arg Glu

118 Asp Gly

122 Ser Glu

123 Ser Gln

129 Thr Asp

(Numbering is that of camel.)

103

Pro

Lys Asp 83 Glu Val

62 Lys Arg 85 Met Leu

106 Ile Val

107 Thr Arg

41

243

LYSOZYME A N D (Y-LACTALBUMIN

15. Axis Deer (Axis axis) Stomach Mucosa Lysozyme

The 129-residue sequence of axis deer stomach mucosa lysozyme was determined by Jollts et al. (1989). There now appear to be two variants. The sequence given in Fig. 10 is that of variant 1. Irwin and Wilson (1990) have concluded that variant 2 differs from variant 1 as follows: Residue Deer 1 Deer 2

66 Asp Asn

90 Asp Ala

88 Asn Asp

94 Thr Ala

117 Gly ASP

16. Langur (Presbytis entellus) Stomach Mucosa Lysozyme

The 130-residue sequence of langur stomach lysozyme was determined by Stewart et al. (1987). 17. Pig (Sus scrofa) Stomach Mucosa Lysozyme

This protein was studied by Jol1i.s et al. (1989) in the mucosa of approximately 20 pigs. They found two variants (1 and 2) with identical mobility at pH 4.3 and a third variant (3) with a slightly higher mobility (0.97 of that of variants 1 and 2). The sequence shown is that of variant pig 3. Note (7): Differences between the three variants are: Residue Pig 3 Pig 2 Pig 1

37 Asn Asp Asp

43 Thr Ile Ile

45 Tyr His Arg

47 Pro

Val Val

50 Gln -

49 Ser -

104 Gln Leu Gln

106 Ile Val Ile

113 Lys Arg Arg

114 Ala Ala Thr

The pig 3 sequence is 130 residues long, and each of the other two variants has 128 residues because of the deletions at residues 49 and 50. 18. Echidna (Tachyglossus aculeatus) Milk Lysozymes Z and ZZ T h e sequences of echidna lysozyme I from the milk of Tachyglossus aculeatus multaaculeatus and echidna lysozyme 11 from the milk of Tachyglossus aculeatus aculeatus have been determined by Teahan et al. (1990; see also Teahan, 1986). Note (8): The sequences for variants I and I1 differ as follows: Residue I I1

13 Val Ala

37 Ser Gly

41 Ser Gln

Both sequences terminate at Cys residue 125. 19. Domestic Hen (Gallus gallus) Egg-White Lysozyme The original sequence was determined independently by Canfield (1963) and by Jolles et al. (1963). The sequences were generally in agreement, but the following differences were evident:

244

Canfield Jollits

HUGH A. MCKENZIE AND FREDERICK H . WHITE, JR.

40 Thr Gln

41

Gln Ala

42 Ala Thr

46 Asn Asp

48 Asp Asn

58 Ile Asn

59 Asn Ile

65 Asn Asp

66 Asp Asn

92 Val Asn

93 Asn Val

On the basis of the electron density map, Blake et al. (1965, 1967a) concluded that the correct assignment for residues 40, 41, 42, 92, and 93 is that given by Canfield (1963). In a later communication Blake et al. (1967a) concluded that residues 58 and 59 were in accord with Canfield (1963), contrary to the opinion given in their first paper. These conclusions were confirmed chemically by Rees and Offord (1972), who also showed that residues 46 and 48 were also in accord with Canfield (1963). Jolles and Jollb (1972) indicated the Canfield (1963) assignments for residues 40,41,42,58,59,92, and 93. Imoto et al. (1972) reported that J. K. Brown (personal communication, 1971) had suggested that residue 103 is Asn, not Asp, as given by Canfield (1963) and by Jolles et al. (1963). This suggestion was later confirmed by E. M. Prager (personal communication to Ibrahimi et al., 1979). T h e given sequence incorporates all of these corrections, and has 129 residues. The disulfide bridges were determined by Brown (1964), Jolles et al. (1964), and Canfield and Liu (1965), the results of the three groups being in agreement. The locations for a-lactalbumins and lysozymes are discussed below.

20. California Quail (Lophortyx californicus) Egg-White Lysozyme The 129-residue sequence is that of Ibrahimi et al. (1979). As indicated above, position 103 in domestic hen egg-white lysozyme was originally considered to be Asp, but was subsequently shown to be Asn. Unpublished experiments by E. M. Prager on the mobilities of equivalent peptides indicated that 103 is Asn in California quail and bobwhite quail lysozymes as well as for the domestic hen protein (see also Section VII,B,23 and 24). 21. Bobwhite Quail (Colinus virginianus)Egg- White Lysozyme

The 129-residue sequence was determined by Prager et al. (1972), and residue 103 is given as Asn on the same basis as that of the corresponding residue in the California quail protein (see above). 22. Turkey (Meleagns gallopavo) Egg- White Lysozyme

The 129-residue sequence determined by La Rue and Speck (1970) is the least satisfactory of the sequences given in Fig. 10, primarily because of the uncertain resolution of the nature of some of the residues presented by the authors as Asx and Glx. However, in Fig. 10 a number of

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assumptions are made in allocating the residues: (1) Residues 18-19 have been given as Asp-Asn, since all c-type hen egg-white lysozymes sequenced to date have this sequence. (2) Residue 35 is given as Glu, since all other c-type lysozymes are Glu. (3) Residue 46 is assumed to be Asn, since all other c-type egg-white lysozymes are Asn. (4)Residue 52 is assumed to be Asp, since all other c-type lysozymes are Asp. (5) Residue 57 is assumed to be Gln, since all other lysozymes and alactalbumins are Gln. (6) Residue 59 is assumed to be Asn, since all other c-type lysozymes are Asn. (7) Residues 65-66 are shown as given by La Rue and Speck (1970) (i.e., Asx-Asx), but are probably Asn-Asp. (8) Residue 74 is shown as Asn, since all other c-type egg-white lysozymes are Asn. (9) Residues 77, 87, 93, 103, and 106 are shown as Asx, since there is doubt about the designation Asp or Asn. (10) Residue 119 is shown as Asp, since all other c-type lysozymes are Asp. 23. Ring-Necked Pheasant (Phasianus colchicus) Egg-White Lysozyme This sequence of 130 residues (note: the amino-terminal residue - 1 is Gly) was determined by Jolles et al. (1979a). Although they give residue 103 as Asp, strong evidence is presented on page 2747 of their paper that this residue is Asn, and it has been so assigned in Fig. 10. T h e sequence for the signal peptide of the prelysozyme of ring-necked pheasant was determined by Weisman et al. (1986) and compared with those of five other species of birds.

24. Guinea Hen (Numida meleagris) Egg-White Lysozyme The sequence of this 129-residue lysozyme was determined by Jolles et al. (1972). 25. Duck (Anas platyrhynchos) Egg-White Lysozyrne The sequence of Kaki duck lysozymes I1 and 111 (designated here KDII and KDIII, respectively) were determined by Hermann and Jolles (1970) and by Hermann et al. (1971). Those of Peking duck lysozymes 1, 2, and 3 (designated here PD1, PD2, and PD3, respectively) were determined by Kondo et al. (1982). In Fig. 10 the sequences of KDII and PDl are shown. Note (9): The differences for the variants were as follows: Residue 4 37 57 71 72 KDII Ser Ser Glu Gly Ser KDIII Glu Ser* Glu Arg Ala PD 1 Ser Ser Gln Gly Ser PD2 Ser Gly Gln Arg Ser PD3 Ser Gly Gln Arg Ser *Residue 37 for KDIII is 70% Ser, 30% Gly.

79 Pro Pro Pro Pro Arg

82 Leu Leu Leu Val Val

93 Arg Lys Arg Arg Arg

116 Arg Lys Arg Arg Arg

122 Lys Arg Lys Lys Lys

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HUGH A. MCKENZIE AND FREDERICK H . WHITE, JR.

Although the revised sequence for residues 65 and 66 of domestic hen egg-white lysozyme was known when the above work was done, the old designations for these residues were given. Also, it was stated that the duck proteins had the same residues in these positions as for the domestic hen proteins. The evidence presented for the residues being Asp-Asn was tenuous. Hence, it is likely that these residues are Asn-Asp. Later, Jolks and Jollks (1984) assigned the duck residues as Asn-Asp, but gave no rationale for this. In Fig. 10 we have shown revised assignments. The assignment for residue 57 was discussed by Teahan (1986). She pointed out that KDII and PDl lysozymes have identical amino acid sequences, except for residue 57, which is given as Glu by Hermann et al. (1971) for KDIII and as Gln by Kondo et al. (1982) for PD1. Prager and Wilson (1972) have shown that both proteins have identical electrophoretic mobilities. Thus, it is likely that KDII has Gln in position 57. This is in accord with the later view of Rodriguez et al. (1987). Further, we note that all other c-type lysozymes and all a-lactalbumins have Gln in this position. Hence, we conclude that KDII and KDIII have Gln in position 57, as shown in Fig. 10. This, then, means that KDII and PD1 are identical. 26. Chachalaca (Ortalis uetula) Egg-White Lysozyme This sequence of 129 residues is that given by Jollks et al. (1976). 2 7 . Pigeon (Columba livia) Egg-White Lysozyme

The sequence of Rodriguez et al. (1985) has a number of surprising features, including termination at residue 127 (Cys),which are discussed below. C. Summary of Important Features of Comparative Sequences

1 . Chain Length

The chain lengths of all but three of the a-lactalbumins, considered in Section B, are 123 residues. One, rat a-lactalbumin, has a chain extension of 17 residues, giving 140 residues total. In their nucleotide sequence study Qasba and Safaya (1984) concluded that this extension arises from a T-to-G base change in the termination codon. Two sequences have fewer than 123 residues: Rabbit has 122 residues and red-necked wallaby has 121 residues. The majority of the mammalian lysozymes (human, baboon, and equine milk; rat urine; and camel, pig 3, and langur stomach) have 130 residues. Bovine, caprine, and deer stomach lysozymes have 129 residues. Although pig stomach lysozyme 3 has 130 residues, two of its variants (1 and 2) have 128

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residues. All but two avian lysozymes have 129 residues. Pigeon has 127 residues terminating at Cys. Echidna milk lysozyme, which resembles the marsupial a-lactalbumin in the Cys termination, has 125 residues.

2 . Amino-Terminal Residues T h e amino-terminal residues of the a-lactalbumins are variable, but four (guinea pig, human, equine, and camel) have Lys. This is in common with all of the c-type lysozymes except ring-necked pheasant, which has a one-residue extension (Gly); however, its next residue is Lys. This arises from a different cleavage point of the prelysozyme.

3 . Disuljide Bridges All of the a-lactalbumins have the structurally important cystine bridges in the same positions: 6-120,28-111,61-77, and 73-91. This is also the case for the equivalent positions in all c-type lysozymeshuman numbering: 6- 128, 30- 116,65-81, and 77-95; domestic hen egg-white numbering: 6- 127, 30- 115, 64-80, and 76-94. However, when the sequence of echidna lysozyme I was determined, this was no longer true (Teahan et al., 1986, 1990). There is no Cys at position 6; it occurs at position 9. The accommodation of this Cys in the structure is discussed elsewhere (Acharya et al., 1989). 4 . Invariant Residues In addition to the invariant positions of the eight half-cystine residues in a-lactalbumins, the following 27 residues are invariant: Glu-25(27), Phe-3 1(33), His-32(34), Ser-34(36), Gly-35(37), Thr-38(40), Val-42(44), Glu-49(53), Tyr-56(54), Gly-51(55), Phe-53(57), Gln-54(58), Ile-55(59), Leu-8 1(85),Asp-82(86), Asp-83(87), Asp-87(91),Asp-88(92), Lys-94(98), Ile-95(99), Gly- loo( 105),Trp- 104(log), Ala- 106(11l),His- 107(112), Leu115(120), Gln-l17(123), and Trp-1 lS(124). (The numbers in parentheses are the equivalent human lysozyme numbers.) When the sequences of pigeon and echidna lysozyme were determined, the number of residues invariant in c-type lysozymes was considerably reduced. In addition to the seven half-cystine residues that are invariant in lysozymes, the following 21 residues are also invariant (human lysozyme numbering, with hen egg-white lysozyme numbering in parentheses): Lys-l( l), Trp-28(28), Glu-35(35), Ser-36(36), Ala42(42), Asn-44(44), Ser-5 1(50), Asp-53(52), Tyr-54(53), Gly-55(54), Gln58(57), Asn-60(59),Trp-64(63), Leu-84(83), Ala-96(95), Lys-97(96), Gly105(104), Trp- 109(108), Ala- 111(1lo), Trp-l12(11l), and Asp-lPO(119). [Residue 7(7) is Glu in all except the camel.] In addition to the seven half-cystine residues, the following seven residues are invariant in all

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HUGH A. MCKENZIE AND FREDERICK H. WHITE, JR.

of the a-lactalbumins and c-type lysozymes (numbering is human alactalbumin, with human milk lysozyme numbering in parentheses): Ser34(36), Tyr-50(54), Gly-51(55),Gln-54(58), Gly-100(105),Trp-104(109), and Ala-106(111).

5. Ligunds for Cu(ZZ) Binding Fallowing the establishment of a-lactalbumin as a Ca(I1)-bindingprotein and the revision of the sequences of several a-lactalbumins (see above), Shewale et al. (1984) suggested that residues 82, 83, 87, and 88 were probably ligands for the calcium. In fact, the X-ray crystallographic studies of Phillips’ group showed that three of the predicted residues (82, 87, and 88) were involved. The Ca(I1) proved to be seven coordinates in baboon a-lactalbumin: In addition to the involvement of the residues shown in Table IX (see also Fig. 8), two water molecules are coordinated. The majority of the c-type lysozymes so far sequenced do not have the residues necessary for the coordination of Ca(I1) (Table TABLE IX Restdues Relevant to Binding of Calcaum a

Residue Binding through

79(83)a C==O

84(88)

(C=O)

87(91) (COO)

88(92) (COO)

Equivalent residues in some a-lactalbumins and lysozymes

Source a-Lactalbumins* Baboonc Humanc BovineC EquineC Rabbit Red-necked wallaby Lysozymesd Human milk Horse milkc Echidna milkC Bovine stomach c2 Domestic hen egg Pigeonc

82(86) (COO)

79 LYS LYS LYS LY s Asn LYS 83 Ala LYS LYS Glu Ala LYS

82 ASP ASP ASP ASP ASP ASP 86 Gln ASP ASP Glu Ser ASP

84 ASP ASP ASP ASP Asn ASP 88 Asn Asn ASP ASP ASP Asn

87 ASP ASP ASP ASP ASP ASP 91 ASP ASP ASP LYS Ala ASP

88 ASP ASP ASP ASP ASP ASP 92 Ala ASP ASP Ala Ser ASP

Number in parentheses is the human lysozyme equivalent number. “Residues identified by X-ray crystallography as ligands for Ca(I1) in baboon a-lactalbumin (Stuart et al., 1986; Acharya et al., 1989) *All a-lactalbumin residues are numbered according to human a-lactalbumin. cKnown to bind Ca(I1). dAll lysozyme residues are numbered according to the equivalent human lysozyme numbers.

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IX). However, the establishment of the sequence of equine lysozyme, and soon afterward pigeon and echidna lysozymes, indicated the possibility that these three lysozymes bind Ca(I1). This was confirmed for the equine and echidna proteins in Canberra, and for the equine and pigeon proteins in Sapporo. However, the echidna lysozyme sequence is the only one in which all residues are identical with the equivalent residues in baboon a-lactalbumin. This matter is discussed further in Section X.

6 . Basic and Acidic Groups The comparison of basic and acidic groups and the ratio of Lys/Arg residues are of importance in discussing the work by Prager, Wilson, and colleagues on the divergence of gastric mucosal lysozymes. Relevant information from Fig. 10 is summarized in Table X and discussed in Section X.

7. Residues in Catalysis We have seen already, in the discussion of various structural approaches to the mechanism of catalysis by hen egg white lysozyme of hydrolytic cleavages, that residues Glu-35(35), and Asp-52(53, human numbering) are considered crucial. These residues are conserved in all c-type lysozymes sequenced to date. Other residues implicated that appear to be invariant are: Trp-63(64), Asn-59(60), Gln-57(58), Asn44(44), and Trp-108( 109). Other residues implicated are not invariant: Asn-103(104), Asp-101(102), Trp-62(63), Ala-107(108), Asn-46(46), Val109(110), Phe-34(34), Asn-37(37), and Arg-114(115).

8. Residues in Galactosyltransferase Interaction Residues that have been implicated in a-lactalbumin-galactosyltransferase interaction do not all occur in equivalent positions in any lysozyme. The nearest similarity is echidna lysozyme: a-Lactalbumin 31 32 35 115 117 118

Phe His G'Y Leu Gln Trp

Echidna lysozyme Equivalent 33 34 37 119 122 123

Phe His Gly Asp Lys Phe

The Ca(1I) potential binding residues are identical for echidna lysozyme and bovine a-lactalbumin (see above).

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HUGH A. MCKENZIE AND FREDERICK H. WHITE, JR.

VIII. GALACTOSYLTRANSFERASE AND THE LACTOSE SYNTHASE SYSTEM A . Gahctosyltramferases: Occurrence, Function, and Isolation 1 . Introduction There has been much interest in recent years in various aspects of galactosyltransferases, especially with regard to their possible use as markers in malignancy and their involvement with cell surface phenomena. We have already seen in Section II,B that lactose is synthesized in the mammary gland through an enzyme, lactose synthase, which consists of two main components: galactosyltransferase and a-lactalbumin. T h e latter acts as a specifier protein modifying the action of the galactosyltransferase. a-Lactalbumin and galactosyltransferase are under hormonal control in the mammary gland during pregnancy (Woods et al., 1977; Ono and Oka, 1980; Turkington et al., 1968). However, unlike a-lactalbumin, which is found only in milk, galactosyltransferase is widely distributed. MorrC et al. ( 1969), for example, studied galactosyltransferase activity in the Golgi fraction of rat liver. Schachter et al. (1970) investigated galactosyltransferase in the Golgi apparatus as it functions in the glycosylation of proteins. Cunningham et al. (197 1) found it in rat seminiferous tubules. Powell and Brew (1974a) demonstrated the presence of galactosyltransferase and glycosyltransferase in the Golgi membrane of onion stem. T h e former showed many similarities to the animal enzyme. It was manganese dependent and gave the same reaction (apparently) as the animal enzyme. It had a K , similar to that of N-acetylglucosamine (5.2 mM) and could be activated by bovine a-lactalbumin. 2 . Galactosyltransferase

Galactosyltransferase is only one member within the broader classification of glycosyltransferases that have been found on cell surfaces; these are involved in contact-mediated cell interactions in general, but more specifically are involved in fertilization, morphogenesis, hemostasis, and cell migration. This topic has been well reviewed by Pierce et al. (1980).

3 . Galactosyltransferaseas a Marker in Malignancy Numerous workers have reported elevations in the total glycosyltransferase level and in the sera and tissues of cancer patients (for a review see Weiser and Wilson, 1987). Podolsky and Weiser (1975),in particular,

LYSOZYME AND a-LACTALBUMIN

25 1

reported the total activity to be higher than normal in malignancy. These workers then isolated a cancer-associated galactosyltransferase I1 (GTII) isoenzyme and characterized it with a molecular weight of 76,000, contrary to the more usual 40,000-44,000 found in milk (Trayer and Hill, 1971). Patients with widespread metastases have elevated GTII levels (Podolsky et al., 1981). Also, of nine patients with Duke’s B lesions, seven had detectable GTII, and in each case it became undetectable after the patients had undergone curative colectomy. T h e authors concluded that GTII was a good marker for malignancy in general, and for pancreatic carcinoma in particular. 4. Present Scope In this review we are concerned primarily with one galactosyltransferase: UDPgalactose: N-acetylglucosamine 4/3-~-galactosyltransferase.For further discussion, see pp. 179 and 180. For a comprehensive review of the galactosyltransferases in general, see Ram and Munjal ( 1985).]Within the lactating mammary gland, galactosyltransferase becomes part of the lactose synthase system and is regulated by the flow of a-lactalbumin through the lumen of the Golgi apparatus (Brew, 1969). Most of the remainder of this section therefore deals with mammary enzyme. 5. Preparation of Galactosyltransferase In the original work on galactosyltransferase, it was isolated by classical procedures of salt fractionation and column chromatography (see the review by Brew and Hill, 1975). Grunwald et al. (1982) have warned strongly against the use of pH <5.0 in any steps involving the separation of casein; otherwise, loss of stability of galactosyltransferase results. An important development in the purification of galactosyltransferase has been the use of either a-lactalbumin or a substrate, bound to an immobile inert substance in an affinity column. With a-lactalbumin, either glucose or N-acetylglucosamine is included in the buffer to enhance the binding of galactosyltransferase to a-lactalbumin. The galactosyltransferase is then eluted with buffer not containing saccharide. Andrews (1970) and Trayer and Hill (1971) were apparently the first to use a-lactalbumin, bound to Sepharose in an affinity column, for the purification of galactosyltransferase. Trayer et al. ( 1974) used a-lactalbumin-agarose as a specific adsorbent for galactosyltransferase. Also for use in affinity chromatography of galactosyltransferase, several ligands structurally related to galactosyltransferase substrates have been immobilized onto CNBr-activated agarose. These include UDP-hexanolamine, N-acetylglucosamine, and galactosyl pyrophosphate (Barker et al., 1972). Of these media, probably N-acetylglucosamineagarose is the best known.

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A major problem in the isolation of galactosyltransferase has been molecular weight heterogeneity of the final product, apparently resulting from proteolysis. Klee and Klee (1972) used the protease inhibitor phenylmethylsulfonylfluoride, but they obtained mainly a low molecular weight form of galactosyltransferase. According to Magee et al. (1976), however, this inhibitor, as used, would give only 25-40% inhibition and is not sufficiently soluble to produce the desired inhibition. The latter authors preferred to use .s-amino-n-caproic acid, together with low tern perature. Kaminogawa et al. (1972) studied the milk protease, which is presumably responsible for the heterogeneity of galactosyltransferase, in comparison with plasmin, and they reported that these two enzymes may be the same. Subsequently, this proposal was confirmed (see Andrews, 1983). Powell and Brew (1974b) concluded that galactosyltransferase occurs in mature milk as a proteolytically degraded form of the galactosyltransferase that first appears in colostrum. Magee et al. (1973) found two different molecular weights for milk galactosyltransferase: 55,000-59,000 and 42,000-44,000. The effect of trypsin on the higher molecular weight form resulted in a product resembling the lower molecular weight form. They reasoned that a trypsinlike protease could therefore be acting to produce the latter in milk. This group further studied the heterogeneity of galactosyltransferase from cow milk, finding multiple forms of this enzyme (Magee et al., 1976). Prieels et al. (1975) found three forms of galactosyltransferase in human milk with molecular weights of 38,000, 43,000, and 50,000. The activity differences found among these forms suggested that conformation at the site of association between galactosyltransferase and the acceptor saccharide had been changed, presumably by enzymatic hydrolysis. In more recent years various factors affecting the stability of galactosyltransferase have been studied. Fraser and Mookerjea (1976) found the use of Triton X-100 to be helpful in stabilizing the enzyme during isolation. Fraser et al. (1980) found that it is also stabilized by albumin and by lysolecithin. Mitranic and Moscarello (1980) studied the effects of lipids on the activity of galactosyltransferase. Thus, phosphatidylcholine, phosphatidylethanolamine, and phosphatidylglycerol stimulated activity, while phosphatidic acid and phosphatidylserine inhibited activity. These observations are particularly relevant to the activity of galactosyltransferase in milk, with its lipid environment. Mitranic and Moscarello (1983) found that bovine serum albumin, as well as some other proteins, has a pronounced activating effect on the enzyme and cautioned against its use in the isolation of galactosyltransferase.

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B . Relationships of Structure to Function in Galactosyltransferase

There is at least one SH group in galactosyltransferase (Babad and Hassid, 1964; Magee and Ebner, 1974; Kitchen and Andrews, 1974). According to O’Keeffe et al. (1980a), an SH (one of three reactive SH groups) is at or near the active site. This point was considered further by Wong and Wong (1984), who studied the inactivation of galactosyltransferase from cow milk by SH-specific reagents. The SH group was found to be located in a nonpolar environment and in a region of nonrestricted rotation. It was found not to be located at the active site, as had been suggested by O’Keeffe et al. (1980a), nor at the proteinprotein interaction site between galactosyltransferase and a-lactalbumin. Various other studies, involving specific alterations of amino acid residues, have been conducted on galactosyltransferase. The results of Powell and Brew (1976~)indicate the involvement of a Lys residue in the activity of galactosyltransferase. Thus, an affinity label for the UDP binding site was created by periodate cleavage of the ribose moiety of UDP. The derivative caused inactivation, which was reversible by nitrogenous bases or stabilized by KBH, reduction. These observations were consistent with their hypothesis that a Schiff base had formed between an aldehyde of the affinity label and the amino group of a Lys residue. Evidence indicating involvement of a Trp residue in the activity of galactosyltransferase was found by Clymer et al. (1976). Thus, UV irradiation of the protein resulted in loss of one Trp with concomitant inactivation. Silvia and Ebner (1980) found Tyr to be essential for activity, since iodination with ICl caused inactivation. However, it was not certain from kinetic studies whether 2 mol of ICl was involved at one site, or one of each at two different sites. Iodination was explored further by Chandler et al. (1980) with lactoperoxidase, which catalyzes iodination of Tyr with I-. Further, they found that galactosyltransferase was inactivated by N-acetylimidazole. This observation was consistent with an essential role of Tyr for activity. However, this reagent also affects the amino groups of Lys residues. Differentiation between these possibilities is afforded by the fact that deacetylation with hydroxylamine will occur for the Tyr derivative, but not for the Lys derivative. Since only about 40% of the activity could be recovered by deacetylation, Lys could also have been involved. Note that this indication is the second for the implication of a Lys residue with galactosyltransferase activity (see Powell and Brew, 1976~). Conformational changes have been observed or suspected for galactosyltransferase. Magee and Ebner (1974) speculated on the occurrence

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HUGH A. MCKENZIE AND FREDERICK H. WHITE, JR.

of a conformational change with the addition of substrates or substrate analogs to the enzyme. They found that such ligands protected against the sulfhydryl reagents p-chloromercuribenzoate and N-ethylmaleimide, as well as against the inactivating action of trypsin, and a protective conformational change appeared to be the most reasonable explanation. Andree and Berliner (1980), using ESR and NMR in a study of the nature of the binding of Mn(I1) and UDP-galactose to galactosyltransferase, proposed a model consistent with their observed proton relaxation rates. Thus, there is a slow conformational interconversion of an initially formed rapidly exchanging conformation of the ternary complex to a second form that contributes negligibly to the relaxation. In addition, their evidence clearly indicated the presence of two Mn(I1) per molecule of enzyme (see also Powell and Brew, 1976a), and they noted that the affinity of galactosyltransferase for this ion is much higher in the presence of UDP-galactose. On the other hand, Klee and Klee (1970) found no compelling evidence that a conformational change from complexing of galactosyltransferase with a-lactalbumin could be responsible for the altered catalytic activity, which they suggested might be due to the “mass action effect” of complex formation (see also Bell et al., 1976). Powell and Brew (1976a) have investigated the effects of various metal ions [Zn(II), Cd(II), Fe(II), Pr(III), and Ca(II)] in combination with galactosyltransferase. All of these can substitute for Mn(II), although they do not produce as much activity as does Mn(I1). Two binding sites were distinguished. First, there is a “tight” binding site, from which Ca(I1) is excluded. Second, there is a “looser”binding site which may bind either Mn(I1) or Ca(I1). Mn(I1) was found to have a specific synergistic effect on UDP-galactose binding. O’Keeffe et al. (1980a) gained further insight into the nature of the metal binding sites of galactosyltransferase. The results from a variety of kinetic, spectroscopic, and affinity chromatography studies suggest specific functions for these sites. Thus, site I appears to be concerned with maintaining structural integrity, either of the active site region or of the protein as a whole, while site I1 is more closely associated with binding of UDP-galactose. As for the metal binding capabilities of these sites, Mn(I1) must be first liganded to site I, prior to a second Mn(II), as well as prior to substrate. Both sites can bind a number of metal ions. However, Ca(I1) and Eu(II1) bind only to site 11. Further, these workers have explored the topography of the galactosyltransferase molecule with fluorescence energy transfer measurements. Thus, transfer between Co(II), bound to site I, and Eu(III), bound to site 11, indicates a distance of 18 f 3 hi between them. An SH group (one of three) in galactosyltransferase was then modified with S-

LYSOZYME AND CY-LACTALBUMIN

255

mercuric-N-dansylcysteine. Transfer measurements between the fluorescent ligand to Co(I1) in site I1 indicated a distance of 19 k 3 A. O’Keeffe et al. (1980b) also studied the a-lactalbumin-galactosyltransferase complex (see below).

C . Interactions of Galactosyltransferase and a-Lactalbumin in the Lactose Synthase System Klee and Klee (1970) found that the A protein of lactose synthase catalyzes lactose production, even in the absence of a-lactalbumin, albeit poorly, because of the high K , for glucose. a-Lactalbumin, complexed with galactosyltransferase, drastically lowers the K,,, values for glucose and N-acetylglucosamine. Depending on the substrate concentration, a-lactalbumin can stimulate or inhibit disaccharide formation, with both N-acetylglucosamine and glucose. The affinities of the two sugars are such that, under the usual conditions of activity determination, the concentration of glucose is optimal for lactose synthesis, whereas that of N-acetylglucosamine is inhibitory. These findings help to differentiate between possible ways in which a-lactalbumin could influence the production of lactose. One of these, which, on the surface, appears plausible, is that a-lactalbumin might accept N-acetyllactosamine as a substrate, this product having arisen from the enzymatic action of galactosyltransferase. The ensuing transglycosylation, whereby lactose would be produced, could then account for the effect of a-lactalbumin in the presence of galactosyltransferase. Brew et al. (1968) showed, however, that a-lactalbumin has no affinity for N-acetyllactosamineand thus could not be involved in this reaction. An alternative possibility involves an induced conformational change in a-lactalbumin as a result of complexing with galactosyltransferase,so as to produce an affinity for N-acetyllactosamine. Although this explanation appears never to have been ruled out, there would be no need to invoke it, since it is already known (Klee and Klee, 1970,1972; Andrews, 1969; Fitzgerald et al., 1970a) that galactosyltransferase has the innate ability to carry out this reaction, even in the total absence of a-lactalbumin. Therefore, the simplest explanation for the action of a-lactalbumin and galactosyltransferase, as suggested by Browne et al. (1969), is that a-lactalbumin, in complexing with galactosyltransferase, modifies the conformational structure of the latter to produce a form of the enzyme that more readily catalyzes the production of lactose, and then does so in preference to N-acetyllactosamine. According to Fitzgerald et al. (1970a,b),lactose may be synthesized at

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maximal rates by galactosyltransferase in the absence of a-lactalbumin, but in the presence of high concentrations of glucose. It was suggested that the lowering of K , for glucose, to permit maximal synthesis of lactose, is the physiological role of a-lactalbumin. In addition, however, according to Schanbacher and Ebner (1970), a-lactalbumin inhibits the transfer of galactose to N-acetylglucosamine. It does not appreciably inhibit transfer to various oligomers tested. A considerable effort has been expended, in several laboratories, on the kinetics of interactions involving galactosyltransferase. The results of Morrison and Ebner (1971a) were not in accord with any mechanism whereby substrates might add in any order, either wholly or partially random. The simplest mechanism, consistent with their kinetic data, was an ordered mechanism, whereby additions to galactosyltransferase proceed in the order Mn(II), UDP-galactose, and N-acetylglucosamine. Morrison and Ebner (197lb) concluded that additions also proceed in an ordered manner when a-lactalbumin is included, and the acceptor is glucose instead of N-acetylglucosamine. Thus, they appeared to add in the order Mn(II), UDP-galactose, glucose, and a-lactalbumin. They regarded a-lactalbumin as a special type of modifier that combines with the enzyme only after the addition of carbohydrate. Also, they found an ordered release of lactose and UDP-galactose and postulated that Mn(I1) does not dissociate during the reaction. Morrison and Ebner (1971~)were concerned with the kinetic effects of a-lactalbumin with either glucose or N-acetylglucosamine. Thus, alactalbumin appears to cause the following: (1) alternate pathways, (2) reductions in K, for either substrate, (3) a decrease in velocity with the N-acetylglucosamine reaction, and (4) an increase in velocity with the glucose reaction. Khatra et al. (1974) studied, by steady-state kinetics, the reactions catalyzed by human milk galactosyltransferase. Whether in the presence or absence of a-lactalbumin, they concluded that the reactants added in the order Mn(II), UDP-galactose, and monosaccharide. They felt, however, that their kinetic results could best be explained with a mechanism whereby a-lactalbumin attaches to the enzyme immediately before the monosaccharide, contrary to the finding by Morrison and Ebner ( 1971b). The attachment of Mn(I1) at site I in galactosyltransferase is essential for interaction with a ligand protein (e.g., ovalbumin) or with alactalbumin (Powell and Brew, 1976a).Also, according to these workers, the presence of saturating concentrations of UDP-galactose potentiates the binding of a-lactalbumin at high Mn(I1) concentration. When the ligand is ovalbumin, the binding of this protein and a-lactalbumin is mutually exclusive.

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Powell and Brew (1976b) found that monosaccharides enhance the binding of a-lactalbumin to galactosyltransferase, which is already saturated with Mn(I1). However, the binding of a-lactalbumin and monosaccharide is random and strongly synergistic. Thus, they felt that the effects of a-lactalbumin on disaccharide synthesis are more satisfactorily explained by random synergistic binding than by the ordered binding proposed by Morrison and Ebner (1971a,b) and by Khatra et al. (1974). Bell et al. (1976) also found that lactose synthesis proceeds after a random equilibrium addition of substrates and a-lactalbumin, since the initial rate parameters obtained with bovine galactosyltransferase at saturating concentrations of Mn(II), and a variety of acceptors, were inconsistent with an ordered addition. They concluded that the large decrease in K , for glucose in the presence of a-lactalbumin is primarily the result of the high degree of synergism in the binding of a-lactalbumin and glucose to the enzyme-Mn(I1) complex. Prieels et al. (1976) studied the binding of glycoconjugates with galactosyltransferase in both the presence and absence of a-lactalbumin. They concluded that, in contrast to its ability to inhibit N-acetyllactosamine production, a-lactalbumin does not inhibit the transfer of Dgalactose to oligomers of N-acetylglucosamine or to glycopeptides. O’Keeffe et al. ( 1980b) studied the galactosyltransferase-cr-lactalbumin complex after dansylation of Glu- 1 of a-lactalbumin. Resonance energy transfer measurements, with cobalt (bound to galactosyltransferase at site I) as the energy acceptor, indicated a distance of 32 A between the dansyl group and the cobalt. Since Glu-1 is close to the cleft region, this observation made it unlikely that this region of a-lactalbumin is involved in acceptor substrate binding. These authors presented a schematic model of the active site of galactosyltransferase and its interaction with a-lactalbumin, summing up their experimental results (O’Keeffe et al., 1980a,b).

D . Structural Requirements of Substrate Andree and Berliner (1978) reported that UDP-glucose was marginally active as a donor substrate for both the transferase and the synthase. Then Berliner and Robinson ( 1982) determined the structural requirements for the donor pyranose moiety of UDP-galactose in either galactosyltransferase or lactose synthase. Thus, an axial 4” hydroxyl group on the pyranosyl moiety is essential for precise substrate alignment, as is an equatorial 6 CH,OH moiety. Where one or the other is lacking, the maximal rate of glycosyl transfer is about 0.05% of that of UDPgalactose. Berliner et al. (1984) further studied the acceptor for lactose synthase.

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They found that the basic requirements are: (1) a pyranose, thiopyranose, or inositol ring structure; (2) equatorial substituents (if any) at C-2, C-3, C-4, and C-5; (3) the aglycone (at C-1) may be either a or P, although a is preferred; (4) in the absence of a-lactalbumin, galactosyltransferase will accept long-chain 2-N-acyl substituents on the glucosamine structure; (5) an equatorial amino or N-acyl substituent (e.g., mannosamine or acetylmannosamine) is also a suitable acceptor in the absence of a-lactalbumin, since both N-acetylglucosamine and N-acetylmuramic acid have complementary binding loci for the N-acyl moiety; and (6) the aglycone moiety must be equatorial ( P configuration). E . Final Remarks

Much has been accomplished, especially in recent years, toward the goal of elucidating the active sites of galactosyltransferase and a-lactalbumin. To this end, alteration of specific residues with observations of consequent effects on structure and activity is enlightening, as are metal ion effects. Where the substrate is concerned, we now have some detailed structural information for both galactosyltransferase and the lactose synthase system. The effect of a-lactalbumin on galactosyltransferase is possibly a mass action effect, culminating in the preference, under normal conditions of activity determination, for glucose over N-acetylglucosamine. Of the conformational changes that occur in galactosyltransferase, particularly concomitant with the addition of Mn(II), much remains to be learned in correlating structural changes with the onset of enzymatic activity. Finally, the weight of evidence from kinetic studies appears to support the point of view that the addition of Mn(II), glucose, and a-lactalbumin to galactosyltransferase, prior to the expression of enzymatic activity, is governed by random synergistic binding with galactosyltransferase. Galactosyltransferase,as was also shown, is a ubiquitous enzyme, while a-lactalbumin is restricted to the mammary gland, and expression of the latter is under hormonal control. The question can be raised as to whether other galactosyltransferases, from their many extramammary sources, might also react with a-lactalbumin or similar proteins, which might result in modifying enzymatic function. This possibility, in fact, occurred to Hamilton (198 1) after he had isolated a galactosyltransferase from the reproductive tract of the male rat (Hamilton, 1980). He then isolated and characterized an “a-lactalbumin-like” protein which, however, differed from its mammary counterpart. Thus, it catalyzed transfer of galactose equally to inositol or glucose, whereas only glucose

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can act as the acceptor in the mammary gland system. We suggest that a continued search for, and characterization of, such a-lactalbumin-like proteins could yield results of considerable genetic and evolutionary impact (see also Section XI). PHYSICAL, CHEMICAL, AND BIOLOGICAL IX. SOMEADDITIONAL COMPARISONS BETWEEN LYSOZYME AND ~-LACTALBUMIN

A . Spectroscopic Studies

Many of the following studies, particularly those that measure changes by absorption or fluorescence, were made for a-lactalbumin, without corresponding studies on lysozyme that may have been used comparatively. One must consider the likelihood that some such studies have been attempted, but without success, since unsuccessful experiments seldom find their way into the literature. It can only be surmised that the conformational structure of lysozyme is sufficiently more resistant to change that such studies would have proved relatively unproductive. It is well established, in fact, that lysozyme does offer more resistance to denaturative change and to chemical alteration in general (Section IX,E and F). 1 . UV Absorption Spectroscopy Kronman et al. (1965) and Kronman and Holmes (1965) appear to be the first to have studied the effects of acid on a-lactalbumin and report that this protein, adjusted to pH values below its isoelectric point, exhibits a hypsochromic shift in its absorption spectrum between 270 and 300 nm. Spectral shifts in this region usually reflect changes in the environment of Trp and Tyr residues. The conformational change is a complex one, involving a series of steps. Because of the nature of the shift, the numbers of Trp and Tyr residues present, and the relative magnitudes of E for Trp and Tyr, Kronman and co-workers concluded that the shift results from environmental alterations for more than one of the buried Trp residues. (At the time of this study, three Trp residues were considered to be buried in bovine a-lactalbumin.) There appears to be no corresponding effect for hen egg-white lysozyme. [However, note the effect of acetic acid studied by Kato et al. (1984).] 2 . UV Difference Spectroscopy

Conventional UV difference spectroscopy and solvent perturbation difference spectroscopy have been used in a wide variety of protein stud-

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ies (McKenzie, 1970; Nicola and Leach, 1976). Both methods have been used in studies on the involvement of Trp residues in various reactions of a-lactalbumin and lysozyme. Efforts with this methodology have been limited, probably because, as pointed out by Imoto et al. (1975), “even a relatively simple-appearing pH difference spectrum reflects a complex set of interactions involving several chromophores and several ionizable groups.” Hayashi et al. (1964) used both methods to study the changes that occur upon formation of the lysozyme-substrate complex. Their results indicated that at least one Trp residue becomes buried in the hydrophobic region produced by formation of the complex. Imoto et al. (1975) used difference spectroscopy to investigate saccharide binding of lysozyme and concluded that Trp-108 makes a principal contribution to this reaction. An early investigation of a-lactalbumin with the solvent perturbation method was undertaken by Kronman and co-workers, who studied the degree of exposure of Trp groups under a variety of conditions, especially the changes at low pH (-4) (Kronman et al., 1965; Kronman and Holmes, 1965; Robbins et al., 1967). When this work was initiated, it was not known that a-lactalbumin is a Ca(I1)-requiring protein (see Sections II1,B and VI,D). As already indicated, the normal form of the protein is now referred to as the N form, and the low-pH form, from which Ca(I1) is lost, is called the A form. Molecular states similar to the low-pH A form may be produced by a variety of other treatments (see Section IX,E). [Molecular states similar to the low-pH A form also may be produced by a variety of other treatments (see Kronman, 1989).] In their initial studies it was thought that the low pH conformational shift in the N + A transition involved the exposure of the buried Trp residues. However, their solvent perturbation difference spectral studies enabled them to conclude that the change at 25°C does not involve unfolding of the molecule in the region of the buried Trp groups and their consequent exposure, but involves alterations in the interactions of these Trp residues with other perturbing groups. At pH 6 and 1°C there is a loss of accessibility of the two exposed Trp groups to sucrose, but they are still accessible to water. In the pH range 1.8-3.0 at 1”C, one exposed Trp group becomes inaccessible to sucrose, but the other is accessible to water and sucrose. Thus, Kronman and co-workers visualized the exposed groups as lying in a “crevice” with contraction that occurs with decrease in temperature, the extent of which was pH dependent. Later, Kronman et al. (1972a) considered that the “crevice contraction” phenomena are probably artifactual. Kita et al. (1976) studied the reversible unfolding of a-lactalbumin in

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guanidine hydrochloride, using difference spectroscopy and pH -jump measurements. They concluded that a-lactalbumin in the native state is less stable than lysozyme, an observation that is in agreement with conclusions from many other studies (see Section IX,D-F).

3 . Fluorescence Spectroscopy Kronman et al. (1971) used both fluorescence spectroscopy and CD in a study of conformational changes occurring upon acylation of alactalbumin. The observed changes in fluorescence were similar to those occurring on acid denaturation. Thus, it appeared likely that the results of acylation, as well as acid and alkaline denaturation, are brought about by conformational changes that give rise to freedom of rotation of Tyr and Trp side chains. Rawitch (1972) reported a difference in the rate of rotational diffusion, determined from the polarization of fluorescence, which suggested that the effective molecular volume of a-lactalbumin in solution is larger than for lysozyme, and this difference suggests conformational differences. Miller and King (1975) studied the differing luminescence properties of lysozyme and a-lactalbumin. For the latter the spectral properties observed were attributed to the proximity of Trp residues to S-S bonds, the reduction of which caused much change, as did acid denaturation. Their work supports the earlier suggestion by Sommers et al. (1973), that energy is transferred from Trp- 109 to Trp-63 in the active site cleft, with subsequent quenching of the latter by neighboring cystine residues. Sommers and Kronman (1980) made further progress in the characterization of Trp chromophores in comparative fluorescence studies of bovine, goat, human, and guinea pig a-lactalbumins by characterization of the environments of individual Trp residues in partially unfolded conformers. In the native state Trp-28 and Trp-109 transfer their excitation energy to Trp-63, whose fluorescence, as suggested earlier by Kronman’s group, is quenched by a pair of vicinal S-S bridges. Changes in fluorescence occurring upon formation of the heat-denatured form are caused by exposure of Trp-63. T h e effect of low pH on a-lactalbumin was studied further by Permyakov et al. (1981). In addition to the spectral shift toward shorter wavelengths (observed by Kronman et al., 1965), they found a decrease in Trp fluorescence quantum yield. They suggested that the replacement of Ca(I1) by H(1) is basically responsible for these effects. Much more recently, Desmet at al. (1987) studied the hydrophobicity of the partially unfolded conformer of a-lactalbumin, which results from removal of Ca(II), in comparison with the native conformer, mak-

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ing use of the hydrophobic fluorescent probe bis-ANS, as it combines with a-lactalbumin. They concluded-not only from these experiments, but also on thermodynamic grounds, as well as from results of adsorption experiments of the apo and bound forms to phenyl-Sepharosethat both Na(1) and Ca(I1) induce a conformational change in alactalbumin, in which hydrophobic regions are removed from the solvent to form a less hydrophobic protein. (For earlier use of bis-ANS in the study of a-lactalbumin, see Section V1,D.) Other uses of fluorescence in examining conformational changes that occur on complexing with metal ions were discussed in Section VI. The results of Kronman et al. (1981), Murakami et al. (1982), Murakami and Berliner (1983), Kronman and Bratcher (1984), and Ostrovsky et al. (1988) have been particularly significant. 4 . Raman Spectroscofi

The Raman spectroscopy of lysozyme dates back more than 50 years, when Edsall(l938) proposed such a study. However, the first spectrum was not obtained by Garfinkel and Edsall(l958) until 20 years later. This was probably the first published Raman spectrum of a biopolymer. More recent studies (e.g., M. C. Chen et al., 1974) resulted from the impetus of the work by R. C. Lord (see Lord and Yu, 1970). The use of Raman spectroscopy in biochemistry was extensively reviewed by Yu (1977),who included a discussion of lysozyme and a-lactalbumin spectra. Yu (unpublished work quoted by Yu, 1974)found that, as would be expected, there was no sign of conformational change for lysozyme, as revealed by the Raman spectrum, as the pH was decreased from 5.2 to 2.0. In particular, there was virtually no change in the amide I11 backbone region (1220-1300 cm-'). On the other hand, there were changes in amide I11 frequencies and the contour of Raman bands for a-lactalbumin upon the same reduction in pH value. Yu (1974, 1977) also showed, by comparison of the environments of the peptide backbone, as reflected in the amide I11 bands, that there was virtually no difference between the spectra of crystals of lysozyme and a-lactalbumin and their respective solutions. However, the spectra were altered by lyophilization, with respect to main-chain conformations (this is considered again in Section XI). Using the 830/850 cm-' doublet as a measure of the negative state of phenolic oxygen and of the tyrosine environment, Van Dael et al. (1987) studied the effect of low pH on bovine a-lactalbumin Tyr groups. They also examined the stabilizing role of Ca(I1) and Na(1) on the structure and the change in state of Trp residues as the molecule unfolds. While considering vibration spectra, mention should be made of the

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study by Careri et al. (1980), who used IR to study the events that occur during the hydration of lysozyme. [For later developments in this work, see the article by Rupley and Careri, this volume.]

5. Circular Dichroism (CD) and Optical Rotary Dispersion (ORD) CD and ORD (generally discussed by McKenzie, 1970, and Simons, 1981) were often used in early comparisons of lysozyme and a-lactalbumin. Although they suffered from limitations in the equipment then available, it is valuable to consider these results here. In Section XI we discuss some future possible studies in this area. Aune (1968) found that the ORD curve for bovine a-lactalbumin (pH 6.1, 0.1 M KC1) was indistinguishable from that of domestic hen egg white lysozyme (pH 4.5,O.l M KC1) in the region 206-233 nm, but that there were large differences in Cotton effects in the 250- to 233-nm region. Similar results were obtained in CD studies by Kronman (1968). Other workers [including one of the authors (H. McK.)] found differences in the 205- to 240-nm region as well as the 250- to 330-nm region. Before considering the conformational significance of these differences, it is necessary to consider the origin of Cotton effects in the 250- to 3 1O-nm region. Glazer and Simmons (1965) were the first to observe a side-chain Cotton effect near 290 nm in the ORD (and later a positive CD band at 294 nm) of domestic hen egg-white lysozyme, and they attributed it to Trp residues. Following publication of the X-ray crystal structure and amino acid sequence of domestic hen egg-white lysozyme, Teichberg et al. (1970) selectively oxidized Trp-108 and found that the positive ellipticity at 294 nm was abolished, leaving negative contributions in the 265- to 300-nm region from other sources, the most prominent extrema being at 293 and 268 nm. It was considered that the oxidation prevented coupling of transitions in residue 108 with those in Trp residues 63 and 111, which are situated nearby. (The positive band is absent in bovine a-lactalbumin, where residues 59 and 107 are not Trp; these residues are 62 and 111 in hen lysozyme sequence numbering.) Comparative studies of CD of human lysozyme and domestic hen egg-white lysozyme have been made by Halper et al. (197l), and of bovine, camel, guinea pig, and human a-lactalbumin, and domestic hen egg, duck egg, goose egg, and human lysozyme by Cowburn et al. (1972). Although the domestic hen egg-white lysozyme has more Trp residues than human lysozyme, Cowburn et al. found the intensity of the CD band near 293 nm was less in domestic hen egg-white lysozyme than in human lysozyme. They concluded that this is due to cancellation of contributions of opposite sign in the domestic hen protein. The 293-nm band in the

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a-lactalbumin is small and is superimposed on larger negative bands from other sources. Cowburn et al. do not agree with the interpretation (based only on the CD of domestic hen lysozyme) of Teichberg et al. (1970) for the 293-nm perturbation and conclude that it involves, at least for human lysozyme, mainly Trp-64 (residue 63 on domestic hen lysozyme numbering). They also consider that the differences in this region between the hen lysozymes and other proteins studied may arise from the adjacent Trp residues 62 and 63 in the hen protein. All of the a-lactalbumins and lysozymes studied, except the egg lysozymes, have negative bands near 270 nm. It is not possible to allocate these bands unequivocally, but they appear to involve Tyr perturbation. Differences in chirality of the disulfide bridges in a-lactalbumin and lysozyme may also cause appreciable contribution to bands in the 240- to 270-nm region. T h e curves in this region cannot be classified simply as typical of mammalian lysozymes, egg lysozymes, and a-lactalbumins. The spectra for bovine, pig, and kangaroo a-lactalbumins are very similar. On the other hand, the curve for human a-lactalbumin is similar to that for human lysozyme, but different from that of domestic hen lysozyme. T h e curve for echidna lysozyme I resembles that of human lysozyme fairly closely. The side-chain effects of red kangaroo a-lactalbumin and echidna lysozyme I resemble those of human lysozyme (Hopper and McKenzie, 1974). On the basis of CD studies, Robbins and Holmes (1970) reported the following tentative fractions for a-lactalbumin structures: 0.25-0.26 (25-26%) of the chain length as a helix, 0.14-0.15 (14- 15%)as /3 structure, and 0.60 (60%)as unordered structure. These values are somewhat different from those reported for lysozyme by Greenfield and Fasman (1969) and by Y.-H. Chen et al. (1974). For the former the fractions were 0.29 (29%) helix, 0.1 1 (11%)p structure, and the remainder unordered. For the latter the values for helix and p structure were 37% and 11%, respectively. X-Ray analysis of lysozyme (Blake et al., 1967a) yielded 28-42% helix and 10% /3 structure. (This range of values for helix indicates regular a helix on the low side, and 3,, as well as distorted helices on the high side, included together with the a helix.) Robbins and Holmes ( 1970) stressed the interpretation that their comparisons meant “similar” conformations and that they had, in effect, corroborated the work by Kronman (1968). On the other hand, Bare1 et al. (1972), using ORD and CD, found slightly more helix for lysozyme than for alactalbumin. More recently, Nitta et al. (1984) reported 33% and 1776, respectively, for a helix and p structure of bovine a-lactalbumin. Thus, while values

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for helix in a-lactalbumin have been within the range found by X-ray analysis of lysozyme, there have been considerable differences in reported values for p structure. Rawitch (1972) found a difference in rotational diffusion of these two proteins and concluded that the effective molecular volume of a-lactalbumin is greater than that of domestic hen lysozyme. Takesada et al. (1973) used CD in a study of the unfolded proteins and concluded that the two differ, with much structure remaining for a-lactalbumin that was not present for lysozyme (see also Section IX,E). CD has been used also in the search for conformational intermediates, in a comparative study of the oxidative refolding of lysozyme and a-lactalbumin (Kuwajima et al., 1985). This topic is dealt with in Section IX,E.

B . Small-Angle X-Ray Scattering Small-angle X-ray scattering measurements yield information relating to protein shape and hydration, as well as radius of gyration, but results based on such studies for a-lactalbumin and lysozyme have been interpreted differently by two groups of workers. Krigbaum and Kugler (1970) concluded that there are appreciable differences in the shape and hydration of the two proteins, but their conclusion has been disputed by Pessen et al. (197 1) and by Achter and Swan (1971). The latter group recalculated the results of Krigbaum and Kugler, making allowance for the effect of a small amount of protein polymerization, mainly dimerization, under their conditions of measurement. When this was done, Achter and Swan concluded that the small-angle X-ray results show the conformations of bovine a-lactalbumin and domestic hen lysozyme to be essentially the same. Although Pessen et al. (197 1) used newly developed apparatus to improve the small-angle X-ray scattering method, they could not reproduce the differences found by Krigbaum and Kugler (1970) and concluded that, except for a small difference in the extent of hydration, the two proteins have essentially identical macromolecular parameters. C . Electron Spin Resonance and Nuclear Magnetic Resonance

ESR and NMR, as is true of fluorescence spectroscopy, have often been used in the study of conformational changes in a-lactalbumin that occur upon complexing with metal ions (Section VI). These methods, however, have been used extensively in studies on lysozyme, as well as on a-lactalbumin. Thus, McDonald and Phillips (1969) used proton

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NMR to study lysozyme (Section VI). They concluded that Co(II), bound at a single site in lysozyme, could perturb resonance positions of protons throughout most of the protein molecule. Cowburn et al. (1970), with use of high-frequency proton NMR as above, as well as ORD, CD, and IR spectroscopy, undertook comparative studies between a-lactalbumin and lysozyme and found results consistent, with close conformational similarity. The observed differences between the spectra of these proteins were considered to be almost entirely explained by sequence differences. Other ESR and NMR studies have also been dealt with in Section VI. Gallo et al. (1971) and Teichberg et al. (1974) used ESR to explore the binding of metals to lysozyme. In addition, Berliner et al. (1983) used NMR as well as ESR to study the Mn(I1) binding site of a-lactalbumin. More recently, Musci et al. (1987)used ESR and NMR to determine certain intramolecular distances between spin-labeled Met-90 and the metal binding site, as well as certain resolvable protons. Bradbury and Norton (1975),on the basis of the model building studies by Browne et al. (1969) and by Warme et al. (1974),were able to make assignments of specific resonances in the proton NMR spectrum of bovine a-lactalbumin to the three His residues. These resonances, after reaction of the protein with iodoacetate under conditions that were nearly specific for the His residues, disappeared from the “native” frequency positions, but did so in a differential manner, consistent with differences in the degrees of exposure to the solvent. This was predicted by the model building studies, particularly those by Browne et al., which were subsequently confirmed by X-ray analysis. Thus, His-68 is the most exposed; His-32, being involved in a helical region (according to Browne et ul., but not to Warme et ul.), is less exposed, while His-107 is the least exposed. NMR has found more recent use in comparative studies of lysozyme and a-lactalbumin. Poulsen et al. (1980), using the nuclear Overhauser effect, were the first to demonstrate the existence of the “hydrophobic box’’ region, in solution, for lysozyme, first noted in the crystalline state by Blake et al. (1967a). Koga and Berliner (1985) applied the nuclear Overhauser effect to the study of a-lactalbumin. They too found a hydrophobic box, similar to that of lysozyme. The study was conducted in both the presence and absence of Ca(II), and there were only “subtle” differences upon removal of the cation. Berliner and Kaptein (1981) used another NMR method, induced dynamic nuclear polarization, to investigate the solvent accessibility of Tyr, Trp, and His residues in five species of a-lactalbumin. This method measures the access of the photo-excited flavin dye to the surface-exposed

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residues. Many differences were noted among the various residues of the protein examined, but Tyr-50 was the only one consistently unexposed. This finding appears to be consistent with the results of Acharya et al. (1989), in which Tyr-50 was placed inside a pocket of residues containing Ile-41, Thr-48, Asn-57, Ser-63, Leu-81, and S-S bond 61-77. The binding of monosaccharide inhibitors to hen egg-white lysozyme was studied with 270-MHz NMR by Perkins et al. (1981) (see Section IV). There is increasing use of NMR in the study of protein conformation (Gronenborn and Clore, 1990). Its application to the study of conformational changes upon modification of a-lactalbumin is considered in Section IX,E.

D . Association and Aggregation The effects of pH on association and aggregation of both lysozyme and a-lactalbumin have long been known. Sophianopoulos and Van Holde (1961) found lysozyme to be monodisperse at pH 5.4 and 20°C; but at more alkaline pH values the protein dimerizes. Sophianopoulus and Van Holde (1964) and Deonier and Williams (1970) reported detailed studies of the dimerization, which is rapid and reversible at Z > 0.0 1, carried out with sedimentation equilibrium and viscosity studies. The results are consistent with the hypothesis that dimerization is favored by the loss of a single proton from the monomer. The viscosity results of Sophianopoulos and Van Holde do not suggest significant changes in tertiary structure of the protein during the dimerization. However, the near-UV CD exhibits concentration dependence on association of lysozyme (Holladay and Sophianopoulos, 1972), and in the region of 256-293 nm reflects changes in the Trp residue. The rapid reversible association and slow aggregation of bovine alactalbumin at pH <4.0 have been studied in detail by Kronman and coworkers (e.g., Kronman and Andreotti, 1964; Kronman et al., 1964). They found that the protein was largely insoluble in the region of pH 4.2-5.2 and soluble at higher pH values. It was very little associated at pH 6.0 and even less at pH 8.5, the conformation being constant in the pH range 6.0-9.5. Kronman et al. (1967) showed that bovine a-lactalbumin undergoes some expansion, without aggregation, above pH 9.5. They concluded that these phenomena are related to a “denaturation-like” process. Robbins et al. (1965) sought to test the hypothesis that the acid aggregation of a-lactalbumin was due to hydrophobic interactions. Their approach was to insert additional nonpolar residues by amidination, and then examine the acid behavior of the product. They found the modi-

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fied protein to be much more susceptible to aggregation than was the native protein. Their results lend support to the hypothesis that hydrophobic interactions play an important role in the aggregation of alactalbumin. When all of the above work is considered, it is obvious that there are marked differences in association and aggregation behavior between lysozyme and a-lactalbumin, but the structural basis for these differences is poorly understood.

E . Denaturation and Renaturation The denaturation of proteins is discussed elsewhere by Tanford (1970); McKenzie and Ralston (1971); and McKenzie (1991). We concern ourselves here with some aspects of a-lactalbumin and lysozyme. Before discussing denaturation it is desirable to recapitulate work on transitions in a-lactalbumin and the variation in nomenclature that has been used. Following their early studies on the effect of pH on bovine a-lactalbumin, Kronman et al. (1972a,b) made more extensive studies of the effect of pH and temperature on both bovine and caprine a-lactalbumin. For the latter they classified the transitions as follows: Transition I: A pH-dependent transition in the pH range 4-8 involves changes in the environment of Trp residues, but does not involve any change in shape, nor is there any extensive conformational shift. Transition 11: In the pH range 2-4 transition I1 involves some conformational shift and expansion of the molecule. This was called the N + U conversion and is now the N + A conversion, as indicated in Section VI. At 25°C there is no increase in Trp group exposure, although there are changes in the environments of these groups. The results at 3°C are ambiguous. Transition IIA: There is an expansion of the molecule above pH 9.5. It is unresolved as to what extent this change is similar to that involved in transition 11. Regardless of whether the protein is subjected to pH values <4.0 or >9.0, is heated above 50”C, exposed to low concentrations of guanidine hydrochloride, or subjected to Ca(I1) removal from the N form, all usually appear to involve dissociation of Ca(I1) in some way. The original use of the term “U” by Kronman et al. (1972a,b) for altered forms of the protein differs from that of other workers more recently, whereby the term has been taken to designate completely un-

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folded protein (see, e.g., Baum et al., 1989, for the unfolded state in 9 M urea at pH 2.0). T h e term “U” with numerical subscripts (e.g., U 1 , U p , and U,) has also been used in various discussions on denaturation of various intermediates in unfolding. For transition I1 and similar types of transitional change, the term “A state” is now used by most workers and appears to have been used first by Kuwajima (1977). Denaturation of a-lactalbumin appears to be a three-state process (Kuwajima et al., 1976, 1985; Kuwajima, 1977). The initial folding of a-lactalbumin to the intermediate stage, assumed to be essentially the same entity as that occurring on denaturation, is dependent on local interactions, followed by hydrophobic interactions, and long-range specific interactions (S-S bond formation and electrostatic attractions). S-S bonds are not important for stabilizing the intermediate. Kuwajima and colleagues point out that their folding model is not inconsistent with the thermodynamic hypothesis of Anfinsen and Scheraga (1975), since the native conformation may yet prove to have the lowest Gibbs free energy; nevertheless, it may be reached through kinetically controlled intermediates. However, according to Rao and Brew (1989), an additional requirement is the presence of Ca(I1) for the refolding of a-lactalbumin, and this cation is essential for the correct pairing of half cystine residues to form disulfide bonds, as well as for the development of native conformation. Dolgikh et al. (1985) suggested a compact intermediate with a slowly fluctuating tertiary structure for a-lactalbumin. Ptitsyn et al. (1983) proposed a model for a-lactalbumin whereby the folding intermediate was seen as a compact globule with fluctuations in its three-dimensional structure (see also Shakhnovich and Finkelstein, 1982). More recently, Gil’manshin et al. (1988) found an early intermediate in the folding of a-lactalbumin that forms in sec after denaturation with 8 M urea. This time is two orders of magnitude smaller than that found by Kuwajima et al. (1985). T h e latter authors maintained that their folding intermediate had essentially the same secondary structure as their denaturation intermediate (the A state). Kuwajima (1989) reviews findings on the “molten globule state” and suggests that it may be a common state among proteins. Baum et al. (1989), through an NMR study of the A state of alactalbumin, produced a more detailed structural picture than has been seen previously. Methods were used that exploit the well-resolved spectrum of the native state to examine the A state indirectly. Some resonances in the latter were considerably shifted from their native positions and were identified through magnetization transfer with the native state.

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HUGH A. MCKENZIE AND FREDERICK H.

WHITE,JR.

Of particular interest was the apparently unchanged helical segment 89-96 in the A state, as indicated by a number of amides that were protected from solvent exchange. It was suggested that the stability of this segment may be brought about by a variety of side-chain interactions. From their results Baum et al. were able to propose a model for the A state, in which much conformational freedom is exhibited, but in which specific elements of the native state are preserved. The denaturation of lysozyme, on the other hand, is a two-state process, according to Tanford (1970). In addition, there is high cooperativity between S-S bonds and conformational structure for this protein, compatible with the hypothesis that the formation of native S-S bonds may occur as an essential prerequisite early in the folding process, prior to the onset of native conformational structure (White, 1982; White and Wright, 1984). The work by Galat et al. (1981), in fact, indicates that, after at least three of the four S-S bonds reform, all measured elements of conformation appear simultaneously. On the other hand, Kato et al. (1981) found spectral evidence for a “rapidly” formed structural intermediate in the refolding of hen egg white lysozyme. It was concluded that the unfolded species assumed its new transient conformation in the mixing process of the pH-jump measurements and that the transformation was complete within the mixing time. Unfolding had been achieved by either 4 M guanidinium chloride or 40% acetic acid. Further evidence for the essential nature of S-S bond formation in the development of native conformational structure, for human lysozyme, comes much more recently from Taniyama et al. (1988). They produced the first evidence that formation of a specific disulfide bond (Cys-56-Cys-28) is a prerequisite for the correct folding of this protein in vivo, expressed in Saccharomyces cerevisiae. Notwithstanding the suggestions of Kuwajima and co-workers for the renaturation of a-lactalbumin (above),it is difficult to see how the renaturation of lysozyme in particular could be governed primarily by thermodynamic influences, in view of the reported requirements for native S-S bonds (for further discussion see White, 1982; White and Wright, 1984). Additional evidence of difference in the refolding process is that the time taken for reoxidation of a-lactalbumin is longer than 25 hr, whereas that for lysozyme is only 90 min (Tamburro et al., 1972). Iyer and Klee (1973) found differences in the rates of S-S bond reduction in these proteins. Reduced a-lactalbumin retained hydrodynamic and optical properties characteristic of folded globular proteins, although its conformation was clearly distinguishable from that of the native protein. Evidence of sameness in the denaturation of a-lactalbumin and lyso-

LYSOZYME AND a-LACTALBUMIN

27 1

zyme comes from Sharma and Bigelow (1974), according to whom these proteins behave similarly on denaturation, and therefore would be expected to have similar backbone conformations (cf. Maes et al., 1969; Bradbury and King, 1971). An indication of sameness on reoxidation comes from Chiaranda et al. (1972), who attempted to reoxidize a reduced mixture of two CNBr fragments of or-lactalbumin; the CD spectra that appeared did not suggest that the native conformation could have been reformed. This observation was interpreted to mean that S-S bonds form early in the renaturation of a-lactalbumin, consistent with the previously mentioned work on lysozyme (Galat et al., 1981; White, 1982; White and Wright, 1984; Taniyama et al., 1988). Thus, the two proteins appear to be similar in this respect. In this picture the differences appear at least as striking as the similarities, in comparing both denaturative and renaturative properties of a-lactalbumin and lysozyme. Such differences are compatible with differences in the conformations of these proteins in the native state, in aqueous solution. T h e behaviors of apo- and Ca(I1)-bound forms of a-lactalbumin differ markedly upon denaturation with guanidine hydrochloride, as shown by Ikeguchi et al. (1986). Thus, at low Ca(I1) ion concentration alactalbumin unfolds to produce a stable intermediate, while at high Ca(I1) concentration the protein unfolds in a manner similar to that of lysozyme. Further studies on the conformers of a-lactalbumin were reported by Hanssens et al. (1984). The thermal transition curve differs between the Ca(I1) and apo forms of a-lactalbumin, although these forms have similar fluorescence characteristics. F. Chemical Reactivities Reactivity differences between a-lactalbumin and lysozyme are given by Lin (1970), where the carboxyls of these proteins are concerned. These differences, however, were considered to be compatible with the model of Warme et al. (1974). Atassi et al. (1970) found S-S bonds in a-lactalbumin to be more susceptible to reduction. Barman and Bagshaw (1972) found six peptide bonds in a-lactalbumin to be digestible by trypsin, but not so in lysozyme, which is therefore relatively resistant to this enzyme. Further, these workers concluded that Trp-26 (also Trp-104 and Trp-108) is not buried-but it is, according to the model of Browne et al. (1969) and the crystal structure. Takesada et al. (1973) made several observations: (1) the number of

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HUGH A. MCKENZIE AND FREDERICK H.

WHITE,JR.

slowly exchanging peptide hydrogens was 35 in a-lactalbumin, but 44 in lysozyme; (2) kinetic profiles of the exchange reaction were different for the two proteins; (3) the midpoints of their thermal transitions were different; (4) effects of pH on the exchange kinetics differed; ( 5 ) a CD study indicated that the unfolded form of a-lactalbumin has an “appreciable amount” of folded structure. A similar conclusion was reached by White (1976) for lysozyme, after full reduction of S-S bonds in 8 M urea, followed by redissolving in dilute buffer. More recently, attention has focused on a specific residue in a-lactalbumin as being essential for its activity in combining with galactosyltransferase to form the lactose synthase system. This is His-32, which is consistently present for all species, as would be necessary for an essential residue. Schindler et al. (1976) specifically modified this residue with diethyl pyrocarbonate, to give the ethoxyformyl derivative, with inactivation of the protein. This His lies within the cleft region of a-lactalbumin and in this position might substitute for Glu-35, which is present in the lysozymes (Fig. lo), thus possibly accounting for the trace of cell lytic activity reported by McKenzie and White (1987). This activity is considered in more detail in Section X. Pfeil (1981) concluded that a-lactalbumin is less stable than lysozyme, with a lower thermal transition temperature, lower denaturational enthalpy, lower heat capacity change, and lower Gibbs free-energy change. Although these generally greater reactivities for a-lactalbumin may suggest a “looser” conformation for this protein, such may not be the case, according to Barman (1970),who suggested two conformations for a-lactalbumin in equilibrium in the native state: one, a relatively tight globular conformation; the other, a more diffuse conformation, which likely would be more accessible to various reagents and to denaturation in general. Thus, when the latter undergoes change, the equilibrium shifts in the direction of the more open conformation.

G. Immunochemical Properties Tanahashi et al. (1968) compared the immunological properties of bovine, water buffalo, ovine, caprine, porcine, guinea pig, and human a-lactalbumins by the method of Oudin. They found that the nonruminant a-lactalbumins do not react with antisera to the bovine protein. This is in accordance with our experience (K. Bell and H. A. McKenzie) using the Ouchterlony and immunoelectrophoretic methods. However, Sakar et al. (1971) found that while bovine, water buffalo, and caprine a-lactalbumins exhibit extensive cross-reaction in the Ouchterlony test, these ruminant a-lactalbumins may be differentiated quanti-

LYSOZYME AND a-LACTALBUMIN

273

tatively in the microcomplement fixation method. Atassi et al. (1970) found that bovine a-lactalbumin and domestic hen egg-white lysozyme do not show cross-reaction in quantitative precipitin analyses. However, weak cross-reaction in hemagglutination determinations was found by Strosberg et al. (1970). On the other hand, Faure and JolKs (1970) found no cross-reaction in similar studies for bovine a-lactalbumin, human milk lysozyme, and various egg lysozymes against anti-a-lactalbumin sera. The only antigenic similarities observed in “assays” against antidomestic hen lysozyme sera were among the several egg lysozymes. Arnheim et al. (1971) found that there was no cross-reaction between the native forms of human and domestic hen egg-white lysozymes, but there was cross-reaction between the reduced carboxymethylated proteins. They discussed several reasons for this and pointed out that antisera produced against unfolded proteins can be useful in evolutionary studies, but they cautioned against pitfalls in this approach. One application is that of Arnon and Maron (1971), who found cross-reactivity between the reduced carboxymethylated forms of a-lactalbumin and lysozyme. They regarded this observation as corroborating evidence that the two proteins evolved from a common evolutionary precursor. Light has been shed by the work of Gavilanes et al. (1984) on the question of why the native forms do not cross-react. Thus, secondary structures of lysozyme and a-lactalbumin, predicted on the basis of the method of Chou and Fasman (1974), are sufficiently different in the region of the antigenic loop (residues 60-83 in hen egg-white lysozyme) that it is not expected that the native forms of these proteins would cross-react. It appears to be generally accepted that, at least for lysozyme, most of the antigenic determinants are assembled topographic determinants. This concept is supported by the observation that little or no crossreactivity occurs between native and denatured lysozymes (Thompson et al., 1972). The antigenic determinants of the native lysozyme molecule appear to include most, if not all, of the surface residues, as evidenced by numerous studies (reviewed critically by Benjamin et al., 1984). This point of view contrasts with that of Atassi and Lee (1978), who claimed a limited antigenicity, based on a study of “surface-simulated peptides.” The sites delineated by the latter workers do not include Arg-68 (Fainanu et al., 19?4), the “loop” region in general, or any of several other segmental regions previously demonstrated to function in this capacity, such as residues 1-20 and 123-129 (see Benjamin et al., 1984). Hopp and Woods (1982) attempted to locate the antigenic sites of a-lactalbumin and found that this activity can be attributed to several peptic fragments and to single Arg and Met residues. They concluded

274

HUGH A. MCKENZIE AND FREDERICK H. WHITE, JR.

that although hen egg-white lysozyme does not cross-react with the a-lactalbumins studied, there are similar distributions of antigenic determinants on the surfaces of these a-lactalbumins and lysozyme. Smith-Gill et al. (1982), with the aid of a monoclonal antibody (HyHEL-5) prepared to domestic hen egg-white lysozyme, were able to identify an antigenic site (i.e., epitope) that was shared by the lysozymes of seven species of galliform birds. Two of these, bobwhite quail and chachalaca, shared only partial identity with epitope defined by this antibody. Duck lysozyme did not react with the antibody. Assuming a lack of long-range conformational changes, Arg-68 was identified as a determining residue. Together with Arg-45, to which it is hydrogen bonded, Arg-68 formed a basic cluster which may be a subsite of the epitope. Further evidence indicated that the epitope extended into the cleft region between Arg-45 and Arg-114 (Fig. 11). Using both the X-ray structure of the Fab :lysozyme complex (Sheriff et al., 1987, 1988; Amit et al., 1986) and the site-specific mutagenesis of

b

LOOP

HyHEL-5 determinant Catalytic Cleft HyHEL-12 HvHEL-7 525.5E4 Hyb.Cl

I

I

HyHEL-9 525.3c7

325.3D1 (residue 121) HyHEL-11

FIG. 11. (a) The amino acid sequence of domestic hen egg-white lysozyme, showing eight peptides that have been shown to be antigenic against anti-domestic hen egg-white lysozyme. N-C peptide is indicated by solid outline; LH,, by a dashed outline; Plb, by stippling; a continuous region (residues 34-54) within Plb, shown as black box; peptide 8, by a heavy black outline; the loop, by black with white lettering; loop 11, by a stippled box; LIII, by a dotted outline. (b) “Space-filling’’model of domestic hen egg-white lysozyme (computer generated). The loop and N-C peptide are dark gray, with residues 1-3 black. Specific residues recognized by monoclonal antibodies are colored or outlined in white: a hypothetical unit determinant for antibody HyHEL-5 is outlined in dotted black. [Reproduced with permission from Benjamin et al. (1984), who give details of the antibodies in their Fig. 2.1

LYSOZYME AND

a-LACTALBUMIN

275

a hen egg-white lysozyme cDNA gene (expressed in yeast), an epitope containing Arg residues 45 and 68 was found by Lavoie et al. (1989b), confirming the original prediction from epitope mapping of evolutionary variants of egg-white lysozymes (Smith-Gill et al., 1982). Site-specific mutagenesis, in particular, is of continuing value to these workers in their exploration of the interaction of monoclonal antibodies with lysozyme. Lavoie et al. (1989a) also compared the crystal structures of two monoclonal antibodies with serological data, and for one of these (their HyHEL-5, complexed with lysozyme) the results agree. However, for their HyHEL-10, complexed with lysozyme, similar comparison does not result in agreement. For the former, however, Arg residues 45 and 68 are implicated as epitopes. Thus, this conclusion is essentially in accordance with that of Lavoie.

H . Conclusions The studies summarized in this section have proved particularly valuable, because they were conducted in (mostly aqueous) solution, enabling comparison with X-ray or neutron diffraction studies, for which crystalline material must be used. Although the conformation of a crystalline protein can be essentially the same as that in aqueous solution, it is not necessarily correct to make this assumption, since there may be large conformational changes in such solution, contingent on the variables of protein concentration, pH, ionic strength, and temperature. In fact, there are many reported differences between a-lactalbumin and lysozyme in solution, in comparison with the similarity between these proteins indicated by high-resolution X-ray analysis of their crystals. On the other hand, UV absorption and fluorescence methods have been involved in relatively few comparative studies between a-lactalbumin and lysozyme. More useful to this end have been CD and ORD, both of which reflect changes in various short-range interactions. Also noteworthy have been the many studies with ESR and NMR. These, in addition, have found considerable use in the study of complexing between a-lactalbumin and metal ions. They will presumably be of value in the future for the study of such reactions for those few lysozymes that coordinate to metal ions. Other methods, including Raman spectroscopy and small-angle X-ray scattering, have yielded results generally supporting similarity between the conformations of a-lactalbumin and lysozyme. There may be two reasons for the reported evidence of dzfferences in conformation and reactivity in aqueous solution: (1) substantial differ-

276

HUGH A. MCKENZIE AND FREDERICK H . WHITE, JR.

ences in conformational structures of these proteins may indeed exist in solution; and (2) the suggestion of Barman (1970; see also Kuwajima, 1977; Dolgikh et al., 1981) may prove to be correct (i.e., a “tightly” folded conformation may exist in equilibrium with a form of a-lactalbumin that is more reactive). Such a phenomenon would explain why a-lactalbumin appears to be more accessible to various reagents than is lysozyme, and why it is more easily denatured, despite indications of a close conformational similarity. Thus, the reactive form of the protein is altered, whereupon equilibrium between the two forms is continually shifted to produce more of the latter form, which then continues to react. If this idea proves to be correct, the question arises as to what specific structural features of the “tightly” folded form of a-lactalbumin (compared with lysozyme) permit transformation to the more reactive form.

X. EVOLUTIONARY ORIGINS OF LYSOZYME AND a-LACTALBUMIN A . Introduction: Molecular Clocks and the Fossil Record As is obvious from a number of recent attempts to confront aspects of evolutionary history and evolutionary mechanisms, there is at present a greater openness to discussion than at any period since the formulation of the “Modern Synthesis”. This is largely the result of the revolution in molecular biology which has removed many of the restrictions on discussion of evolutionary mechanisms accepted since the development of that synthesis. But it has also followed on a period of reinterpretation of the fossil record as a source of new historical data, and as a testing ground for mechanisms. The claim of the palaeontologists to exclusive rights to the interpretation of the sequence and dating of evolutionary events has been under siege by advocates of cladistic taxonomy and, more effectively, from proponents of molecular clocks. Finally, the much publicised attack on the logical status of many of the explanatory concepts of Neo-Darwinism has pushed many workers into re-examining possible explanations for evolutionary phenomena that would have had few advocates five years ago. In the resulting turmoil, and it is indeed a turmoil, the once dominant fields of population genetics and development biology have provided a stabilising influence, rightly pointing to experimental data on the factors influencing evolutionary processes to counter some of the less well grounded and more speculative views.

From the preface by Campbell and Day (1987) to a ‘Rates of Evolution’ symposium

1 . Molecular Clocks The above comments reflect the trepidation that we feel as chemists, who do not profess to be either paleontologists or evolutionary biologists, in discussing this wide-ranging problem. It is evident in what has already been presented in this article that a-lactalbumin and lysozyme have important evolutionary relationships that involve divergence and/

LYSOZYME AND (Y-LACTALBUMIN

277

or convergence. Before discussing specifically their relationships, we must consider some important general background aspects. Even 30 years ago paleontology was virtually the only source of information about the periods when common ancestors lived. Indeed, the mammalian fossil record was not particularly good. Early writers on comparative biochemistry, such as Baldwin and Florkin, were limited in their sources. Perhaps the first person to perceive the unique value of the molecular evolution of proteins and nucleic acids was Anfinsen (1959), who wrote of this subject in his important book. However, it appears to have been Zuckerkandl and Pauling who, in 1960-1965, introduced the concept of the molecular clock (for a historical review which is part of a group of important critical papers in an issue of the Journal of Molecular Evolution, see Zuckerkandl, 1987). Zuckerkandl and Pauling (1962) and Margoliash (1963), by making sequence comparisons of hemoglobins and cytochromes c, respectively, concluded that the number of point mutations needed to account for the differences in amino acid sequences is correlated linearly with estimates from paleontology of the time elapsed since the species compared had a common ancestor. In discussing the molecular evolutionary clock, it is important to stress that it does not have the accuracy of the National Bureau of Standards standard of time, let alone that of radioactive decay. Furthermore, it is not absolute, but must be calibrated against fossil records. Many workers have reported that the amino acid sequences of proteins usually diverge at fairly constant rates. Often, the point mutations have no effect on protein function. Wilson et al. (1977), in their important review, indicated that each functional class of proteins tends to have its own specific rate of change. They used the term “unit evolutionary period,” which is the time, in units of lo6 years, required for the accumulation of a 1% difference in amino acid sequence. Concomitant with such studies, Kimura developed his neutral theory of molecular evolution. He asserted that the great majority of evolutionary changes at the molecular level, as seen in comparative studies of protein and DNA sequences, are the result not of Darwinian selection, but of random drift of selectively neutral (or nearly neutral) mutants (for reviews see Kimura, 1983, 1987). While such views were an anathema to some biologists and geologists, perhaps even greater concern was expressed over the views of Wilson and others on the time dependence evident in quantitative immunological comparisons of mammalian albumins and also of lysozymes (Wilson et al., 1977). One important factor in this lack of acceptance was the faulty view of the antigenic structure of proteins existing in the early 197Os,when much of this work was done (as we have already seen for lysozyme and a-lactalbumin in Section

278

HUGH A. MCKENZIE AND FREDERICK H. WHITE, JR.

IX,G). However, with more recent ideas on antigenic structure, as reviewed by Benjamin et al. (1984), apparently there is evidence that a good correlation may exist between immunological distance and sequence difference for some globular proteins. Hence, valuable information can be obtained about rates of change for many mammalian groups, as well as some plants, bacteria, etc. (see also the general review by Wilson et al., 1987). Despite the considerable body of evidence that Kimura accumulated in support of his neutral theory, he did find it necessary to reconsider some departures from clockwise progression of molecular evolution and to suggest future experimental programs in an attempt to settle certain issues (Kimura, 1987). In addition to comparison of amino acid and nucleotide sequences of a-lactalbumin, and their rates of change as molecular clocks, a considerable amount of comparative information has accumulated on the three-dimensional structure of these proteins; their physical properties in solution; effects of amino acid substitutions, in both genetic and cloned variants; and their functions and immunological properties. In assessing this information it is important not to lose sight of the known paleontological information on the origin and evolution of mammals. 2. Mammalian Evolution and Paleontology

The evolution of mammals has not been without controversy: Over 20 years ago Crompton (1968) entitled an important article, “The Enigma of the Evolution of Mammals.” Nevertheless, the majority of paleontologists would accept the phylogenetic relationships of the major groups of amniote tetrapods-reptiles, birds, and mammals-summarized diagrammatically in Fig. 12a. Clemens (1989) has maintained that, although one recent comparative study resulted in Gardiner’s (1982) proposal for a closer relationship between birds and mammals (see Fig. 12b), the weight of the evidence, particularly that from the fossil record, supports the interpretation given in Clemens’ article (see also other references in Clemens, 1989; for general discussions see Benton, 1984, 1990; Carroll, 1988; Gauthier et al., 1988). A critical feature of Clemens’ view (summarized in Fig. 12c) is the basal dichotomy between the group including modern reptiles and birds on the one hand and that including modern mammals on the other, and now appearing to date back some 300 million years. The Monotremata are seen as part of the other major branch of the diagram. The discovery of Steropodon in Australia extended the record of Monotremata back to -100 million years ago (Archer et al., 1985). A theme underlying many mammalian classification studies has been the desire to determine better the relationships of the modern monotremes, marsupials, and eutherians. The origin of the monotremes

I &' Ha

othermia

E"+

A

T

iota

I Tetrapoda

(b)

Mammalia

Mammal like "reptiles" A (ca. 300) C

FIG. 12. Some evolutionary relationships based on paleontological evidence. (a) and (b) Two views of the evolution of tetrapods (amphibians, reptiles, birds, and mammals) (a) shows a 'conventionay view (see, e.g., Benton, 1984, 1990); (b) shows the view of Gardiner (1982). Common names are shown on (a), systematic names in (b). (c) Clemens' (1989) view of the phylogenetic relationships of major groups of amniote vertebrates (birds, reptiles, mammals), especially showing monotremes, marsupials, and eutherian mammals. Divergence at A probably occurred about 300 MY ago (late Carboniferous). Divergence at B being about 135 MY ago (late Jurassic). Date of divergence at C is not known (although some consider 200 MY ago, late Triassic). D: Steropodon, dated about 100 MY ago (early Cretaceous). E: 90- 100 MY ago.

280

HUGH A. MCKENZIE AND FREDERICK H . WHITE, JR.

and their relationships to Marsupialia and Eutheria, in particular, were discussed by Griffiths (1978, 1989). From these and other studies (see, e.g., Kemp, 1983; Clemens, 1989) the therian mammals, including Marsupialia and Eutheria, still appear to be regarded reasonably as a monophyletic group. However, the former differentiation placing monotremes in a separate group may no longer be valid. [For general background information on marsupials and monotremes, see the articles by Dawson (1977) and by Griffiths (1988), respectively.] B . Evolution of Lysozyme and a-Lactalbumin: Divergence andlor Convergence? Even when Brew and Campbell (1967) suggested, on the basis of similarities in amino acid composition, that a-lactalbumin and lysozyme have diverged from a common precursor, proposals for the evolutionary development of homologous proteins were not new. Examples then known included pancreatic zymogens (Hartley et al., 1965), hemoglobin and myoglobin (Ingram, 1963), and immunoglobulins (Hill et al., 1966). However, none of these has the wide appeal of a-lactalbumin-lysozyme divergence because of the development of their contrasting biological functions. Subsequent comparisons of a wide variety of a-lactalbumins and ctype lysozymes revealed 35-40% identity in amino acid sequences; similarity, but not identity, in three-dimensional structures; high conservation of disulfide bridges; and similarity in many other properties. Such studies have resulted in the general acceptance of divergence of a-lactalbumin and c-type lysozymes, rather than their convergence. In the latter type of evolution, two unrelated genes may have evolved until arriving at a stage at which homology exists in the corresponding proteins, with the resulting sequences and conformations sufficiently adapted to perform their respective biological functions. The argument against this convergent process appears to be strong, and it has been further strengthened by the study of introns and exons. 1 . Zntrons and Exons In contrast to the genes of prokaryotes, for which the coding sequences are continuous, those of eukaryotes are present in blocks (exons) separated by intervening noncoding sequences (introns). Gilbert (1978) who introduced these terms, suggested that exonhntron structure could provide a mechanism for increasing the rate of evolution. It was pointed out that if, for example, exons corresponded to units of protein function, recombination within introns could reassort protein functions to

28 1

LYSOZYME AND a-LACTALBUMIN

produce new proteins from parts of existing ones. Furthermore, Blake suggested that if exons also corresponded to integrally folded substructural domains, there would be a higher probability that this type of recombination would result in a folded viable protein molecule (for a review see Blake, 1983). Comparisons of lysozyme and a-lactalbumin have been made at the transcriptional level. Thus, cDNA clones and nucleotide sequences of rat (Dandekar and Qasba, 1981; Qasba and Safaya, 1984), guinea pig (Craig et al., 1981; Hall et al., 1982), human (Hall et al., 1987), caprine (Kumagai et al., 1987), ovine (Gaye et al., 1987), and bovine (Vilotte et al., 1987; Hurley and Schuler, 1987) a-lactalbumins are now known, together with much of their flanking sequences. It can be seen from Table X that the genes of the four a-lactalbumins shown consist of four exons, separated by three introns. The introns occur at identical positions in all four species. Although the sizes of the exons are highly conserved, those of the introns are not. As would be anticipated, DNA sequence comparisons reveal much homology within the exons, but less conservation in the introns. If domestic hen egg-white lysozyme (Jung et al., 1980), human lysozyme (Peters et al., 1989), and bovine stomach (Irwin et al., 1989) are included in the comparison, introns occur at positions in the genes for the two lysozymes corresponding to those for the four a-lactalbumins (Acharya et al., 1989, comment that such results do not appear to agree TABLE X

Comparison of Exon and Intron Lengths" of a-Lactalbumin and Lysozyme Genes

Gene a-Lactalbumins Human Bovine Guinea pig Rat Lysozymes Domestic hen Human lk Bovine st Mouse M

(1) (2) (3)

(4) (5) (6) (7) (8)

159 160 165 165

648 32 1 338 34 1

159 159 159 159

489 473 48 1 429

76 76 76 76

499 504 50 1 1016

333 330 314 328

165 164 >151 166

1270 1563 -2100 -1300

162 165 165 165

1810 1938 1900 -2000

79 79 76 79

79 853 -2700 -450

180 1094 553 854

-

"In base pairs.

bHen,Hen egg white; lk, from leukemic material; st, stomach mucosa. c ( l )Hall et al. (1987), ( 2 ) Vilotte et al. (1987), (3) Laird et al. (1988), (4) Qasba and Safaya (1984), (5) Jung et al. (1980), (6) Peters et al. (1989), (7) Irwin et al. (1989), (8) Cross et al. (1988).

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HUGH A. MCKENZIE AND FREDERICK H. WHITE, JR.

entirely with the domain theory). Hall and Campbell (1986) point out that the acquisition of introns preceded the gene duplication event that produced a-lactalbumin. The variation of intron lengths among these genes, however, indicates divergence within these regions following gene duplication (see Table X, which also includes results for the mouse M gene). In further examination of these structures, exon 2 has proved to be the most highly conserved and appears to be involved with substrate binding in both proteins, in addition to active site residues in lysozyme. Exon 4 plays a role in galactosyltransferase binding for a-lactalbumin and accordingly shows little similarity to exon 4 for lysozyme, which does not bind with galactosyltransferase. Also, the conserved Asp residues corresponding to exon 3 are consistent with the relatively high affinity of a-lactalbumin for Ca(I1). In their comparison of the nucleotide sequence of the coding region of human pre-a-lactalbumin cDNA with that of domestic hen egg-white prelysozyme cDNA, Hall et al. (1982) found, after maximizing alignment, that there was a greater nucleotide (53%) than amino acid (38%) identity. They also noted that, of the codons that differ by only a single base, more than half represent “silent” substitutions. These observations add further weight to the concept that lysozyme and a-lactalbumin arise from divergent evolution from a common gene, as opposed to convergent evolution from two distinct genes. In their recent study of multiple genes for ruminant lysozymes, Irwin et al. (1989) noted that, throughout evolution, the positions of the introns in the lysozyme gene have remained at identical locations within the lysozyme family. However, differences have been observed in both the lengths of introns and the total gene length. In comparison with a-lactalbumin, lysozyme genes show greater differences, both in the sizes of their introns and in gene length, from each other and from the alactalbumins examined, than was observed within the latter group of proteins. Thus, they suggested the possibility that evolutionary change in size of the genes, from lysozyme to a-lactalbumin, may be associated with the change in gene expression, which may have involved the insertion and deletion of enhancer elements. 2 . Chick-, Goose-, Phage-, and Insect-Type Lysozymes

The homology of a-lactalbumin with lysozyme, the similarity in threedimensional structure and molecular size, etc., are for the well-known c-type (chick) lysozyme. However, there are other forms of lysozyme that catalyze the same reaction as c type. These include insect lysozymes, which are essentially of two types: the c type, (Jolles et al., 1979b; Eng-

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strom et al., 1985) and a type having a high level of sequence identity with phage lysozyme and a high chitinase/muramidase activity ratio (Fernandez-Sousa et al., 1979). Other forms of lysozyme include phage lysozyme (Remington et al., 1978), g-type (goose) lysozyme (Isaacs et al., 1985), and bacterial lysozyme from Streptomyces erythraeus (Harada et al., 1981; see also the review of Jolli3 and Jolli.s, 1984). These non-c-type lysozymes are not our concern here, but some brief comments are pertinent. Three-dimensional structures of the latter three have been determined, and two of them (goose and bacteriophage T4) have been intensively compared with c type by Weaver et al. (1985). Even though their amino acid sequences appear to be unrelated (for early work see Tsugita and Inouye, 1968), Grutter et al. (1983) suggested that the nature of their structural correspondence indicated that c, g, and phage types diverged from a common evolutionary precursor (see also Matthews et al., 1981, and the review by Bajaj and Blundell, 1984). Weaver et al. (1985) noted some similarities in the active site of the three lysozymes, but with the following striking difference. Residue 73 (Glu) in goose corresponds with residue 35 (Glu) in chick and with residue 11 (Glu) in bacteriophage T4. On the other hand, there are two Asp residues at positions 86 and 97 in the goose active site, neither of which corresponds exactly with Asp-52 of chick nor Asp-20 of T4. The implications for potential differences in the mechanism of catalytic action by the three lysozymes were discussed by Johnson el al. (1988) and by Weaver et al. (1985). T h e latter authors discussed the unresolved question as to whether the c-type lysozyme exons correspond to distinct structural and/or functional entities that are conserved during evolution of the three types of lysozyme considered. Phylogenetic analysis of amino acid sequences of c-type egg-white lysozymes from a variety of birds is generally in accord with taxonomic classification. However, there are some differences: For example, the chachalaca is classified normally in the order Galliformes, but its lysozyme differs more in sequence from those of pheasantlike birds than do the c-type lysozymes of ducks (for a discussion of this and other examples, see Jolles and Jolles, 1984). 3’. Stomach Lysozymes: Convergence for the Ruminant?

During the past 15 years there has been a fascinating array of contributions to understanding the evolution and properties of c-type lysozymes from Wilson, Prager, and their colleagues at the University of California-Berkeley. In the course of their studies, they made a number of interesting observations on stomach lysozymes from ruminants and colobine monkeys (particularly the Hanuman langur, Presbytis entellus).

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Further, they stressed that the convergent evolution of a fermentative foregut in these two groups of mammals has provided them with a unique opportunity to study adaptive evolution at the protein level. In a survey of 23 mammalian species, Dobson et al. (1984) found that c-type lysozyme activity per gram of stomach mucosa is many times higher for ruminants and a leaf-eating (colobinae) monkey than for animals without a foregut. This activity reflects an increased level of lysozyme (molecules) rather than higher specific activity (per molecule). These lysozymes have a narrow pH range of activity, their optimum activities occurring at pH values slightly less than 5.0 for physiological ionic strengths ( I 2 -O.l), in contrast to other c-type lysozymes, whose activity range is broader and optimal at pH -6.5. The ruminant and colobine enzymes are also resistant to pepsin digestion. In light of these and other observations, Dobson et al. (1984) concluded that lysozyme has adapted to function as a digestive enzyme in the true stomach, where it probably degrades the cell walls of bacteria entering from the foregut (for another view see Prieur and Froseth, 1986). Three enzymes (c, , cq, and c Q ) were found to occur in the bovine stomach; the sequence of one of these was determined by Jolles et al. (1984) (see Fig. 10 and Section VII). Subsequently, Stewart et al. (1987) also determined the sequence of the langur stomach lysozyme (see Fig. 10) and further developed the hypothesis of convergent or parallel amino acid replacements (so-called homoplasy). They compared the sequences of the bovine and langur lysozymes with those of the rat, baboon, human, and equine in relation to the biological tree and constructed parsimony trees. If these lysozymes had evolved predominantly by divergence, then the parsimony tree built from them should have matched the branching order of the species. However, the tree placing bovine stomach lysozyme with the langur enzyme was as parsimonious as the biological tree. In the opinion of Stewart et al., homoplasy was the most plausible explanation for this result. This interpretation, involving convergent evolution, and the method of data analysis have been disputed by Cornish-Bowden (1988), and his criticisms were subjected to a spirited reply by Stewart et al. (1988). One feature of the ruminant and Old World monkey stomach lysozymes is their low isoelectric points (pH -7.0-9.0) compared with the high pH value (10.0-12 0’ for many lysozymes. Arg appears to have selected for, during the recent evol.,?‘ :been selected against, ant. ary history of the ruminant lysozymes (Stewart and Wilson, 1987). Lys and Arg are considered generally to be the “epitome of conservative, neutral replacements” (Zuckerkandl and Pauling, 1965; Jukes, 1978).

.

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Stewart and Wilson (1987) particularly stressed the lowering of the Argl Lys ratios in these lysozymes. However, the values of Arg/Lys ratios, compared in Table XI, lead us to the conclusion that low ratios are not uniquely associated with the evolution of the ruminant lysozymes. Recently, JolKs et al. (1989) determined amino acid sequences of stomach lysozymes from deer and pig (1,2,3) and compared them with those of stomach lysozymes of bovine ( c 2 ) and langur, and human, baboon, rat, mouse M, chicken, chachalaca, and duck (1) lysozymes. (These sequences are compared in Fig. 10 and Section VII.) They constructed many phylogenetic trees in comparing the sequences of the 13 lysozymes chosen. No tree was (statistically) significantly more parsimonious than the biological tree. They concluded (see their Fig. 4)that the most convergent events took place early in the cow lineage, before it diverged from the deer lineage. The divergence times taken in the comparison were: M (early placental mammals), 60-80 million years ago; A (divergence of pig), 50-60 million years ago; and B (divergence of deer and cow), 20-30 million years ago. The rate of change along AB for lysozyme was considered to be 1.2 changes per million years, or approximately three times the rate for other mammalian lysozymes. In contrast, the average rate from B to present times falls to 0.2 changes per million years, or approximately one-half the “normal” rate for other mammalian lysozymes. Thus, Jolles et al. (1989) confirmed their view that during the period of what they call “recruitment” of lysozyme as a major digestive enzyme in ruminants, the rate of sequence change was accelerated, and later, TABLE XI Comparison of Basic and Acidic Groups and ArglLys Ratios in c-Type Lysozymes No. of residues per molecule

Species

Arg

Lys

Cow stomach c2 Deer stomach Langur stomach Pig stomach 3 Baboon milk Human milk Horse milk Echidna milk Rat urine Domestic hen egg

3 4 6 7 8 14

11 10 9 13 5

4

15 15 6 6

aA

=

3 12 11

5

ArgILys ratio

0.29 0.40 0.67 0.54 1.6

2.8 0.27 0.2 2.0 1.8

No. of residues per molecule Lys

+ Arg

+

3 4 9 + 6 13 + 7 5 + 8 5 + 14 15+ 4 15+ 3 6 + 12 6 + 11 11

10+

Asp 7 7 9 10 9 8 10 9 9 7

+ Glu + 8 +9 + 3

+3

+ 3 + 3 +6 + 3 + 3 + 2

(Lys + Arg) - (Asp + Glu). Note: Asp and Glu do not include Asn and Gln.

Aa -1

-2 +3 +7 +I +8 +3 +6 +6 +8

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after it was established in its new role, the rate of change was diminished to less than the normal rate. Irwin and Wilson (1989) have shown in a survey of bovine lysozyme clones that lysozyme cp, the most abundant form in bovine stomach mucosa, is encoded by at least two genes, whereas c1 and c3 are possibly encoded by only one gene. They believe that the recruitment involves an early regulatory event followed by a 4- to 7-fold increase in expression allowed by gene amplification. In an interesting paper, Irwin and Wilson (1990) considered the contradictory evolutionary histories of ruminant lysozymes that have been predicted by analysis of genomic blots (Irwin et al., 1989) and sequences of bovine stomach lysozyme cDNAs (Irwin and Wilson, 1989). They characterized stomach lysozyme cDNAs from domestic sheep and axis deer and compared them with one another and with those of the cow. It was found that different parts of the ruminant stomach lysozyme genes have had different evolutionary histories. The 3’ untranslated region has evolved in a divergent fashion since the original duplications 40-50 million years ago, supporting the genomic blot interpretation, whereas the coding region has evolved in a concerted fashion (i.e., the multiple sequences within a species evolved in unison). 4. Divergence of a-Lactalbumin and c-Type Lysozyme

In Section VII,B and Fig. 10 we compared the sequences of 13 alactalbumins (if the bovine A variant, equine B and C variants, and ovine variant are included), 23 mammalian c-type lysozymes (if donkey, mouse M, bovine stomach 1 and 3, caprine 1 and 2, ovine 1-3, camel 1, deer 2, echidna 11, and porcine 1 and 2 are included), and 13 avian c-type lysozymes (if KDIII and PD2 and PD3 are included). Analysis of the sequence differences indicates that, with the recent considerable increase in the number of lysozymes sequenced, there has been an appreciable decrease in the numbers of residues that are invariant in lysozymes as well as for both proteins. Nevertheless, there is still significant overall homology (-35%) between a-lactalbumin and c-type lysozyme. From the similarities in amino acid sequences, three-dimensional structures, intron-exon patterns, etc., there can be little doubt that the concept of divergence is still valid for these proteins. What is controversial are the rate of evolution and the details of the way in which alactalbumin arose, although it is conceded generally that the mechanism involves gene duplication. There are essentially two theories for the divergence, each of which has undergone some modification since it was proposed originally. The first of these, Model I (so called by White et al., 1977, but designated Model I1 in the recent discussion of Prager and Wilson, 1988),

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appears to have originated with Dickerson (see Dickerson, 1971; Dickerson and Geis, 1969). In this model the a-lactalbumin is considered to have arisen about 170-200 million years ago, which Dickerson dated as the start of mammalian development. This duplication is more recent than when those reptiles from which ‘mammallike’ reptiles (and later mammals) diverged from those reptiles whose later divergence produced modern reptiles and birds. With the limited number of a-lactalbumin and mammal and bird lysozyme sequences then available, it appeared that mammalian lysozyme sequences were more similar to those of bird lysozymes than to a-lactalbumins. Hence, Dickerson, in order to make Model I plausible, was led to the proposal that the a-lactalbumin gene underwent accelerated evolution as it acquired its new function and lost the old lysozyme function. As more sequences became available it was evident to Wilson and others that the average rate of sequence change in mammalian lysozymes was virtually the same as that in a-lactalbumin from the time the placental mammals arose. They found that the unit evolutionary period (for a definition see Section X,A,l) for lysozyme was 2.5 X lo6 years and for a-lactalbumin was 2.3 x lo6 years. Thus, Wilson and colleagues (see White et al., 1977; Wilson et al., 1977) were led to propose Model I1 (referred to as Model I by Prager and Wilson, 1988), the essential feature of which is that the a-lactalbumin-lysozyme duplication occurred long before the mammary gland evolved and before the above reptilian split. They believed that this model was in accord with the known sequence resemblances, did not need to invoke rate acceleration, and was, therefore, consistent with the molecular evolutionary clock. In 1984 Brew and colleagues (Shewale et al., 1984) published the sequence of a-lactalbumin from the milk of the red-necked wallaby (Macropus rufogriseus). This paper was of interest in its own right as the first report of the sequence of a marsupial a-lactalbumin, but it was also important in making revisions in the sequences of several other alactalbumins. As a result of the latter, they were able to make proposals for potential binding sites for Ca(I1) to a-lactalbumin (which had recently been shown to be a metalloprotein) and to reassess the evolutionary relationships of a-lactalbumin and lysozyme. Shewale et al. also proposed a modification of Model I, with the gene duplication occurring before the mammary gland arose, but after the reptile split (see Fig. 12). Over the next 2 years (1985- 1986) a number of other developments occurred rapidly. T h e amino acid sequences of echidna lysozymes I and I1 were determined by Teahan et al. (1986, 1991b; see also Griffiths et al., 1985; Teahan, 1986). T h e sequences differed in three residues (Fig. lo),

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WHITE, JR.

but both had several important features: each was (and still is) the only a-lactalbumin or lysozyme that does not have a Cys at position 6, being at 9 instead; and each has all of the residues essential for binding Ca(I1) as in a-lactalbumin (see below), and several other residues normally considered unique to a-lactalbumin. At about the same time Rodriguez et al. (1985) determined and later discussed (Rodriguez et al., 1987) the sequence of pigeon egg-white lysozyme, and, to our surprise, it had residues that indicated potential ability (since confirmed by Nitta et al., 1988) to bind Ca(I1). Further, its sequence terminated at Cys-127, as do echidna lysozyme and wallaby alactalbumin (residue 120, a-lactalbumin numbering). In a sense, a protein such as pigeon lysozyme had been anticipated earlier by White et al. (1977). It is interesting to note that, although lactation is a specific mammalian adaptation, a few birds (e.g., pigeons, emperor penguins, and greater flamingos) secrete a fluid analogous to milk from their gullets. Soon afterward, Stuart et al. (1986; see also Acharya et al., 1989, and Sections V-VII) identified unequivocally the binding sites for Ca(I1) in baboon a-lactalbumin. An examination of Table IX and Fig. 10 indicates that all a-lactalbumins so far sequenced have identical residues in the binding site region, with the exception of rabbit a-lactalbumin, for which residue 79 is Asn instead of Lys and residue 84 is Asn instead of Asp. While it may still be sterically possible for Ca(I1) to be bound in a chelate ring-type arrangement to rabbit a-lactalbumin, it may not be bound as strongly as to other a-lactalbumins. The lack of charge on residues 79 and 84 in the rabbit may be of importance in weakening the binding: Linse et al. (1988) suggested that surface charge may be more important in the binding of metal ions to proteins than is realized generally. As far as we are aware, the extent of binding of Ca(I1) to rabbit a-lactalbumin has not been determined qualitatively nor quantitatively. Of the lysozymes sequenced so far, only echidna has the residues 79, 82, 84, 87, and 88 (a-lactalbumin numbering) in common with a-lactalbumin for Ca(I1) binding, and there is evidence that it does bind Ca(I1) (D. C. Shaw and R. Tellam, personal communication). Equine and pigeon lysozymes have all of the equivalent residues, except for 88, which is Asn instead of Asp. We have already seen in Table XI that both of these lysozymes bind Ca(I1) (Nitta et al., 1987, 1988). Zeng et al. (1990) recently reported the crystallization of equine lysozyme in a form that they consider suitable for X-ray studies. In view of such developments, it is not surprising that there have been several attempts in 1985- 1989 to reconsider the evolutionary relationships. The approach in many studies has been to construct parsimony trees using methods and computer programs based essentially on the maximum parsimony methods of Farris (1970, 1972) or Fitch and Mar-

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goliash (1967). These are distance methods, the starting point being a matrix of pairwise distances, such as the number of amino acid residue differences or minimal mutation distances. In contrast, Prager and Wilson (1988) used character state parsimony analysis. They treated nucleotides as cladistic characters and then looked for shared states at phylogenetically informative sites (for details see page 328 of their paper). An important feature of their work is their attempt to apply tests of statistical significance to the results. Here, we consider briefly three of the recent studies. Prager and Wilson (1988) performed (character state) parsimony analyses of DNA and amino acid sequences for c-type lysozymes of vertebrates and insects and a-lactalbumins. They considered the results presented to provide statistically significant evidence in support of ancient gene duplication, and stated that the period in which this occurred was ‘before the bird-mammal divergence.’ (Presumably, this refers to the type of divergence at A shown in Fig. 12c, one branch of which led to the mammallike reptiles and ultimately the mammals, the other of which later led to divergence of reptiles and birds.) With respect to their model, Prager and Wilson cautiously state, “Although such a demonstration makes this model plausible, we should not consider it established until lactalbumin-like genes (or pseudogenes) or proteins are shown to be present in non mammals.” In the event that the search for lactose and a-lactalbumin in nonmammals is negative, they believe that a long period occurred between gene duplication and the acquisition of specifier activity for lactose synthesis. They included in their analysis only those c-type lysozymes that they call “conventional” and did not include “unconventional” lysozymes (although they presented a justification for this and, at the end of their paper, briefly mentioned the possibility that alactalbumin is related more closely to the unconventional [Ca(II)] binding lysozymes). We believe that preferable terms are “non-calciumbinding” and “calcium-binding” c-type lysozymes, respectively. The Hokkaido group has presented two papers: “The Evolution of Lysozymes and a-Lactalbumin” (Nitta and Sugai, 1989) and “Evolution of Metal Binding Sites in Proteins” (Sugai et nl., 1988). In both studies they included a-lactalbumins and mammalian, avian, and moth (Hyalophora) lysozymes. Nitta and Sugai (1989) examined comprehensively a wide array of evolutionary trees, many of which only prove to be interesting computational exercises. However, some, they concluded, are of evolutionary significance. Their preferred model involves an initial gene duplication before bird-mammal divergence, leading to calcium-binding lysozymes on one lineage and non-calcium-binding lysozymes on another. They then involved a second duplication after the bird-mammal divergence, giving

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HUGH A. MCKENZIE AND FREDERICK H . WHITE, JR.

rise to mammalian calcium-binding lysozymes in one lineage and, ultimately, a-lactalbumins on the second lineage. They postulated a period of rapid evolution of a-lactalbumin. Three aspects were considered in the development of the model: (1) a-Lactalbumin occurs only in mammals; hence, it is reasonable that it has arisen from a mammalian lysozyme lineage; (2)since Hyulophoru lysozyme does not bind calcium, it is reasonable to assume that the ancestral type did not bind calcium, and if maximum parsimony is observed with respect to acquisition (or loss) of the capacity to bind calcium, the best evolutionary tree groups all of the alactalbumins and calcium-binding lysozymes together; (3) sequence results were analyzed by the Fitch-Margoliash distance method, and also by character state parsimony analysis. Nitta and Sugai (1989) differed from Prager and Wilson (1988) in identifying a period of rapid evolution of a-lactalbumin “not in the process of acquisition of the activity of a-lactalbumin, but after the loss of lysozyme activity.” In both papers the Hokkaido group assumed that echidna lysozyme possesses dual activity and the monotreme lineage is independent of the marsupial and eutherian lineages. These assumptions may not be valid. In the paper by Sugai et al. (1988), it is stated, “The hypothesis of ancient divergence must be abandoned because it is not compatible with a fossil evidence that the lineages of insects and vertebrates diverged long before the radiation of vertebrates.” It is not clear what time scale is involved in this statement. The new studies, especially that by Prager and Wilson (1988), appear to support Model I1 rather than Model I (in the original nomenclature). However, further work is needed to resolve all of the issues involved. C . Are the Functions of Lysozyme and a-Lactalbumin Mutually Exclusive?

The study of the functions of these two proteins is important in gaining an understanding of the evolution of milk proteins and their relationships. Since there seems to have been misunderstanding among some authors about the work on these proteins in the laboratory of one of the authors (H. McK.), we will give a brief history of the findings of these studies to provide clarification. In the early 1960s Mervyn Griffiths, an authority on the biology of monotremes and marsupials, stressed the importance of extending comparative studies to include their milk proteins. Soon after the proposal by Brew and Campbell (1967) of a common ancestor for a-lactalbumin and lysozyme, the studies were extended to include these proteins. It was hoped that the milk of the monotremes, echidna and platypus, might have a primitive a-lactalbumin. Nevertheless, McKenzie was surprised when a graduate student, K. E. Hopper, found a lysozyme in one

LYSOZYME AND a-LACTALBUMIN

29 1

subspecies of echidna that appeared to have weak specifier activity for the lactose synthase system as well as having lytic activity. By 1970- 1971 preliminary characterization of echidna lysozymes I and 11 had been made (Hopper et al., 1970; Hopper and McKenzie, 1974). No evidence was found for the presence of a “classical”a-lactalbumin. The work was also discussed briefly in a review note, entitled “Milk Proteins in Retrospect and Prospect” (McKenzie, 1971). Hopper and McKenzie (1974) gave details of the lytic activity, weak lactose synthase activity of echidna lysozyme I, the fact that bovine galactosyltransferase could substitute for echidna galactosyltransferase, the isoelectric points of echidna lysozymes I and 11, and the amino acid composition of echidna lysozyme 1. A surprising similarity of echidna lysozyme to equine lysozyme was also noted. Nevertheless, some authors subsequently stated erroneously that alactalbumin, but not lysozyme, was found by Hopper and McKenzie in echidna milk, and it has also been alleged incorrectly that no evidence had ever been presented for the properties of the isolated protein. For logistic reasons, difficulty of locating lactating female monotremes, other urgent priorities, and surprisingly, a lack of financial support and encouragement for this work in Australia (despite its unique fauna), it was not possible to characterize further the echidna lysozymes until the early 1980s. The studies by Teahan et al. (1986, 1990), to which we have already alluded, confirm and extend all but one of the findings of the earlier studies. T h e occurrence of two echidna lysozymes (I and 11) of differing isoelectric points was confirmed. Further, the sequences were found to have unusual features, including certain similarities to equine lysozyme, such as residues for binding Ca(I1). There were weak lactose synthase and lytic activities in the milk samples. However, although the isolated lysozymes possessed lytic activity, they did not act as specifier in the lactose synthase system. The reasons for this are unknown. The obvious possibility is that the original experimental observations of Hopper and McKenzie are in error; however, careful controls were used. A different procedure for isolation was used by Teahan in order to get high yields of lysozyme to enable the determination of the sequence. Commercial galactosyltransferase and a different method were used in the determination of lactose synthase activity of the isolated protein. Nevertheless, it is puzzling, when echidna lysozyme appears to have virtually all of the structural attributes necessary to enable specifier activity, that it was not observed in the recent preparation. This discrepancy and the nature of the specifier protein in the platypus, obviously, must be the subject of further study. It is commonly believed that lactose is the major sugar in milk. However, studies in our laboratory, as well as the more extensive and wideranging studies by Messer and colleagues, indicate that this is not so for

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HUGH A. MCKENZIE AND FREDERICK H . WHITE, JR.

marsupial and monotreme milk (see, eg., Kamerling et al., 1982; Messer and Green, 1979; Messer and Kerry, 1973; Messer and Mossop, 1977; Messer et al., 1983). For example, echidna milk contains a small amount of free lactose, but much larger amounts of oligosaccharides, such as sialyllactose and fucosyllactose; the major oligosaccharide of platypus milk is 3,2’-difucosyllactose. Thus, either an a-lactalbumin or a lysozyme with specifier activity would be expected in monotreme milk. In addition to the evidence for lactose oligosaccharides in marsupial milk, the occurrence of a-lactalbumin throughout lactation has been demonstrated, for example, in the milk of the red kangaroo [Macropus wfus (Meguleia rufa)] (Bell et al., 1980; McKenzie et al., 1983) and the grey kangaroo (Macropus gzganteus) (McKenzie et al., 1983). An additional a-lactalbumin in the milk of M. gzganteus also appears in late lactation. Lysozyme has also been demonstrated in its milk. These c-type lysozymes and a-lactalbumins have been only partially characterized (see also Brew et al., 1973). T h e only mammal for which the presence of lactose in its milk has not been reported appears to be the California sea lion (Jenness, 1982). No nonmammalian occurrence of a-lactalbumin has been reported. In their theory of the evolution of a-lactalbumin, Hayssen and Blackburn (1985) considered that the duplication of the genetic material for lysozyme occurred as long as 300 million years ago, and that the duplicated material evolved subsequently via an intermediate form with both functions. They also suggested that the protolacteal secretion (from mammary gland precursors) enabled the survival of the eggs or young by virtue of its antimicrobial properties. If the evolution of a-lactalbumin did not occur until after the split of Monotremata and Marsupialia, it is possible that echidna lysozyme could have both functions. [Incidentally, the work by Whittaker et al. (1978) on monotreme hemoglobins and myoglobins does not support a constant evolutionary rate.] It has been conventional wisdom that lysozyme is not active in the lactose synthase system and that a-lactalbumin does not have lytic activity. T h e essential residues for interaction of specifier protein with galactosyltransferase have not yet been unequivocably defined, nor has the role of Ca(I1) in this system. Thus, it is not, at present, possible to rule out weak specifier activity for lysozyme in the lactose synthase system. On the basis of the known structure for c-type lysozyme and the nature of the groups involved in its catalytic activity, and early models of a-lactalbumin structure, there were good structural reasons that militated against a-lactalbumin having lytic activity. This point of view was well developed in the useful reviews by Hill and Brew (1975) and by Brew and Hill (1975). Recent X-ray structural determinations for a-

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lactalbumin by Phillips and co-authors added strength to this point of view (see Acharya et al., 1989), but they did not eliminate the possibility of trace lytic activity for a-lactalbumin and specifier activity for lysozyme. McKenzie and White (1987) studied the question of trace lytic activity in a-lactalbumin. This protein was isolated from a variety of species, and samples were studied for trace lysozyme activity by a sensitive method, previously developed by McKenzie and White ( 1986). All samples exhibof the specific activity of hen eggited trace lytic activity at a level of white lysozyme. Considerable effort was made to avoid contamination by lysozyme in the preparations used. Thus, it was concluded tentatively that the trace lytic activity was due to a-lactalbumin, not to lysozyme contamination. XI. CONCLUSIONS AND

THE

FUTURE

We have tried in this article to integrate the array of investigations on a-lactalbumin and lysozyme. Their range is overwhelming: T h e methods of chemistry, physics, molecular biology, genetics, genetic engineering, comparative biochemistry, enzymology, mathematics, and computer science have been used. Our emphasis has been on the comparative: If we have failed in the integration, at least we hope we have conveyed the spirit of adventure, and that the reader can see that there are further areas to explore. Broadly, there are two aspects in these comparative studies: first, fundamental studies aimed at determining the structure of the proteins, their evolutionary changes, and the structures necessary to maintain their functions and the mechanism of their actions; and second, determination of those biological properties which may lead to their use in pharmacology, medicine, and food science and technology. A tremendous amount has been achieved by the application of X-ray crystallography in determining the three-dimensional structure of alactalbumin and lysozyme, and in the case of lysozyme, the mode of its catalytic action. Nevertheless, despite the tremendous advances, there are still areas of this mechanism that are not fully understood. This is especially true of the comparative mode of action of c-, g-, and phagetype lysozymes. In the case of a-lactalbumin, its mode of interaction with galactosyltransferase and the nature of the critical residues involved have not been clearly defined. The primary and tertiary structures of galactosyltransferase are not known. The mechanism of the catalytic action and the precise role of metal ions are still the subject of disagreement.

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It is evident that there are subtle differences in the structures of c-type lysozymes and a-lactalbumins, beyond what has become evident from Xray crystal structure studies. Scheraga (1988) indicates some of the limitations in current approaches to conformational energy calculations. The procedures used by Scheraga and co-workers for prediction of the protein conformational structure involve a search for the global minimum of the potential energy, allowing additionally for hydration and conformational fluctuational entropy (Gibson and Scheraga, 1987,1988; Kang et al., 1987). The “multiple-minima” problem thus far has hindered computation of the native conformation. However, Scheraga (1988) has made progress in surmounting this problem with peptides u p to 20 residues long. Current efforts are reportedly in progress in studies on peptides of 100-200 residues. Many years ago Linderstr@m-Lang(1952) drew attention to the importance of motility of protein structure. Further work needs to be done on the dynamics of the structures of lysozyme and a-lactalbumin (see also Artymiuk et al., 1979). Kossiakoff (1985) pointed out that the most useful attribute of neutron diffraction studies of proteins (compared with X-ray diffraction) is their ability to locate hydrogen (or deuterium) experimentally. Recent advances in apparatus and data acquisition mean that this method will become increasingly valuable in the study of a-lactalbumin and lysozyme, especially in the location of water molecules and the dynamics of these proteins. An example of a recent application is that by Lehmann et al. (1985). Much of the solution conformational work was done in 1960-1972, when CD, ORD, and Raman spectroscopic apparatus were not very satisfactory. Johnson ( 1988)indicated the scope of secondary structural investigation of proteins with modern CD apparatus. With the increased accessibility of the UV region, such measurements need to be done on a-lactalbumin and lysozyme in a variety of environments over a wide temperature range. Kauzmann (1987) indicated that, in considering the thermodynamics of the unfolding of proteins, we are tending to avoid the hard experiments. Thus, spectroscopic studies of the effects of high pressure on these proteins are sorely needed. Many differences in solution properties between a-lactalbumin and lysozyme are not compatible with prediction and X-ray results, and thus it is surprising that these differences exist. This paradox is consistent with the suggestion by Barman ( 1970), whereby the native tightly folded conformation of a-lactalbumin exists in equilibrium with a “looser” form that is more subject to the various reactions studied, including denaturation. It would also be consistent with the tightly folded but “slowly fluctuating” intermediate of Dolgikh et al. (1981). This equilibrium could

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have contributed to observed difficulties in obtaining crystals suitable for X-ray analysis (until baboon a-lactalbumin was used). There is, moreover, another problem to be considered, assuming the above equilibrium, which is the question of what differences in structural features might permit an equilibrium to exist for a-lactalbumin, while it does not exist for lysozyme. More work is needed to elaborate on this question. For the present it appears that an equilibrium, whereby an alternative and more reactive form of a-lactalbumin is produced, could be a contributing factor in the ability of a-lactalbumin to express traces of cell lytic activity, as well as exhibit the various other unpredictable activities. The use of site-directed mutagenesis in the investigation of a-lactalbumin and lysozyme is in its infancy. Interesting studies are now in progress in the laboratory of A. C. Wilson in collaboration with the laboratory of J. F. Kirsch, and we look forward to future results. Although protein chemists often feel that they now know a good deal about the nature of residues that are critical for proteins to exercise their functions, this may be partially illusory. The study by Luntz et al. (1989) on the structural significance of an internal water molecule studied by sitedirected mutagenesis of Tyr-67 in rat cytochrome c is salutary. While site-directed mutagenesis has a bright future in the study of alactalbumin and lysozyme (see, e.g., Alber et d , 1987; Muraki et d , 1987; Matsumura et al., 1988), this will in no way diminish the need for the study of variants of these proteins from a variety of species. We have stressed in the investigations in our laboratory over the years, and we stress again here without apology, the need to pay special attention to the isolation of these proteins for structural, immunological, and activity studies. There have been unexplained variations in parameters of crystals of a-lactalbumin isolated on separate occasions in some laboratories. Also, the Raman studies by Yu and others [see, e.g., Yu (1977)l indicate appreciable spectral changes on freeze-drying of both a-lactalbumin and lysozyme. We caution against the use of freeze-drying in structural studies. Many of the variants of lysozyme and a-lactalbumin now being studied are available only in small amounts, and there is naturally a temptation to “purify” the protein by HPLC. While this method has considerable advantages, we caution against its use for some purposes. T h e nature of the high pressures involved, and in many cases of the solvents used, can ’bring about irreversible changes. While this may not always be of great importance in amino acid sequence studies, it can be disastrous for conformation and activity studies. Furthermore, many published separations purport to give ‘pure’ peptides and proteins; the nature of the patterns displayed in many publications is testimony to the optimism of the workers involved. Again, we stress that homogeneity cannot be demon-

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strated-only absence of heterogeneity by a particular test. Hence, care should be taken in the choice of such tests, as well as methods of isolation. For both purposes capillary electrophoresis may be the preferred method. It is essential that further work be done on the isolation and characterization of echidna and platypus (monotreme) proteins as well as those of the grey and red kangaroos (marsupials),in order to resolve the issues discussed in Section X. The above remarks on purification are particularly pertinent to such studies. If younger readers are unaware of the traditions of purification of proteins, especially those established in the Carlsberg Laboratory and the former Department of Physical Chemistry at Harvard Medical School, and the rewards for the sheer hard work involved in isolation, we refer them to the recent autobiography of Arthur Kornberg (1989). It has frequently been assumed that lysozymes isolated from different fluids (or organs) from a given species will be identical. Unless the sequences are determined completely, without assumptions from peptide maps, one cannot be certain of this. Again, the elucidation of whether residues are Asp or Asn or Glu or Gln can be dependent on the care taken in treatment of the protein and in sequence determination. The studies by White et al. (1988) on cow milk lysozyme show that the sequence of the milk protein differs from that of bovine stomach lysozyme cp. This indication, that lysozymes from different secretions and tissues of the same species may be different, has been further substantiated by joint studies from two of the most prolific laboratories in lysozyme research: those of Allan Wilson and of Pierre and Jacqueline Jolks. Amino-terminal sequence determinations for caprine stomach lysozymes 1 and 2 and caprine tear lysozymes 1, 2a, and 2b were made by Joll&s et al. (1990). Their results show considerable differences in sequence between the caprine stomach and tear proteins. The 40-residue amino-terminal sequences of the latter bear a striking resemblance to that of cow milk lysozyme. They concluded that their results indicate that the caprine tear family of lysozymes has diverged from the stomach family by an ancient duplication and that later duplications may be responsible for the multiple forms of tear and milk lysozymes in ruminants. Such comparative studies of lysozymes from different secretions and tissues of a given species need to be extended. As well, the genetic association between lysozyme levels in bovine serum and colostrum found by Lie et al. (1986) and the variation in milk found by White et al. (1988) need further study. Great care should be exercised in the isolation of the more labile a-

297

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lactalbumin if definitive information is to be obtained on the activity of a-lactalbumin in the presence and absence of various metal ions. Above all, the number and nature of metal ion binding sites must be determined and the apparent differences between the results of Phillips’ X-ray group at Oxford University and the views of Kronman (1989) must be resolved. In this connection NMR studies will be very important (see the approach of Adebodun and Jordan, 1989). We have already referred to the considerable discrepancies between values of stability constants from different laboratories. These are summarized in Table XII. The determinations need to be repeated by apTABLE XI1 Association Constantsfor Binding of Ca(II) to a-Lactalbumins and Lysozymes “ ~~

Protein a-Lactalburnin Bovine“

~

Log KsJ 6.4 (log Ks,2 = 4.5 6.4 (pH >6.0)

8.7 9.8 8.0

7.3 9.6 Human Caprine Lysozyme Equine Pigeon

6.9 8.5 8.4 6.3

7.2

Method

Reference

Direct binding, K(I) absent Direct binding, 0.1 M K(I) Fluorometry, chelate presentd Fluorornetry, chelate presentd CD, chelate present: for 25°C by extrapolation Ca electrode, pH 8.0, 37”c CD, chelate and NaCl absent 0.1 M NaCl CD, chelate presentd CD, chelate presentd

1

Dye titration, 0.1 M KC1, 20°C Dye titration, 0.1 M KCI, 20°C

2 3 4 5

6

7 5 5 8 8

“First association constant, Ks,l; second association constant, Kr,2. at 25°C unless indicated. *References: (1) Kronman et al. (1981), ( 2 ) Bratcher and Kronrnan (1984), ( 3 ) Perrnyakov et al. (1981), (4) Murakami et al. (1982),(5) Segawa and Sugai (1983), (6) Hamano et al. (1986), (7) Mitani et al. (1986), ( 8 ) Nitta et al. (1988). “The list for bovine a-lactalbumin is not exhaustive, but is illustrative of the variation in Ks,l values. dChelate is EGTA or EDTA.

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HUGH A. MCKENZIE A N D FREDERICK H . WHITE, JR.

propriate procedures using carefully prepared samples of a-lactalbumin and lysozyme. It is not surprising, because of the antibacterial properties of lysozyme and the variation in levels of occurrence of both a-lactalbumin and lysozyme in various tissues and fluids, that attempts have been made to exploit these properties. An example of this is that lysozyme only occurs at very low levels in cow milk, and hence cow milk-based infant formulas are deficient in this antibacterial agent. Proposals have been made to add domestic hen egg-white lysozyme to boost the level of lysozyme. One of the authors (H. McK.) has advised against this because of its not having identical antigenic properties to human lysozyme and because many commercial samples of hen egg-white lysozyme are contaminated with ovalbumin, a powerful allergen. The revolution in animal breeding in which foreign genes can be substituted for that of lysozyme could be exploited to produce cows that have human lysozyme in their milk (for a general *discussionon the revolution in animal breeding, see Wilmut et al., 1988). Jolles and Jolles (1984) have reviewed the use of lysozyme as a marker in certain diseases. Serum lysozyme levels have been used extensively in the diagnosis of leukemias. Jolles and Jolles discussed some of the reasons for increased and decreased serum levels in various diseases, such as acute or chronic granulocytic leukemia, myeloid metaplasia, and aplastic anemia, and decreased levels in tears in keratoconjunctivitis. They have also considered the interaction of lysozyme with sulfated proteoglycans and its role in the calcification of epiphyseal cartilage. It is to be expected that such studies will yield valuable information, giving rise to further applications in the future (see also Fett et al., 1985). Lysozyme will continue, of course, to serve as a prototype protein for the investigation of the specificity of immune recognition. As Hall and Campbell (1986) have stressed, about one-third of all human metastatic breast carcinomas regress in response to some form of endocrine therapy; yet, despite much research, there is still no reliable way of identifying this group prior to treatment. One approach has been to search for milk proteins, particularly a-lactalbumin, within breast tumors or serum. Despite much effort, Hall and collaborators were unable to find a-lactalbumin being expressed in any of the breast tumors examined (see, e.g., Hall et al., 1981). However, they did find a peptide that was similar, but not identical, to pre-a-lactalbumin. It is to be hoped that the precise nature of the peptide will be determined. Hamilton and collaborators have made extensive studies on the complex processes involved in sperm maturation. We noted in Section VIII that, in the course of this work, they found rat rete testis and epididymal

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fluids to be rich in galactosyltransferase activity. Also, they found an alactalbumin-like protein to be present (see Hamilton, 1981). With a rat mammary gland a-lactalbumin cDNA clone as a hybridization probe, RNA sequences homologous to a-lactalbumin mRNA were detected by Qasba et al. (1983) in the total RNA from rat epididymus. This is taken to mean that a-lactalbumin-like protein is similar in structure to that of a-lactalbumin from the mammary source. In more recent work, De Geyter et al. (1989) suggest that mouse sperm is decapacitated by bovine mammary a-lactalbumin. There is action of a-lactalbumin on the sperm head, inhibiting binding to the zona pellucida. All of this implies a similar function of a-lactalbumin-like protein(s) in the male reproductive tract. It is possible that the a-lactalbumin/lysozyme gene family has a third member. The need for further studies of this member has been stressed in Section VIII. Finally, there are temporal differences in the expression of milk proteins (e.g., casein and a-lactalbumin) among species, exemplified by the rat, guinea pig, and kangaroo (see, e.g., Hall and Campbell, 1986; Burditt et al., 1981). T h e precise causes of these differences remain to be elucidated. ACKNOWLEDGMENTS Work commenced on this article while one of us (H. McK.) was Head of, and the other (F.H.W.) was Visiting Fellow at, the Protein Chemistry Group, John Curtin School of Medical Research, Institute of Advanced Studies, Australian National University, Canberra, ACT 2601, Australia. Warm thanks are expressed to Professors David Phillips and John Edsall for many helpful discussions, and to Dr. Margaret McKenzie for invaluable bibliographical assistance and help. Thanks are due to Drs. Ellen Prager and Allan Wilson for valuable discussions and for generously making available over the years results in advance of publication. H. McK. wishes to thank especially Dr. Mervyn Griffiths for stimulating his interest in the proteins of marsupial and monotreme milk and for invaluable cooperation and help. The skilled help of Panit Thamsongsana in preparing the tables, figures, and manuscript is gratefully acknowledged. One of us (F.H.W.) thanks Robley Light, Chemistry Department, Florida State University, for his generous donation of space and facilities.

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