The Proteins Of The Exocrine Pancreas

THE PROTEINS OF THE EXOCRINE PANCREAS By P. DESNUELLE and M. ROVERY Labaratoire de Chirnie Biologique, Faculti der Sciences, Marseille, France

I. Introduction.. .......................................................... 11. Biosynthesis of Pancreatic Enzymes. .................................... A. General Techniques for Determining Pancreatic Enzymes . . . . . . . . . . . . . B. Biosynthesis and Localization of Enzymes in Acinar Cells 111. Recent Advances in the Chemical Characterization of Some Proteins of Exo.............................. enclature. .................... B. Bovine Chymotrypsinogen A . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Bovine Chymotrypsinogen B . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Porcine Chymotrypsinogen A , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. Bovine Trypsinogen and Trypsin.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . F. Porcine Trypsinogen.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G. Bovine Procarboxypeptidase A and Carboxypeptidase A . . . . . . . H. Bovine and Porcine Carboxypeptidases B . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I. Porcine Lipase.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . J. Bovine Ribonuclease., . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . K. Sheep and Mouse Ribonucleases.. . . . . . . . . . . . . . . . . . . . . . . References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

139 141 141 148 153 153 154 163 165 167 171 173 175 176 187 191

I. INTRODUCTION^ A brilliant success of modern biochemistry has been to show that all or almost all proteins of bovine pancreatic juice are enzymes (1).It may be said in a cursory way that this juice is a solution of enzymes in bicarbonate and that its composition is strictly utilitarian. Bicarbonate is present in order to neutralize hydrochloric acid coming from the stomach and to keep enzymes in solution. Enzymes are present for digesting alimentary products in the intestine. On the other hand, pancreatic juice seems to have been created for the protein chemist’s delight, since its proteins are biologically active, relatively simple, and endowed with unusual properties. Intraluminar digestion consists of a series of hydrolyses converting large molecules with specific structures into smaIler molecules able to force their way into the intestinal mucosa. In many species, pancreatic juice contains 1 The following abbreviations are used in the text: DEAE-cellulose, diethylaminoethyl-cellulose; CM-cellulose, carboxymethyl-cellulose; DFP, diisopropylphosphofluoridate; ATEE, acetyl-L-tyrosine ethyl ester; BAEE, benzoyl-L-arginine ethyl ester; Tris, tris(hydroxymethy1)aminomethane.

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one or several hydrolases for each main component of the diet: a series of proteolytic enzymes for proteins, a lipase, a phospholipase, and a cholesterolesterase for lipids, an amylase for polysaccharides, a ribonuclease and a deoxyribonuclease for nucleic acids. The fact that pancreatic enzymes are exclusively hydrolases interests protein chemists as well as enzymologists. Most oxidoreductive enzymes consist of a protein carrier and a small coenzyme to which the largest part of the catalytic effect may be attributed. On the contrary, many hydrolases merely consist of a holoprotein in which the active site is formed by a tridimensional and specific arrangement of amino acids residues. One of the most challenging problems of molecular biology is to find out a correlation between catalytic activity and protein structure. Even when a metal ion is involved in the catalytic activity of hydrolases or in their stability, these ions appear to be bound in a specific way to some groups of the protein molecule and their action can be discussed in terms of protein structure. The study of pancreatic enzymes at the molecular level has been facilitated by two circumstances: (a) pancreatic juice and pancreas extracts are simpler than many other biological fluids; (b) most of their proteins have a relatively low molecular weight. The proteins behave well during fractionation procedures based on molecular kinetics and some hope exists of determining their structures with the available techniques of protein chemistry. The enzymatic activity of pancreatic proteins may be regarded as an additional attraction and a help in tracing these proteins during purification and in demonstrating their homogeneity. The external secretion of the pancreas has other connections with protein chemistry. In the first place, it is known to be stimulated, not only by nerves and cholinergic drugs, but also by two hormones, secretin and pancreozymin. Secretin, discovered in Ivy’s laboratory by Hammarsten and Agren, has been purified to a considerable extent from hog intestine (2). I t mainly promotes a flow of water and mineral salts into the secretory ducts. The enzyme content of pancreatic juice seems, however, to be controlled by nervous stimulation and by pancreozymin. This second hormone, discovered by Harper and Raper in 1943, is not yet fully characterized (3). But it may be easily understood that the mobilization of enzyme proteins stored behind the membranes of zymogen granules (to be discussed later) and the passage of water molecules through the walls of the secretory ducts are two distinct processes requiring two different types of stimulation. Although the whole problem of the stimulation of the exocrine pancreas is not discussed here for obvious reasons, it will be pointed out that it induces a relatively large protein output to which must correspond a very active protein biosynthesis. Cannulated steer pancreas for instance excretes

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an average of 1 gm of protein per hour (4).Mouse pancreas, which represents only 0..5-0.6% of the total body weight of the animal, concentrates about 10% of injected radioactive valine ( 5 ) . Again, the activity of pancreatic enzymes and the relative ease with which they can be fractionated facilitate an accurate study of their biosynthesis in intact animals and their possible variations with external factors. Finally, the presence in pancreas acinar cells of cytoplasmic components and enzymes able to hydrolyze them requires some protective devices which can be partly elucidated by the techniques of protein chemistry. The first part of this review will be devoted to enzyme biosynthesis by pancreas and the second part to chemical characterization of certain pancreatic enzymes. Some of these enzymes have already been comprehensively discussed in recent volumes of this series (6, 7).

11. RIOSYNTHESIS OF PANCREATIC ENZYMES A . General Techniques for Determining Pancreatic flnxymes The intrrest of experiments designed to investigate enzyme biosynthesis by pancreas strongly depends upon the accuracy of available techniques for the determination of enzymes in such complex mixtures as pancreatic juice, pancreas homogenates, or lysates of zymogen granules. Significant technical advances have been made recently in this direction. They have been facilitated by previous investigations carried out in vilro with purified enzymes of bovine pancreas. 1. Chromatography

I n 1953, Hirs (8) found that well separated peaks of chymotrypsinogen A and ribonuclease A could be directly obtained by chromatographing I
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proteins should present themselves in an order of decreasing isoelectric points. This is actually the case except for ribonuclease which is the last protein to be eluted from Amberlite IRC-50 under the conditions of the experiment, although it is isoelectric at a lower pH than trypsinogen and chymotrypsinogen A. The data of Fig. 1 appear to be of fundamental importance for a number of reasons. After two chromatographic steps, 90% of the total proteins of bovine juice has been fractionated in a series of discrete peaks. Almost all peaks can be associated with known enzymatic activities. Thus, a qualitative picture of the enzymatic content of the juice is obtained at once. It

I

-

ChTg B

+Cotion

exchange+Anion -Effluent

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FIG.1. Chromatography of bovine pancreatic juice on DEAE-cellulose (anionic proteins) and Amberlite IRC-50 (cationic proteins) (1). RNAase, ribonuclease; ChTg-a, chymotrypsinogen A; Tg, trypsinogen ; ProCp-B and Cp-B, procarboxypeptidase B and carboxypeptidase B; DNAase, deoxyribonuclease; ProCp-A, procarboxypeptidase A.

will be interesting to locate on the diagram somewhat ill-defined pancreatic activities named elastase (9, lo), insulinase (lo), protaminase (ll),pankrin (12), and “esterases” by applying their specific tests to each fraction. It should be possible in t,his way to see whether or not the corresponding enzymes really exist. On the other hand, quantitative information about the relative and absolute proportions of pancreatic enzymes can be obtained by determining the peak areas. But, for such information to be correct, the enzymes must be eluted from the column completely. They should also have been obtained previously in a pure form so that their extinction coefficients are known. Finally, the peaks should be pure. All these rather drastic requirements seem to be fulfilled in the case of bovine pancreatic juice. A quantitative balance sheet (1) can therefore be established with the following results:

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chymotrypsinogen A, 16 % of the proteins of the juice; chymotrypsinogen B, 16 %; trypsinogen, 14 %; procarboxypeptidase A, 19 %; procarboxypeptidase B, 7 %; ribonuclease, 2.4 %; deoxyribonuclease, 1.4 %; amylase, less than 2 %; lipase, very low; unidentified, 10 %. I n a more recent work (4) , it has been found by the same techniques that the proportions of the cationic proteins of bovine juice remain fairly constant during a 5-hr period of secretion and that they are 15% for chymotrypsinogen A, 16% for trypsinogen, and 3.3% for ribonuclease (weight ratio, 5 : 5 :1; molar ratio, 3:3:1).

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0,250

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FIG.2. Chromatographic diagram of porcine pancreatic juice (13, 14). Abbreviations used for enzyme names are the same as in Fig. 1. The anionic proteins are fractionated on a DEAE-cellulose column equilibrated with 0.005 M phosphate, pH 8.0 and eluted a t the same p H with a concentration gradient. The cationic proteins are fractionated on a CM-cellulose column by stepwise elution with buffers of increasing pH's. Ordinates, optical density of the fractions at 280 mp. Abscissas (on the right diagram), volume of eluate expressed in number of interstitial volumes of the column.

These results give, for the first time, reliable information about the general composition of bovine juice. Its content of proteolytic enzymes is strikingly high (about 72 % of the total proteins). Chymotrypsinogen B, which was formerly considered as a minor constituent, is very abundant. On the other hand, bovine juice is quite poor in amylase and lipase. Figure 2 gives the diagram obtained with porcine juice collected by a permanent fistula (13, 14). I n this case, Amberlite IRC-50 has been replaced by CM-cellulo,se for the fractionation of the cationic proteins. But the other conditions are the same as in the preceding experiment. The first remark suggested by Fig. 2 is that porcine juice contains relatively large amounts of amylase and lipase. Pig amylase (15) and pig lipase (16, 17) have already been purified. The difference existing in this

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respect between pig and cattle juices is quite striking. As stated above, the main components of bovine juice are proteolytic enzymes. The porcine secretion is much more diversified. The physiological significance of this difference is still unknown. Since preliminary experiments have shown that dog (see below), rat, and human juices also contain large amounts of amylase and lipase, it may be postulated that the bovine secretion represents a special case associated with rumination. I n other words, fats and carbohydrates are perhaps hydrolyzed by the bacteria of the rumen before reaching the intestine. Figure 2 shows further that very large amounts of promrboxypeptidasm

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PlOCpA Amylase

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Fro. 3. Protein and activity peaks obtained by chromatography of porcirio p:tiicretltic juice on DEAE-cellulose (13). The solid lines give the enzymatic activities of the fractions. The dotted lines give the protein background taken from the corresponding zones of Fig. 2.

A and B are present in porcine juice. However, thc order of emergence of these precursors is not the same as for bovine juice. Moreover, a single chymotrypsinogen peak is present instead of two as in bovine juice. This peak is found on the cationic side, although the isoelectric point of thc corresponding protein is 7.2 (to be discussed later). It will be referred to as porcine chymotrypsinogen A. The possible existence of a second porcine chymotrypsinogen is discussed later. Finally, the order of emergence of chymotrypsinogen A and trypsinogen is not the same here as for bovine j uice , Figure 3 gives the correlation existing between protein peaks as determined by ultraviolet absorption and the enzymatic activities of the fractions (13). The correlation is good. A single activity is found under most of the peaks but ribonucleolytic activity is located, not only under the

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ribonuclease peak, but also in other parts of the diagram. Furthermore, utilization of the diagram for quantitative purposes is rendered uncertain by two facts: (a) some porcine enzymes have not yet been purified. Except in the cases of amylase, chymotrypsinogen A, and trypsinogen (to be discussed later), their extinction coefficients are not known. (b) The specific activities found under some peaks are lower than normal. The porcine

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0.750

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FIQ.4. Chromatography of dog pancreatic juice on DEAE-cellulose (13, 14).The column is equilibrated with 0.005 M phosphate, pH 8.0 and eluted by a concentration gradient of phosphate indicated in the figure by a straight line. (1) Unfractionated cationic proteins; (2) amylase; (3) lipase; (4)deoxyribonuclease; ( 5 ) anionic chymotrypsinogen; (6), (9), and (10) carboxypeptidase A and its precursor; (7) and ( 8 ) carboxypeptidase B and its precursor. Ordinates on the left, optical density of the fractions at 280 mp. Ordinates on the right, molarity of the phosphate. Abscissas, volume of eluate expressed in number of interstitial volumes of the column.

enzymes are apparently more labile than their bovine analogs during chromatography on DEAE-cellulose at pH 8.0. Figure 4 gives the result of a single and preliminary experiment with dog juice (13, 14). Its main interest is to show that a “map” of the enzymatic equipment of the pancreas of various species can be obtained by chromatography in a relatively short time. Similar chromatographic techniques have been recently used for the fractionation of liver extracts (18). About sixteen peaks with known activities have been identified. But the liver enzymes are very numerous indeed, and their systematic fractionation represents a more difficult problem.

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Thus it seems that, at least for the time being, a complete and quantitative diagram can only be obtained with bovine juice. But chromatography gives a very good opportunity of drawing qualitative maps of the enzymatic

0

25

10

20

hr.

FIG.5. Kinetics of chymotrypsinogen(s) and trypsinogen activations in pan-

creatic juice and pancreas homogenates (22). Upper diagram: activation of chymotrypsinogen(s) in rat pancreas homogenate. Pancreas (1 gm) is homogenized 2 min a t 0°C and 2000 rpm in a Potter-Elvehjem apparatus with 9 ml of 0.25 M sucrose. One milliliter is diluted with 9 ml of 0.2 M phosphate buffer, pH 7.6 and incubated a t 0°C (white circles) or 35°C (black circles) with trypsin (1mg/100 mg of total protein). The chymotryptic activity is measured against acetyl-L-tyrosine ethyl ester. Abscissas, incubation time in minutes. Ordinates, specific activity of chymotrypsin (~-equivalents/min/mgof protein). Lower diagram: activation of trypsinogen in pig pancreatic juice. A lyophilized sample of juice is dissolved in a p H 7.9 buffer 0.005 M in Tris, 0.04 M in NaC1, and 0.02 M in CaClz , The solution (25 mg of protein/100 ml) is incubated with trypsin (4 mg/100mg of protein). The tryptic activity is measured against benzoyl-L-arginine ethyl ester. The activity of added trypsin is deducted from each result. Abscissas, incubation time in hours. Ordinates, specific activity of trypsin &-equivalents/min/mg of protein). Temperature: 0°C.

pattern of other species. These maps give valuable information for further purification. Chromatography is also extensively used for isolating enzymes from juice, pancreas, and its subcellular fractions (see later). These isolations are easier than complete chromatography, since the most suitable technique can be selected in each special case.

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2. Direct Determination of Enzymatic Activities

When quantitative chromatography gives inconclusive results, the general composition of the mixture can sometimes be ascertained by direct determination of enzymatic activities. If the specific activities of the pure enzymes are known, an estimation of their absolute proportions (grams of enzyme in 100 gm of total protein) can be made. In the reverse case, possible variations in the enzymatic pattern of a given species can at least be detected by determining activities for a certain volume of juice or for a certain weight of pancreas. The main difficulty of course is in finding satisfactory techniques for measuring enzymatic activities in complex mixtures. For obvious reasons, this latter problem will not be fully discussed here.

FIG.6. Activation of chymotrypsinogen(s) and trypsinogen as a function of added trypsin (22). On the left: chymotrypsinogen(s) activation in pig juice. Incubation time, 120 min. The other conditions are the same as in Fig. 5 . On the right: trypsinogen activation in pig juice. Incubation time, 18 hr. Abscissas, milligrams of added trypsin per 100 mg of total protein. Ordinates, specific activity (p-equivalents/ min/mg of protein) after deduction of the activity of added trypsin.

But an important point is to realize that: (a) comparisons at the molecular level cannot be established between species, since the same enzyme formed by various species may have different specific activities and require different cofactors. (b) Very large errors can be made, and actually have been made in the past, by using poor techniques for the determination of enzyme activity. At the present time, directly active enzymes such as amylase (19, 20), lipase (16), and ribonuclease (21) are measured with a reasonable degree of accuracy. Good techniques and specific substrates are also available for chymotrypsin, trypsin, carboxypeptidases A and B. But the special problem here is that proteolytic enzymes must be activated and that the activation step must be carefully controlled in complex mixtures (Figs. 5 and 6). Under the conditions selected (22), a well-defined and reproducible activity plateau is reached in Fig. 5 for chymotrypsin and trypsin. The height

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of this plateau is proportional to the amounts of homogenate or juice used. Figure 6 shows that there exists a lower limit for added trypsin below which trypsinogen activation does not start and chymotrypsinogen activation is very slow. This limit probably reflects the existence of trypsin inhibitors and may give an interesting expression of the stability of pig juice. When full activation is obtained, the amount of added trypsin may be varied within large limits without any detectable influence on the final activity values. Since the specific activities of pure porcine amylase (23), lipase (16), and trypsinogen (Section 111,F) are known, approximate values for the percentage of the three enzymes in pig pancreatic juice can be estimated as stated above. These values are respectively 7.5, 2.5, and 24% of the total proteins. By taking into account the specific activity of pure pig chymotrypsinogen A (Section 111, D), a value of 14% is found for this class of precursors. However, the value is preliminary since the specific activity and the amount of the second rhymotrypsinogen of pig are not yet known.

B. Biosynthesis and Localization of Enzymes in Acinar Cells The acinar cells of pancreas synthesize, not only structural proteins and enzymes for their own metabolism, but also exocrine or “exportable” enzymes which are carried into the duodenum for digestive purposes. To this dual function corresponds a rather complicated system involving, besides biosynthesis itself, segregation, transport, storage, and final excretion. 1. Histological Observations

It is generally assumed that protein biosynthesis occurs in or on small rihonucleoprotein particles named ribosomes. The rough-surfaced regions of the endoplasmic reticulum of pancreas acinar cells are studded with ribosomes (24). After disruption of the cell structure by homogenization, the endoplasmic reticulum is converted into microsomal vesicles which contain the molecules formerly present in the reticulum and to which the ribosomes are still bound. These ribosomes can be spun down as a separate fraction when the microsomes are dissolved by sodium deoxycholate (25). On the othcr hand, when guinea pigs are starved and killed 1 hr after feeding, the cavities (cisternae) of the endoplasmic reticulum of acinar cells contain granules and the microsomal fraction is more dense. When the starved animals are killed before feeding, the cavities are empty and the microsomal fraction is lighter. It is therefore tempting t o postulate that the intracisternal granules are formed by enzymes which have just been synthesized under food stimulation (25-27). Finally, it was already noted by Heidenhain (28) in 1875 that pancreatic acinar cells contained relatively large granules which occupied a large part

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of the cytoplasm during starvation, but became fewer in number after stimulation of the exocrine secretion. These granules appeared to act as enzyme “stores” and were called zymogen granules. More recently, beautiful electron micrographs have confirmed that zymogen granules are associated with the exocrine secretion of pancreas. They are seen to coalesce between themselves and with the walls of the secretory ducts, and thus to discharge their content into the duct lumen within the limits of a closed system (27). On these observations is based the Palade and Siekevitz theory according to which pancreatic enzymes are synthesized in ribosomes, segregated into “exportable” and “nonexportable” enzymes in the reticulum cisternae and the Golgi apparatus. The exportable enzymes are then stored in zymogen granules until the next stimulation induces them to pass into the juice. It will be of interest to see to what extent this theory can be confirmed by chemical techniques. 2. Chemical Observations

a. Site of Biosynthesis and Localization. When C14-labeled amino acids are injected into animals, the trichloroacetic acid insoluble proteins of the pancreas rapidly incorporate radioactivity (29, 30). This observation has been confirmed and considerably extended by Morris and Dickman (5) and by Siekevitz and Palade (31, 32). The first authors injected C14-valine into mice and counted radioactivity in ribonuclease A separated in an apparently pure form from some subcellular fractions and supernatant according to the chromatographic technique of Hirs et al. (33). They found that radioactivity was already very high in microsomal ribonuclease 5 min after injection, that it reached a maximum in 15 min, and then declined. The rate of radioactivity incorporation was lower for supernatant and zymogen granules and still lower for nuclei. After 120 min, the highest radioactivity was found in zymogen granules. Similar results were obtained b y Siekevitz and Palade when guinea pigs received Cl4-leucine. After homogenization of the pancreas in 0.88 M sucrose, the zymogen granules were carefully separated from mitochondria by centrifugation in a discontinuous density gradient (26). The microsoma1 fraction was further fractionated into ribosomes, intracisternal granules, microsomal content, and two postmicrosomal fractions. The proteins of each fraction were separated into a n acid-soluble part which was considered as containing most of the exportable enzymes and an acidinsoluble part. Finally, chymotrypsinogen A was isolated from the acidsoluble part by chromatography on Amberlite IRC-50 (8). Radioactivity was determined in each fraction and each sample of chymotrypsinogen A. To restrict the discussion to chymotrypsinogen, radioactivity was highest

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after 1-3 miri in the samples prepared from ribosomes and then in the samples from intracisternal granules. It began to appear in the zymogen granules and the supernatant after 3 min and became highest in the zymogen granules after 45 min. The real significance of the radioactivity found in the supernatant cannot be estimated since the number of subcellular particles which were broken or lysed during homogenization and further treatment is unknown. With this restriction, the results were fully compatible with the assumption that exocrine enzymes are produced a t a very high rate in ribosomes, are packed together in intracisternal granules, and finally are stored in zymogen granules over a certain time. b. Correlation between Zymogen Granules and External Secretion. When zymogen granules are suspended in water or in a solution more alkaline than pH 7.2, they lyse (34) and their content may be subjected to chromatography in the manner described earlier for pancreatic juice. The diagrams obtained with the lysates of bovine granules and with bovine juice are qualitatively and quantitatively very similar (35-37). The same enzymes are found in nearly the same proportions. This very interesting similarity strongly suggests that the enzymes present in the juice come directly from the granules and it is therefore fully compatible with the view that exocrine enzymes are stored in the granules. But it must be recalled here that the main components of bovine juice are proteolytic enzymes and that intragranular localization of exocrine enzymes has only heen proved for this group so far. Deoxyribonuclease and ribonuclease are also present in zymogen granules. A special investigation has even been carried out to show that the granules contain ribonucleases A and B (37). But] the amounts of the nucleolytic enzymes are too low in bovine juice for an accurate quantitative comparison to be established and nothing is known so far for amylase and lipase. It would be interesting in this respect to isolate porcine zymogen granules and see how much lipase and amylase they contain. The problem of the real extent of intragranular localization of pancreatic mzymes is a rather academic one, since it is fairly certain that enzymes exist outside the granules. Some convincing arguments have been prewilted suggesting that ribonuclease, for instance, is not strictly localized. The enzyme extracted from mouse pancreas supernatant is sometimes more radioactive than samples extracted from granules and the excess of radioactivity of the first cannot be attributed to unsedimented microsomes ( 5 ) . Some “free” ribonuclease in the cytoplasm of guinea pig (26) and cattle (38) acinar cells seems to be able to bind a fluorescent antibody. On the other hand, proteolytic activity, as measured against casein after an uncontrolled activation, appears to be more strictly localized inside dog granules than amylase and lipase (34). The same is likely to be true for

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amylase when compared with ribonuclease (39, 40). Finally, whcn mouse granules are mobilized by pilocarpine stimulation, the loss in the whole homogenate is 55 % for amylase and only 33 % for ribonucleaee (5). But the important problem which can be solved quickly by existing and reliable techniques is whether or not all exocrine enzymes are localized inside granules. Keller and Cohen (36) also subjected to chromatography acidic extracts of cattle pancreatic microsomes and ribosomes. In microsomes they found the expected amounts of all enzymes which are known to be stable in acid, viz., chymotrypsinogen A, trypsinogen, deoxyribonuclease, and ribonuclease. The amounts of chymotrypsinogen B were abnormally low and ribonuclease B was perhaps not present. The results concerning ribosomes were made somewhat uncertain by the ability of these particles to incorporate proteins from the medium. Nevertheless, a series of characteristic enzymes could be isolated from what appears to be the very site of their biosynthesis. 3. Biological Significance of Enzyme Localization

When a cell synthesizes an enzyme able, as pancreatic enzymes are, to hydrolyze its own constituents, some kind of protection must be provided. The first assumption is that the cell contains inhibitors which afford a temporary protection (41) until the enzyme is evacuated. The second is that the cell at first synthesizes inactive molecules which are activated later in the intestinal tract. The third is that digestive enzymes are not mixed in situ with cell constituents but separated from them b y impervious membranes. The existence of zymogen granules in pancreatic acinar cells is consistent with the third assumption. Enzymes would be synthesized and localized inside granules in order to provide, not only a possibility of storage between two secretions, but also a protection for the cellular constituents. I n other words, zymogen granules would be, at the same time, a storehouse and a jail. But the general significance of this assumption strongly depends upon the extent of intragranular localization for each individual enzyme. This extent is not yet known with absolute certainty. On the other hand, even if localization of proteolytic enzymes were complete these enzymes could be expected to digest the other enzymatic proteins in the granules. An additional protection must therefore be provided against them. Pancreas contains a series of proteins able to inhibit active trypsin by a reversible and stoichiometric reaction (31). Rut, their exact role is still unclear and it is not known whether they are really present in the granules. Quite obviously, the most efficient protection against proteolytic enzymes is the one discovered 25 years ago b y Northrop, Kunitz, and

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IIerriott (42)-their synthesis by pancreas in the form of inactive precursors which are activated later by a limited proteolysis induced b y trypsin (7, 43-45). It is very striking indeed that inactive precursors have only been identified so far in the case of proteolytic enzymes and that all known proteolytic enzymes of pancreas exist in the form of inactive precursors: chymotrypsinogen A (42), chymotrypsinogen B (46), trypsinogen (42), procarboxypetidase A (47, 48), and procarboxypeptidase B (49-53). This type of protection is not restricted to pancreas, but is used for instance for blood [prothrombin (54), plasminogen (55)]. It is also striking that all pancreatic precursors are activated by trypsin which represents the driving force of the whole system and for which a special activator named enterokinase is available in the duodenum (,56, 57).

4.Net Synthesis Several experiments have been performed in the past for measuring the rate a t which the normal enzyme content of pancreas is restored after depletion by stimulation of the external secretion. This type of study is of value only when enzymatic activities are determined by reliable techniques. On the other hand, it has the advantage of measuring a net protein synthesis instead of merely relying upon incorporation of radioactive amino acids. It will perhaps be useful in the future to reinvestigate the whole problem with improved techniques. Morris and Dickman (5) for instance have recently published interesting curves showing the variations of amylase and ribonuclease content in mouse pancreas after pilocarpine sttimulation. Activity decreases at first, as stated above, b y 55% for amylase and 83% for ribonuclease, and then increases. The rate of increase however is much higher for amylase (normal level after 10 hr) than for ribonuclease (normal level after 21 hr) . Moreover, amylase content rises well above the normal level to reach 200% after 20 hr. Another interesting problem is the study of the enzymatic content of the pancreas, not only as a function of time after stimulation, but also as a function of various external factors such as the composition of the diet, and the nutritional state of the animals. Several laboratories have already attempted to establish the existence of an “adjustment” of enzyme biosynthesis to the main component of the diet and to find out the mechanism by which the digestive process may influence enzyme biosynthesis (58-60). Pancreatic juice collected over an extended period of time by a permanent fistula may be considered an ideal material for studying the net synthesis of a series of well-defined proteins. As soon as radioactive proteins are present in zymogen granules they may appear in the juice after a certain delay, which depends upon the conditions of the next stimulation. When rats in which a continuous flow of juice is promoted by secretin or

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carbamoyl choline receive CI4-glycineby intravenous injectlion,the proteins of the juice do not become radioactive during the first 50 min. Radioactivity appears later and becomes maximal after 90 min (61). In more recent and comprehensive investigations (4,62), S35-cystineor C14-arginine were injected into yearling steers bearing a permanent fistula and the juice was collected in 1-hr periods during 8 hr or more frequently just after the injection. The catioriic proteins (trypsinogen, chymotrypsinogen A, and riboiiuclease) of each sample were separated by chromatography and the radioact,ivity was counted in each peak. The main difficulties in this type of experiment is that the pool of the radioactive amino acids is not known, that only a part, of the enzymes probably appear in the juice, and that it has not yet been definitely established that all exocrine enzymes follow the same route from the ribosomes to the juice. However, interesting differences have been found between chymotrypsinogeii and trypsinogen, on one hand, and ribonuclease on the other. Both proteolytic precursors seem to be synthesized a t proportional rates from common intermediates and also transported a t proportional rates to the juice. I n contrast, radioactivity appears more slowly in ribonuclease after the injection of radioactive amino acids, and the specific radioactivity of this protein is definitely lower.

111. RECENTADVANCES I N THE CHEMICAL CHARACTERIZATION OF SOME lJROTEINS OF EXOCRINE IJANCREAS

A . Introductory Remarks Concerning Nomenclature It, is perhaps risefril to discuss briefly at the beginning of t'his chapter the nomenclature used for describing the structure of a protein molecule and the proteolytic enzymes found in the external secretion of pancreas. 1. Protein Structure

It is now generally agreed that proteins contain several types of bonds inducing several types of structures. The simplest, idea is to correlate each structure with the corresponding bond type. But the choice of the best correlation seems to be somewhat difficult. From a purely chemical point of view, the fundamental fact is that proteins contain covalent bonds which are split by rather drastic means and more labile, noncovaleiit bonds which may be split or a t least altered by denaturation alone. Two types of covalent structures exist, involving peptide bonds and disulfide bridges respect,ively. Both structures should be clearly differentiated since they are destroyed by quite different chemical treatments (hydrolysis for the first, oxidation or reduction for the second). They will be called for this reason, respectively, the primary and the secondary structures. Conversely, several inter- or intrachain, noncovalent

154

P. DESNUELLE AND M. R0VEH.Y

structures may be expected to exist. Since, in the present state of our knowledge, their common characteristic is their noncovalent nature, it seems permissible to use a single expression for both of them, namely tertiary structure. An important fact is that the tertiary structure of a protein may be easily modified either by denaturation or by alteration in any one of the covalent structures. 2. Nomenclature of the Proteolytic Enzymes of Pancreas

Northrop and co-workers (42) characterized in bovine pancreas a single chymotrypsinogen which gave rise by activation to a certain chymotrypsin. This chymotrypsin seemed to be converted later into other active products. It was named a-chymotrypsin in order to underline its apparently direct filiation with the precursor. The other active products were named @- and y-chymotrypsins. When Jacobsen (63) discovered that a-chymotrypsin was not the primary active compound arising during chymotrypsinogen activation, he called this compound ?r-chymotrypsin and used for its autolysis product the first available greek letter (B-chymotrypsin). Finally, when a second chymotrypsinogen was found in bovine pancreas (46), it was called chymotrypsinogen B and one of the corresponding enzymes, chymotrypsin B. The first precursor was then called a-chymotrypsinogen or chymotrypsinogen A. This kind of nomenclature needs revision. It is proposed to term chymotrypsinogen A the more cationic, and chymotrypsinogen B, the more anionic of the proteins present in bovine juice which, under well-specified conditions, give rise to an ATEE-splitting activity. This rule may be easily extended to other species. Furthermore, it is felt that the available information (7, 43, 44, 64) about the chemical events associated with chymotrypsinogen activation should be used in chymotrypsin nomenclature. Thus, r-chymotrypsin, arising by the breakdown of a single peptide bond, would become chymotrypsin A1 In the same way, 6- and a-chymotrypsins would become chymotrypsins A:, and Ad. Proteins of the a-family which are likely to be intermediates during the formation of the a-compound (65) would be named chymotrypsins Aa In order to avoid confusion, the classic and the new nomenclatures will be used simultaneously in this review.

.

.

B. Bovine Chymotrypsinogen A 1 . COOH-Terminal Residues

The study of the terminal residues of bovine chymotrypsinogen A is beset with unusual difficulties. The iTHz-terminal half-cystine cannot be found unless the protein is oxidized by performic acid (66). It is not split

PROTEINS OF EXOCRINE PANCREAS

155

by aminopeptidase even when 100 enzyme units are used per mole of protein (67). The COOH-terminal residue cannot be detected by the usual hydrazinolysis technique and it is unavailable to carboxypeptidase A in the native protein. After denaturation of the protein by urea (67), and after performic acid oxidation or reduction of S-S bridges (68), carboxypeptidase A splits large amounts of several amino acids. A careful analysis of the kinetic curves and the negative results obtained by hydrazinolysis suggest that asparagine is COOH-terminal. All these observations show how cautiously the results given by the so-called end-group techniques must be interpreted. Bovine chymotrypsinogen A is not, as believed earlier, a cyclic protein. It contains an “open” chain beginning with half-cystine and ending with asparagine. The existence of a single chain is confirmed by the fact that reduction of the S-S bridges does not induce any change in the molecular weight (52). Since the protein contains only one chain, the carboxypeptidase technique may give some information about the COOH-terminal sequence. The common practice in the use of this technique is to determine the order in which the amino acids are liberated and to draw kinetic curves for each amino acid. The ideal case is met when large differences exist between the rate of splitting of the bonds. Ambiguous results are sometimes obtained either when all bonds are split rapidly or one bond near the carboxyl end is split much more slowly than the following ones, or, finally, when a given residue is present more than once in the available sequence. As already stated above, denatured or reduced chymotrypsinogen A in the presence of carboxypeptidase liberates many amino acids in large amounts. More than 1 mole of alanine is liberated from each mole of chymotrypsinogen. The sequence has been formulated in two slightly different ways: -Try(Ser,Ala) Val.Thr .Leu .Ala.Asp(NH,) (68, 52) or -Thr .Val.Leu.Ala .Asp(NHZ) with a second alanine residue somewhere in the chain (67). The COOH-terminal sequence of other chymotrypsinogens is also difficult, to ascertain by conventional techniques. Nevertheless, investigations concerning this sequence reveal two interesting facts: (a) The s-sulfo derivatives obtained by sulfite treatment of chymotrypsinogen and trypsinogen (52), although completely denatured, can be kept in solution in the absence of denaturing agents. Absorption spectra and amino acid analysis show that tryptophan and tyrosine are unaffected by the treatment. End-group determinations show further that no peptide bonds are being split. Thus, S-sulfo derivatives appear to lend themselves remarkably well to physicochemical investigations and structural work using enzymatic degradation (to be discussed later). (b) The availability to carboxypeptidase of the COOH-terminal sequence of chymotrypsinogen A is strictly proportional to the extent of denaturation as

150

P. DESNUELLE ANI) M. ROVERY

found by viscosity determinations. Moreover, once exposed by complete denaturation in concentrated urea, this sequence remains available when the protein is renatured by dialyzing away urea at acidic pH (67). Thus, although soluble at its isoelectric point and almost fully activatable, the renatured form of chymotrypsinogen is not identical with the native one. This point will be discussed further in the section on ribonuclease. It is likely that renaturation is made possible in some cases b y the rigid network of S-S bridges which prevents full extension of the peptide chain(s) and thus allows restoration of at least a part of the tertiary structure. In the case of chymotrypsinogen, the COOH-terminal sequence represents a kind of “frce tail” which is not, involved in a “ring” closed by S-S hridges. Such a tail may remain in an extended form even aft’erthe rest, of t8hemoleculc is renatured in a more or less strict way.

2. The Three Chains of a-Chymotrypsin (Ad) It is now known that activation of chymotrypsinogen A involves hydrolysis of a total of four peptide bonds (65). Activation itself is hrought about by the t,ryptic hydrolysis of an arginyl-isoleucine bond which forms r-chymotrypsin (A,). Then, three other chymotxypsin-catalyzed splittings occur: (a) of a leucyl-serine bond leading to d-chymot>rypsin(A2) and (b) of a tyrosyl-threonine bond and an asparaginyl-alanine bond leading either to neochymotrypsinogens or to chymotrypsins of t,he a-family (A, and A*). Since two dipeptides (serylarginine and threonylasparagine) are liberated during the process, these four splittings must have cut the single chain of the precursor into three parts. I n other terms, the final product, a-chymotrypsin (A4),must contain three open chains (Fig. 7). These chains have actually been identified by Meedom (69, 70) after performic acid oxidation, and their terminal residues are those expected from the activation scheme. a-Chymotrypsin (A4) represents an interesting example of a protein having three open chains (A, B, and C) held together by disulfide bridges. The fractionation technique originally used by Meedom involves dissolution of performic acid-oxidized chymotrypsinogen in 0.1 A; ammonia, precipitation of chain B by acetic acid, and chromatography of t’hesolut.ion on Dowex 50. Chain A is not adsorbed under these conditions. Chain C is eluted by 5 M ammonia. Pure chain A is readily obtained in this way. It is a tridecapeptide beginning with cysteic acid and ending with leucine. The NH2-terminal cysteic acid proves that chain A is the NH2-terminal sequence of the precursor. Since chain A contains only 13 residues, the activating split which converts chymotrypsinogen into the primary chymotrypsin occurs very close to the amino end, namely between the fifteenth and the sixteenth bond. This bond is actually the first bond of the sequence which fulfills the structural requirements of trypsin. A quite similar situa-

157

PROTEINS OF EXOCRINE PANCREAS

tion is encountered during trypsinogen autoactivation, when trypsin splits the sixth bond (lysyl-isoleucine bond) of the KHz-terminal sequence (71, 72).

n

TYr

I I Asp* I Ala Thr

ST

Leu

AI

Ileu Arg

Thr

I Ser

I

Asp*

I

I

7 I

Ala' (Asp*-

+

cy< -Neo-ChTg -

+ Thr.Asp*

Ala

'

'Asp*-

T.C.

C.C.

+Cys

7-

-

ChT-6

TY

€tL€52

FIG.7. Stepwise formatioil of the three chains of a-chymotrypsiri (A,) (64). ChTg, bovine chymotrypsinogen A. ChT-a, -8 and -a, respectively, r-chymotrypsin (Ad, 8-chymotrypsin (Az), and a-chymotrypsin (AJ. Neo-ChTg, neochymotrypsinogens (degraded and still activatable forms of chymotrypsinogen A). and -, NH2- and COOH-terminal residues, respectively. T.C. and C.C., hydrolysis catalyzed by trypsin and chymotrypsin, respectively. Asp*, asparagine residue. A, B, and C, chains A, B, and C of a-chymotrypsin (Al).

+

In both cases, activation is associated with a sharp decrease in optical rotation (73, 74) which suggests that the chain has become more tightly coiled. In contrast, when chymotrypsinogen A is converted into neochy-

158

P. DESNUELLE AND M. ROVERY

motrypsinogens by the splitting of a single bond in another point of the molecule, the optical rotation remains constant (75). Therefore, a certain correlation can be immediately established between activation and some rpodifications of the tertiary structure. This correlation is of course in full agreement with the accepted theories concerning the catalytic center of serine-enzymes (76). But the correlation between the precise location of the strategic bond and the structural modifications is as yet unknown. It has been postulated in the case of trypsinogen that the four negatively charged aspartic acid residues of the NH2-terminal sequence induce a local extension by electrostatic repulsion and that the chain coils further like a released spring when the residues are split off (77). Nothing indicates thus far that this hypothesis or a similar one can be applied to the chymotrypsinogen case. A second point must be raised concerning the location and general environment of the strategic bond. The main characteristic of a precursor is of course the ability to give an active enzyme after suitable modifications of its structure. But when the conversion involves, as in the present case, the splitting of a single bond, high yields of active enzyme are only obtained when the splitting of this bond is quite specific. Chymotrypsinogen A and trypsinogen contain, respectively, 19-20 basic bonds (4 arginines, 2 histidines, 13-14 lysines) and 19 basic bonds (2 arginines, 3 histidines, 14 lysines) which a t first sight fulfill the structural requirements for tryptic cleavage. Nevertheless, the strategic bond is split quickly arid quantitatively, whereas the splitting of the others, which would probably inactivate the molecule, does not occur or occurs to a very slight extent. This unique characteristic is again likely to be under the control of the tertiary structure which may expose the strategic bond and bury the others. The location of the strategic bond a t the amino end of a peptide chain is perhaps significant in this respect, although the amino end in chymotrypsinogen is not a free tail as in trypsinogen, but a ring closed by the NHP-terminal half-cystinc. To return now to chain A, Meedom (78) almost completely established the structure of this chain by the classic techniques of partial degradation. The single doubtful detail concerned the relative location of proline and alanine in the fourth and fifth positions. It is now known (67, 75) that, aminopeptidase splits cysteic acid and glycine quickly and then valine much more slowly. This fact suggests that proline may be the fourth resi-. due. When the DNP-derivative of the main peptide left behind by amino-. peptidase is submitted to partial degradation by acid, the dipeptide DNP.Val.Pro is obtained. Thus the complete sequence may be written: S

I

C y.GIy.Val.Pro.Ala.Ileu.Val.Pro.Glu.(NHa).Leu.Ser.Gly.Leu.

159

PROTEINS O F EXOCRINE PANCREAS

where Cy-S is cysteic acid in chain A and half-cystine in the NHz-terminal sequence of chymotrypsinogen A. This latter sequence may be further extended by placing on the right : Ser.Arg.Ileu.Val.Gly- which corresponds to the dipeptide split-off during activation and to the amino end of chymotrypsin. Thus, the arrangement of the first 18 residues of the NHz-terminal sequence of bovine chymotrypsinogen A is now determined. However, the situation is far from being so clear for chains B and C. These chains have been reported by Meedom to contain 180 and 50 residues respectively. But the technique described does not give pure products,

1

FIG.8. Purification of chain C by chromatography on Sephadex G-50 (79). Sephadex G-50 column (1.8 X 35 cm) equilibrated with 0.05 M HCI. Lyophilised aqueous extract of 30 mg of DFP-inhibited, performic acid-oxidized a-cbymotrypsin (A,) is dissolved in 1 ml 0.05 M HCI, put into the column, and eluted (5 ml/hr) b y 0.05 M HCl. Solid line, absorption of the fractions at 280 mp. Dotted line, absorption at 230 mp. Ordinates, optical density. Abscissas, volume of eluate in milliliters. A, chain A; C, chain C.

mainly because the chains associate strongly and are degraded in ammonia by some unknown mechanism. Better results are obtained for chain C, which corresponds to the chymotrypsinogen COOH-terminal sequence, when aqueous extracts of oxidized a-chymotrypsin (A4) are chromatographed on Sephadex G-50 equilibrated with 0.05 M HC1 (79). Figure 8 shows that three well-separated peaks emerge. The first one is an ill-defined mixture containing chains B and C. The second is chain C in an apparently pure form. The third is the small chain A which does not absorb at 280 mp. Chromatographically purified chain C can be obtained in an over-all yield of 30%. It contains alanine as the single NH2-terminal residue and all amino acids except histidine and phenylalanine. Nothing is known thus far about the further purification of chain B.

1 GO

r.

DESNUELLE AND M. ROVERY

3. Treatment with Sulftte

The specific splitting of protein disulfide bridges is an int'erestirig prohlem in several respects. (a) Cystine is partially destroyed during acid hydrolysis. An accurate determination of this amino acid requires it,s previous transformation into another derivative. (b) Intrachain and interchain bridges slow down enzymatic attack and the chromatography of tJheresult,ing cystine peptides is difficult. (e) In most proteins, disulfidt? bridges represent the only interchain link. When the bridges are cut, t,he chains tiecome independent and can be isolated. Performic acid oxidation, which converts each bridge into two sulfonic acid groups, has been successfully used in a number of cases; hut it has serious limitations. Even when special precautions are observed for minimizing chain splittings and tyrosine halogenation, destruction of tryptophan seems to be unavoidable. Besides, the oxidized material is often poorly soluble. Reduction preserves the integrity of tryptophan. All the att,empts made for split,t,ing protein disulfide bridges by metal hydrides, cyat,eine, thioglycolate, and mercaptoethanol havc heen already comprehensively reviewed. The SH groups appearing during reduction are labile and quickly reoxidize. They must therefore be converted a t once irit,o more st,ahle derivatives by alkylation. When iodoacetate or iodoacetamide is the alkylat,ing agent,, 8-carboxymethylcysteine residues are obtained which arc stable during acid hydrolysis and may be easily chromatographed. A different approach has been used recently for chymotrypsinogen A and trypsinogen. It involves treatment of the protein by sulfite in the presence of a n oxidizing agent. Sulfite (80) splits each bridge by forming an organic thiosulfate (S-sulfonic or S-sulfo compound) and a thiol. The oxidizing agent [o-iodosobeneoate, tetrathionate (81), or cupric ions (82)], transforms the thiol again into disulfide and so on until the reaction is complete. S-Sulfocysteine is unfortunately labile during acid hydrolysis. But the extlent of the reaction may be followed either hy ampernmetric titration of the thiol formed in the absence of oxidants (81), by polarographic determinatiori of t,he cuprous ions appearing when thiols are oxidized by cupric ions (52), or by radioactive techniques when S35-sulfiteis used (52). Each protein represents a new problem since certain S-S bridges are specially resistant even in denatured proteins. Under well-selected conditions, however, all bridges of chymotrypsinogen and trypsinogen appear to be split. Results obtained by cuprous ion polarography agrec with those obtained by direct determination of cystine after conversion into cysteio acid (52). Nevertheless, the possibility that the splitting is incomplete in some cases should not be neglected since it may give int,eresting information about the general structure of the molecule arid about, t,he relations between secondary structiire and activity.

PROTEINS OF EXOCRINE PANCREAS

161

Most denatured proteins are insoluble in water as a result of strong interactions between fully extended peptide chains. In the special case of S-sulfochymotrypsinogen and S-sulfotrypsinogen, aggregation may also occur. But, when special precautions are taken, water-clear solutions are obtained which are homogeneous in the ultracentrifuge (52). Under other circumstances (81), however, the S-sulfo derivatives appear to be poorly soluble in the pH range 3-9. Insolubility increases when reduction becomes more complete. No attempts have been made thus far to separate the chains of a-chymotrypsin (A4) after sulfite treatment, although this treatment is successful in the case of insulin (81). Sulfite-treated insulin is chromatographed on Dowex 50-X2 equilibrated with p H 2.2 buffer 0.2 M in citrate and 8 M in urea. Chain A passes unretarded whereas chain B is eluted by 0.2 M phosphate buffer, pH 7.6 containing the same amount of urea. Both chains have the expected compositions.

4. Primary Structure Table I gives the amino acid composition of bovine chymotrypsinogen A. Quite similar results for most amino acids have been obtained for this protein in two laboratories by the usual chromatographic technique in its manual or automatic (83) form involving all the necessary corrections and cxtrapolations to zcro time. Cystinc has been determined as aysteic acid 2Lfter pcrformic acid oxidation. Valucs of tryptophan are probably less satisfactory since they are derived from spectrophotometxic or miorobiological determinations. The protein contains only 2 histidines, 2 methionines, 4 arginines, 4 tyrosines, and 5 cystines per mole. As pointed out above, the first 18 amino acids in the NHz-terminal sequence of this protein are already known. The COOH-terminal sequence is also known, a t least provisionally, for six or seven residues. The region where chymotrypsin specifically attacks the molecule during neochymot,rypsinogen formation is -Tyr.Thr.Asp. (NH2).Ah- (65). The specific point of attachment of the diisopropylphosphoryl radical in DFP-inhibited a-chymotrypsin (A4) is G1y.Asp.Ser.Gly.Gly.Pro.Leu (89). Thus, the arrangement of 35-36 residues out of about 240 has been determined almost by chance while investigating other problems. Since bovine chymotrypsinogen A is one of the best characterized proteins available so far, it is important to launch a more general attack by conventional t,echniques. However, the enormous labor needed for the complete analysis of much smaller proteins, such as ribonuclease and lysozyme, is a good warning against excessive optimism. When digested by chymotrypsin (90-92), chain B gives an ethanol-insoluble acidic “core” with about 130 residues and 14 soluble basic peptides which seem to derive from the carboxyl end.

TABLEI Amino Acid Composition of Some Chymotrypsinogensa Bovine Ab

(84)

(85)

Bovine Bc (86) (87)

Alanine Arginine Aspartic acid Glutamic acid Glycine Histidine Isoleucine Leucine Lysine Methionine Phenylalanine Proline Serine Threonine Tyrosine Valine

22 4 22 14 23 2 10 19 13 2 6-7 9 30 23 4 22

22 4 22 14 23 2

18 4 17 17 18 2

19 13-14 2 6 9 27 23

15-16 10 28 6 12 17 17 3 20

Tryptophan Half -cystine' Amide

7 ' 10 24

10 23

Molecular weight

25,100

-

Eikmat 280 mp N% Isoelectric point

20.0

21.0 16.5

Amino acid

16.5 9.1

8

10

4

23

-

20-21 5 19 16 20 2 8 17 10 4 6 12 18-19 20 3 21

20-21 5 18 12 19 2 9 17 10 26 5 12-13 21-22 19 4 22

6h

-

-

89

Porcine Ad (85)

8 15

79

14

21,380f 613j 24,OOOi 22,336 f 441j 22,500k 21,800 f 3%" 22,700' 18.0 18.0 16.2 16.1 5.2 7.2

NHz-Terminal residue

Half -cystine

Numbers of residues are given per 25,100gm for bovine chymotrypsinogen A. They are calculated for bovine chymotrypsinogen B and porcine chymotrypsinogen A by using the molecular weight values given b y chemical analysis (21,400or 24,000 for bovine chymotrypsinogen B ; 22,300for porcine chymotrypsinogen A). * Results obtained by ordinary chromatography for the first column and by use of an amino acid analyzer (83) for the second. Results obtained by use of an amino acid analyzer. Some of the results of the second column have been confirmed by independant methods, Results obtained by use of an amino acid analyzer. a Not included in the estimation of the molecular weight. Microbiological technique. 0 Spectrophotometric technique. Chromatography after alkaline hydrolysis. As cyst,eic acid after performic acid oxidation. j Values estimated from chemical analysis. Value obtained by sedimentation-diffusion on an impure sample (88). Values kindly determined by Mr. D. M. Brown and Prof. E. L. Smith on a sample prepared in this laboratory. The values have been obtained, respectively, b y the Archibald method of sedimentation equilibrium and by sedimentation-diffusion, assuming a partial specific volume of 0.721for the protein. J

162

PROTEINS OF EXOCRINE PANCREAS

163

A detailed discussion of the results is unnecessary since chain B has not yet been obtained in a pure form. Experiments with chymotrypsinogen are not open to the same objection. When S-sulfochymotrypsinogen is digested by trypsin (92) a core is again obtained as well as a series of soluble peptides. The core can be further digested by other proteolytic enzymes. A large number of peptides has already been identified and assembled in a preliminary way to give an unique sequence of 242 residues (93). Peptides derived from chymotrypsinogen and trypsinogen have also been extensively studied by Keil, Sorm, and their collaborators. The first experiments were mostly designed for finding out some structural similarities between both proteins. Short peptides formed by partial acid hydrolysis were isolated and compared (94-97). The usefulness of short peptides in sequential analysis is unfortunately limited. More recently, however, various larger peptides obtained by peptic degradation of chymotrypsinogen (98) and trypsin (99) and by tryptic degradation of oxidized chymotrypsin (100) have been isolated and partially identified. A list of known sequences has also been published (101).

C. Bovine Chymotrypsinogen B The second, anionic bovine chymotrypsinogen (chymotrypsinogen B) , crystallized by Laskowski et al. (46), was initially thought to be of minor importance. This impression probably arose because of heavy losses during purification. Direct chromatography (1) proved later that the amounts of chymotrypsinogens A and B in bovine pancreatic juice were in fact very similar. Therefore it became important to compare as closely as possible the structures and modes of activation of two chymotrypsinogens synthesized by the same species. End-group determinations showed some years ago that, although electrophoretically homogeneous at a single pH, crystalline chymotrypsinogen B is probably impure. It has been reported to contain chymotryptic activity and neochymotrypsinogen (87). Attempts have been made, either to crystallize the precursor in the presence of DFP (87), or to purify further the crystalline material by chromatography (102). Amino acid analysis (87) and end-group determinations (103, 104) have been carried out on these purified preparations. Another approach for purification purposes is to abandon the crystallization step. After a preliminary fractionation of 0.25 N sulfuric acid extracts of fresh pancreas, the enriched fraction is directly chromatographed (105) on a CM-cellulose column equilibrated with 0.05 M citrate buffer, pH 4.2. As shown in Fig. 9, chymotrypsinogen B is eluted by a 0.05 M citrate buffer, pH 4.6, The best fractions (specific activity, 2.5-2.6) are homogeneous as shown by an equilibrium chromatography on DEAE-cellulose in 0.05 M phosphate buffer, pH 8.0.

164

r.

DF,BNUELT,F, ANI) M. ROVERY

The amino acid composition of chymotrypsinogen B (Table I) has been recently determined in two laboratories with slightly different results. Full discussion must be postponed until more is learned about the molecular weight and exact number of residues of chymotrypsinogen B. But the general impression already emerges that both bovine precursors belong to the same protein family with low and similar amounts of arginine, histidine, methionine, phenylalanine, and tyrosine. The most striking difference is in

2c

t 3 Fractions

FIQ.9. Preparative chromatography of bovine chymotrypsinogen B (105). CMcellulose column (3.0 X 9.0 cm) equilibrated with 0.05 M citrate, pH 4.2. Arrow indicates change to 0.05 M citrate, pH 4.6. Solid line, potential activity. Dotted line, proteins. The values along the peaks indicate the specific activity of some fractions. 1. Chromatography of the precipitate obtained in 0.4 saturated ammonium sulfate (specific activity, 0.9). 11. Second chromatography of the fractions having the lowest activity in diagram I. Ordinates, activity or proteins found in each fraction (1 ml) and expressed in per cent of the total activity and total proteins introduced into the column. Abscissas, volume of eluate in milliliters. the amide content which explains a t least partly the anionic character of component B. The same holds for porcine chymotrypsiriogen A which is presented below. Bovine chymotrypsinogens A and B have the same NHz-terminal residue, namely half-cystine. Both are activated by trypsin a t about the same rate. The best technique thus far available for differentiating chymotrypsins A and B is to determine their activities on acetyl-L-tryptophan ethyl ester in the presence of 30% methanol (1). Table I1 shows that the much lower activity of chymotrypsin B on tryptophanyl esters is due to a stronger depressing effect of methanol (106).

165

PROTEINS OF EXOCRINE PANCREAS

D . Porcine Chymotrypsinogen A Comparisons between chymotrypsinogens of various species are a t least as important as between the two chymotrypsinogens of the same species. Some information is now available about porcine chymotrypsinogen and trypsinogen. Porcine pancreas is extracted as usual with 0.25 N sulfuric acid and the clear extract is fractionated by ammonium sulfate between 0.2 and 0.4 saturation. When this fraction is chromatographed on CM-cellulose, the diagram given in Fig. 10 is obtained (85). This simple technique gives a t once nearly pure products. After a second chromatography, performed under equilibrium conditions, homogeneous peaks are obtained (Fig. 11). After two chromatographic runs, porcine chymotrypsinogen A behaves as a homogeneous substance during free electrophoresis a t various pH’s and TABLEI1 Action of chymotrypsins A and B on Tyrosyl and Tryptophanyl Esters in the Presence of Methanol (106) Specific activities measured on:

ATEE”

Activated protein

A B

ATry EEa

5

30

5

30

3.0 2.2

0.81

0.92 0.70

0.78 0.05

0.60

a ATEE, acetyl-L-tyrosine ethyl ester; ATryEE, acetyl-L-tryptophan ethyl ester. The numbers below each ester represent methanol concentrations by volumee.

ionic strengths. The instability of trypsinogen prevents a closer study of its homogeneity by physicochemical means. Some of the chemical properties of porcine trypsinogen will be discussed later. The first question concerning the chymotrypsinogen is whether porcine pancreas contains one or two proteins endowed with potential chymotryptic activity. Figure 12 gives two chromatographic diagrams (85) obtained with porcine pancreatic juice collected by a permanent fistula. A potential chymotryptic activity is invariably found in the “anionic” as well as in the “cationic” fractions of the juice. When the anionic proteins obtained in both cases are further chromatographed on DEAE-cellulose a t pH 8, a large chymotryptic activity is again found in the anionic fraction. Since, although isoelectric a t p H 7.2, the chymotrypsinogen described above behaves as a cationic substance on DEAE-cellulose a t p H 8, it seems likely that porcine pancreas does contain two different chymotrypsinogens and that, the more anionic variety is inactivated, not extracted by sulfuric acid or not eluted by the phosphate gradient. For this reason, the purified precursor is named chymotrypsinogen A.

0

FIG.10. Chromatography for the purification of porcine chymotrypsinogen A and trypsinogen (85). CM-cellulose column (0.9 X 9.0 cm) equilibrated with 0.05 M citrate buffer, p H 4.3. Stepwise elution with 0.05 M buffer, p H 4.6 and pH 5.0, 10-4 M in DFP. Solid line, potential activity against ATEE (chymotrypsinogen, ChTg) and BAEE (trypsinogen, Tg). Dotted line, proteins. The values along the peaks indicat : the specific activity of some fractions. Wider columns (3.0 X 9.0 cm) are used for

preparative runs, with the same results. Specific activity of the preparation put into the column, 0.9 for chymotrypsinogen and 0.19 for trypsinogen. Ordinates, activity or proteins found in each fraction (1 ml) and expressed in per cent of total activity or total proteins introduced into the column. Abscissas, volume of eluate in milliliters.

FIG.11. Second chromatography of porcine chymotrypsinogen A and trypsinogen (85). Both elutions are performed with buffers of constant composition (equilibrium chromatography). On the left : chymotrypsinogen A (specific activity, 2.9). CM-cellulose column equilibrated and eluted with 0.03 M citrate, pH 5.0. On the right: trypsinogen (specific activity, 0.350.37). CM-cellulose column equilibrated and eluted with pH G.0 buffer 0.015 M in citrate and lo-' M in DFP. Ordinates and abscissas are the same as in Figs. 9 and 10. Solid line, activity. Dotted line, proteins. 1GG

167

PROTEINS OF EXOCRINE PANCREAS

As in bovine chymotrypsinogens A and B, the single NHz-terminal residue in porcine chymotrypsinogen A is half-cystine. The amino acid composition and the molecular weight of the protein are given in Table I. 10

%

.-

% r\

-

DEAE C

41%

CM-C

5

40

0

20

40

Fractions

____c

FIG.12. Cationic and anionic proteins of porcine pancreatic juice (85).On the left: DEAE-cellulose column equilibrated with 0.005 M bicarbonate, pH 8.0 and eluted after the dotted vertical line by 0.3 M bicarbonate, pH 8.3. Solid line, proteins. The figures near the peaks give the percentages of potential chymotryptic activity associated with each peak. On the right: CM-cellulose column equilibrated with 0.02 M citrate, pH 6.02 and eluted stepwise at the same pH by citrate buffers of increasing molarities (0.06,0.12, and 0.25). The figures near the peaks give again the percentages of chymotryptic activity found under each peak. Ordinates and abscissas are the same as in Figs. 9-11.

E. Bovine Trypsinogen and T r y p s i n 1. Trypsinogen Activation

An excellent correlation has been recently established, during bovine trypsinogen activation in the presence of calcium, between appearance of BAEE-splitting activity, opening of a single bond as measured by the pHstat technique, appearance of a trypsin-trypsin inhibitor peak during electrophoresis, and optical rotation changes (74). Since the same correlation is known to exist between the extent of activation, NHz-terminal isoleucine, and hexapeptide formation (72), it can now be accepted with considerable confidence that activation of the precursor is determined by some changes in the tertiary structure associated with the splitting of the sixth bond of the chain. No COOH-terminal residue has ever been found in trypsinogen and trypsin by hydrazinolysis or carboxypeptidase digestion

108

P. DESNUELLE A N D M. ROVERP

of the native or denatured proteins. Carboxypeptidase fails to liberate any amino acid even in S-sulfotrypsinogen (68). The influencc of calcium ions during t,rypsiriogen autoactivation is well known. In thc presence of calcium, activation is quantitative, in thc sensc that a given weight of precursor gives nearly the activity displayed by the same weight of crystalline trypsin. In the absence of calcium, trypsinogen gives rise to inactive proteins that are called “inert proteins” by Kunitz. The formation of these proteins has recently been investigated in some detail (107). Calcium exerts two opposite effects: (a) it speeds u p the split,ting of the strategic lysyl-isoleucine bond by a factor of more than 10 and thus great,ly accelerates the activation process. It would be int,eresting to know whether this specific influence on the sixth bond of the tail is due to a local effect, for instance on the four aspartic acid residues present nearby, or to a general modification of the tertiary structure which would expose tho bond in question still more. (0) In trypsinogen, as in many proteins, calcium protects some bonds against tryptic attack. This second effect is generally considered as brought about by a strengthening of the tertiary st8ructture which shifts the equilibrium: native form denatured form further t’otbo left. The inactive proteins formed in t,he absence of calcium have not, yct~ been fully identified, but they appear to contain additional end groups (NHZterminal serine for instance) which probably correspond to additional splits in the interior of the chain. The latter should not be considercd as merely inactivating preformed trypsin. They prevent the opening of the sixth bond by some changes in the tertiary structure of trypsinogen (107). It has also been shown by Kunitz that trypsinogen is activated b y firstorder reactions induced by other enzymes such as enterokinase and mold proteases. As in autoactivation, activation by purified enterokinase runs parallel with the appearance of an NH2-terminal isoleucine (57). But the hexapeptide has not been isolated in this experiment and enterokinasc acts in a pH range whcre autoactivation occurs a t a quick rate. More work is clearly needed before the identity of both processes is firmly established. In contrast, accurate experiments have been carried out to clear np tha mechanism of trypsinogen activation by mold proteases. These enzymes act a t acidic pH’s a t which autoactivation and further trypsin autolysis do not occur. A crystalline protease from Aspergillus saitoi fully activates bovine trypsinogen a t p H 4.5 by splitting the same lysyl-isoleucine bond as trypsin (108). Owing to the broader specificity of the mold enzyme, however, some other bonds are split in the hexapeptide as well as in the proteins formed during the process. Since activation is quantitative, this latter fact, suggests that some degraded, still active, and so far unidentified forms of trypsin may exist, Split products of the hexapeptide also appear when trypsinogen is activated by an enzyme from Penicillium janthindlum (109, 210).

PROTEINS OF EXOCRINE PANCREAS

109

2. Activation of Acetyltrypsinogen

Can enzyme molecules be simplified? If the hypothesis of a narrowly restricted catalytic center is correct, the rest of t,he molecule may he regarded as a nonessential, removable piece of “junk” (76), as long as the enzyme-substrate connections and the stability of the center are retained. Large parts of some enzyme molecules may actually be removed without impairing activity. But the existence of active split products in t,he specific case of trypsin is still doubtful. The possibility that acetyltrypsin may undergo aiitolysis without losing activity is now denied. Inactive acetylated molecules are merely digested by active ones (111). But it has been postulated recently that acetyltrypsinogen, which cannot be activated by trypsin since the t-NH2 of the sixth lysine is blocked (74), may give rise to active products under the influence of pepsin at pH 3.4 (112, 113). These products are eluted in a well-defined peak during chromatography on DEAE-cellulose. They have been reported to have a molecular weight of 6000, a single NHz-terminal residue of phenylalanine, and no histidine. This last point would shatter the accepted theories concerning esterolytic centers. For the time being, it can merely be pointed out that acetyltrypsinogen is heterogeneous (74, 113) and that the specific activity reached during peptic activation is low. But nothing of course absolutely requires that the active center of t>rypsinmust be built in a single way. 3. Chromatography

Bovine trypsinogen and trypsin are still prepared by the tedious and time-consuming technique of Northrop and Kunitz involving ammonium sulfate fractionations and crystallizations at alkaline pH. Trypsinogen can be crystallized only once and is obtained in rather impure state. Crystalline trypsin also is not pure. Besides NHz-terminal isoleucine, it contains some other end groups which are eliminated by further crystallizations of the DFP-inhibited derivative. However, bovine trypsinogen has been successfully chromatographed on Amberlite IRC-50 at pH 6.0 (1, 114) or on CM-cellulose at p H 3.2 in a gradient of increasing ionic strength (1 15). The starting materials used are commercial crystalline preparations (114, 115), acid extracts of fresh pancreas (114), or pancreatic juice (1). Results obtained with the porcine precursor (see Figs. 10 and 11) suggest that better resolution would be achieved on CM-cellulose at a somewhat higher pH. Bovine trypsin can also be chromatographed on CM-cellulose a t pH 3.2 in an ionic strength gradient (115). I n this way, active trypsin begins to separate from inactive proteins present in commercial preparations or formed during trypsinogen activation in the absence of calcium. After

170

P. DESNUELLE AND M. ROVERY

repeated chromatography, it appears to be essentially pure, but the yield is rather low and a poor scparation of trypsin and trypsinogen is obtained under these conditions. A crystalline sample of bovine trypsin has recentiy been chromatographed on CM-cellulose at pH 6.02 in the presence of calcium ions (116). The diagram of Fig. 13 show that, after the emergence of an unretarded inactive peak (25 %), trypsin can be eluted in an apparently homogeneous form by raising the molarity of the citrate buffer

I

%

20 -

0

10

0.05M

20

Fractions

4O.IOM

FIG. 13. Chromatography of active trypsin on CM-cellulose (116). CM-cellulose column (0.7 X 9.0 cm) equilibrated with pH 6.02 buffer 0.05 M in citrate and 0.013 M in CaCl2. Arrow indicates change to buffer 0.1 M in citrate and 0.013 M in CaC12. Commercial sample of crystalline trypsin (specific activity, 0.23). Solid line : proteins (25% in the first peak and 64% in the second). Activity against BAEE, 0% in the first peak and 80% in the second. Figures along the solid line give the specific activity of the fractions against BAEE. Dotted line: activity against ATEE (13y0 in the first peak, 48% in the second, and 33% in the third). Figures along the dotted line give the specific activity of the fractions against ATEE. Ordinates are the same as in Figs. 9-12. Abscissas, number of fractions having a volume of 1.4 ml.

from 0.05 to 0.1. When ATEE is used instead of BAEE for testing the activity of the fract.ions,three peaks are obtained. The first two probably represent chymotrypsin or chymotrypsin-like enzymes which are not eliminated by crystallization. The third is exactly under the trypsin peak. This latter observation suggests that pure trypsin is able to split tyrosyl esters at quite a noticeable rate. The ratio of the activities displayed by pure trypsin against ATEE and BAEE seems to be 1:6.5. On the other hand, the ratio of the ATEE-splitting activities of pure trypsin and pure chymotrypsin seems t o be about 1:50. Trypsin and chymotrypsin are extensively used for the determination of

PROTEINS OF EXOCRINE PANCREAS

171

the primary structure of proteins. Their well-known specificity enables them to split a limited number of bonds and to form relatively simple peptide mixtures. Trypsin in particular is believed to be specific essentially only for “basic” bonds and any peptides formed by this enzyme are expected to have a basic COOH-terminal residue. The total number of peptide bonds split by trypsin in a given chain is expected to be, and sometimes is (117), equal to the number of basic residues. However, trypsin as well as chymotrypsin sometimes split peptide chains at “wrong” points. These disturbing anomalies are commonly attributed to the presence in each enzyme preparation of small amounts of the other. Attempts are often made to eliminate what is considered as an undesirable impurity. But trypsin is often tested on “aromatic” substrates, such as p-nitrophenylacetate, 0-naphthyl esters of N-acylphenylalanine (1 18), fatty acid esters of m-hydroxybenzoic acid (1 19), and p-nitrophenyl esters of carbobenzoxyamino acids (120, 121). Trypsin is inhibited by aromatic derivatives of orthophosphoric acid. This suggests that cross reactivity may exist with chymotrypsin. Cross reactivity between both enzymes has actually been demonstrated by kinetic experiments in the presence of specific inhibitors (122). Figure 13 confirms this fact in the case of trypsin. About two-thirds of the chymotrypsin-like activity of the sample belong to a contamination and may therefore be eliminated, for instance by chromatography. But the rest appears to belong to trypsin itself, since the ATEE-splitting, as well as the BAEE-splitting, specific activities are constant all along the trypsin peak.

F. Porcine Trypsinogen Figures 10 and 11 show how porcine trypsinogen can be prepared in high yield by chromatography on CM-cellulose. The amino acid composition of this protein is given in Table 111, with two sets of values for the bovine precursor. Analytical results have also been obtained with a commercial sample of bovine trypsin (123). It would certainly be premature to compare the general composition of bovine and porcine trypsinogens. However, three specific differences have already been noted (126) : (a) the NHz-terminal residues of porcine trypsinogen is phenylalanine instead of valine. (b) Even in its native state, the porcine protein is attacked by carboxypeptidase A. After urea denaturation, large amounts of alanine and asparagine are liberated, followed at a slower rate by isoleucine, glutamine, threonine, and leucine. Since hydrazinolysis by the usual procedure gives negative results, asparagine is likely to be COOH-terminal in porcine trypsinogen. More experiments are needed for the rest of the sequence. (c) As in the case of bovine trypsinogen, a single significant peak is obtained by chromatography of the dialyzable compounds formed during autoactivation. This peak, however, contains the

172

P. DESNUELLE AND M. ROVERY

octapeptide l’he.l’ro.Thr.(Asp)4.Lys, instead of the hexapeptide Val(Asp),.Lys. It is quite interesting to note that the two trypsinogens available at the present time are activated by a similar mechanism, that the TABLEI11 Amino Acid Composition of Bovine and Porcine T r ypsinogensa Bovine trypsinogen Amino acid

(124)

Alanine Arginine Aspartic acid Glutamic acid Glycine Histidine Isoleucine Leucine Lysine Methionine Phenylalanine Proline Serine Threonine Tyrosine Valine

Residues in 24,900 gm 15-16

-

62.5 16.5 112.1 67.7 101.2 15.3 60.4 64.9 43.1 8.1 20.9 43.5 101.2 44.3 32.2 63.3

12

-

12 35

-

13 2 24 10 21 3 12

2 25

11

3

-

la

14 1 4 7 38 9-1 1 9 15

14-15

-

3 8 39-40 11 4

Half -cystine Amide

23 f 3

Molecular weight E:im at 280 mp N% Isoionic point

23,800 13.9 9.3

NH2-Terminal residue

Residues (10-8) in 100 gm

(125)

--

Valine

Porcine trypsinogen (126)

23,300 -

-

-

4

28 17 25 4 15 16 11 2 5 11 25 11 8 16

24,909 f 549b 13.9 16.9 Phenylalanine

a Numbers of residues are given per 23,800 gm for bovine trypsinogen. They are given per 24,900 gm for porcine trypsinogen and itlso per 100 gm, since the molecular weight of this protein calculated from chemical analysis alone is still preliminary. b Preliminary value estimated from chemical analysis.

characteristic structure (Asp),.Lys on the left of the strategic bond is the same in both proteins, and that the other, more distal residues of the NH2terminal sequence are different. In a family of proteins elaborated by various species for a given biological function, there are probably “essential”

PROTEINS OF EXOCRINE PANCREAS

173

parts, closely related with the function, which have a predetermined structure and “unessential” parts in which some variations are permissible.

G . Bovine Procarboxypeptidase A and Carboxypeptidase A 1. Purification and Activation of Procarboxypeptidase A

In 1935, Anson (127) crystallized what is now called carboxypeptidase A from autolyzed bovine pancreas and noted that fresh pancreas did not contain the active enzyme, but an inactive precursor now named procarboxypeptidase A. It has been reported in the preceding sections that pancreatic juices of other species also contain large amounts of procarboxypeptidase A which can be separated by chromatography on DEAE-cellulose at pH 8.0 in a buffer of increasing molarity. For preparative purposes, it is perhaps more convenient to start from pancreas acetone powder (pancreatin). Ninty-five per cent pure bovine procarboxypeptidase A has been obtained by ammonium sulfate fractionation of pancreatin extracts and isoelectric precipitations (47). When the proteins precipitated by 0.39 saturated ammonium sulfate are chromatographed on DEAE-cellulose in a concentration gradient of pH 8.0 phosphate buffer, the two last and most acidic peaks contain procarboxypeptidase A in an electrophoretically homogeneous form (48). The molecular weight of the protein determined by light scattering and sedimentationdiffusion is 94-96,000. Its isoelectric point in univalent buffers of 0.2 ionic strength is below 4.5. The tryptic activation of bovine procarboxypeptidase A into carboxypeptidase A has already been discussed in a series of comprehensive reviews (7, 128). It will be recalled here that in sharp contrast to trypsin and chymotrypsin, carboxypeptidase A is formed by only one-third of the precursor molecule, the rest being split off during activation. But the most interesting point of the activation process is probably the transitory appearance of a ATEE-splitting, DFP-sensitive enzyme which accumulates at low temperature in the presence of low amounts of trypsin (48). When the temperature and the amount of trypsin are raised, a part of this chymotrypsin-like enzyme disappears, whereas carboxypeptidase activity, measured against carbobenzoxyglycylphenylalanine,appears. It had been assumed that procarboxypeptidase A gave rise in a first step to the ATEEsplitting enzyme, which was converted later on into carboxypeptidase A by autolysis and further degradation by trypsin. However, it has been shown more recently (129) that procarboxypeptidase A is split by concentrated urea, buffer solutions at pH 10, and chelating agents such as 1 ,lophenanthroline into three subunits with the same sedimentation rate as carboxypeptidase A. It is also now known that procarboxypeptidase A

174

P. DESNUELLE AND M. ROVERY

contains three NHz-terminal residues, aspartic acid (or asparagine) , lysine, and half-cystine. Therefore, the possibility exists that the ATEE-splitting enzyme and carboxypeptidase A do not arise, as believed earlier, successively from the same molecule, but from two different subunits of this molecule. Bovine carboxypeptidase A is likely to be formed by a single chain beginning with an asparagine residue (130) and terminated by the sequence (Glu,Leu,Thr,Val) Asp (NH2) ( 131). 2 . Role of Metals in Carboxypeptidase A

Unlike chymotrypsinogen and trypsinogen, carboxypeptidase A is not inhibited by DFP and cannot therefore be considered as a serine enzymp. It is actually a metallo enzyme containing one zinc atom per molecule.2 When zinc is removed by dialysis against acidic buffers or 1,10-phenanthroline (132, 133), an inactive apoenzyme is obtained which does not differ from the enzyme itself, except for activity and zinc content. Activity is gradually restored by addition of zinc up to one equivalent (133, 134). Some other metals of the first transition period (Mn++, Fe++, Co++, Ni++) can also restore activity in the apoprotein (134). Relative affinities of the apoenzyme for various metal anions have been investigated by equilibrium dialysis against solutions of radioactive salts (134). It is now demonstrated that zinc is bound in the bovine enzyme to an SH group probably belonging to a cysteine residue (135). I n contrast to the native or reconstituted carboxypeptidase, the apoenzyme has one titratable SH group. A good correlation is found between titratable SH group, zinc content, and activity. Long storage induces simultaneous loss of SH groups and loss of reactivability by zinc. Both are partially restored by a limited addition of cysteine. Furthermore, apocarboxypeptidase A treated by zinc liberates two protons, indicating zinc binding to a second group thought to be nitrogen (136). Thus, the active center of the enzyme is likely to include one sulfur, one nitrogen, and one zinc atom. Some observations also suggest that the nature of the metal bound to the apoprotein may influence the activity of the enzyme against different substrates. Cadmium, mercury, and lead carboxypeptidases have been reported to be active on esters only (136). On the other hand, when the zinc-containing, active carboxypeptidase is incubated with cobaltous ions, activity towards the specific peptide substrates increases two-fold whereas esterase activity to wards hippuryl-L-phenyllactic acid remains unchanged (137). While acting in this interesting way, cobaltous ions are much less tightly bound to the protein than zinc ions. Original activity is quickly regained by dilution or dialysis. A bibliography of this subject is found in a recent and comprehensive review by Neurath (128).

PROTEINS O F EXOCRINE PANCREAS

175

H . Bovine and Porcine Carboxypeptidases B Certain bonds are split by proteolytic enzymes, not merely because they belong to a given protein, but because their structure, locatJion, and/or availability fulfill certain requirements. Thus, the nomenclature of proteolytic enzymes should not be based upon the name of the proteins of which these enzymes have been tested for the first time, but upon factors directly connected with the bonds. A good illustration of this important principle is given by the following case: It was claimed that pancreas contains a “protaminase” because autolyzates of the gland displayed considerable activity against protamines (138, 139). It turned out later that pancreas actually contains a second carboxypeptidase which easily splits the basic bonds of these peculiar proteins. The specific range of this second carboxypeptidase has been unambigously defined (49-51). It hydrolyzes COOH-terminal basic bonds in synthetic substrates such as a-N-benzoylglycyl-L-lysine and hippuryl-Larginine as well as in proteins. In contrast to carboxypeptidase A, it is competitively inhibited by eaminocaproate and 6-aminovalerate (50). Thus pancreas contains a basic carboxypeptidase (carboxypeptidase B) which exclusively splits off the COOH-terminal basic residues appearing during tryptic hydrolysis, and a carboxypeptidase A which preferentially splits off the COOH-terminal aromatic residues resulting from previous chymotryptic digestion. Like carboxypeptidase A, carboxypeptidase B exists in pancreas and pancreatic juice in the form of an inactive precursor, procarboxypeptidase B. Bovine procarboxypeptidase B has been purified from pancreatin extracts (50). However, after a twenty-fold purification according to the technique described, the preparation still contains large amounts of chymotrypsinogen B ( 5 2 ) .Final purification must therefore involve chromatography as well as fractional precipitations and extractions. Porcine procarboxypeptidase B presented in the chromatographic diagram of Fig. 2 has not yet been further characterized, though an apparently good purification technique is now available for porcine carboxypeptidase B (53). This technique includes water extraction of an acetone powder of autolyzed pancreas, fractionation with ammonium sulfate, and chromatography on DEAE-cellulose columns eluted with a linear gradient of 0.0 to 0.2 M NaCl in 0.005 M Tris buffer, pH 7.5. It is quite significant that all procedures recently described for the purification of pancreatic proteases are based upon some preliminary fractionation with ammonium sulfate and a highly efficient ion-exchange chromatography under suitable conditions. When purified in this way, porcine carboxypeptidase B is electrophoretically homogeneous in a 0.05 2cI phosphate buffer, pH 7.02. Sedimentation-

176

P. DESNUELLE AND M. IE0VEH.Y

diffusion gives for its molecular weight the same value as for bovine carboxypeptidase A, namely about 34,000. It also contains one zinc atom per mole (53) which can be removed and replaced by other metal ions. I n the case of carboxypeptidaseB also, the natureof the metal bound to the enzyme appears to influence the ratio peptidase activity: esterase activity. Incubation with cobalt enhances peptidsse activity and incubation with cadmium TABLE IV Aniino A c i d CoinpositiorL of Bovine liarboxypeptidase A and Porcine Carboxypeplidase B Number of residues per 34,400 gm of: Amino acid

Bovine carboxypeptidase A (141)

Alanine Arginine Aspartic acid Cystine (half) Glutamic acid Glycine Histidine Isoleucine Leucine Lysine Methioiiine Phenylalttniiie Proline Rerine Threonine Tryptophan Tyrosine Valine

20.0 10.0 30.3 4.0 25.0 23.2 7.7 20.1 24.7 18.4 1.0 14.9 11 .0 33.1 26.6 6.1 19.7 16.4

25.1 10.0 32.4 7.6 24.8 23.0 5.8 17.2 22.7 17.5 5.1 11 .n 13.2 17.5 30.2 9.2 20.4 10.8

312.2

304.4

-

Porcine carboxypeptidase B (53)

cnhzlnaes csterasc activity (140). Table I V gives the amino acid composition of bovine carboxypeptidase A and porcine carboxypeptidase B.

I . Porcine Lipase The purification and properties of porcine pancreatic lipase have been discussed in a recent review (142). Only the most significant facts related to the protein nature of the enzyme will therefore be presented here. 1 . Purification

After many unsuccessful attempts, porcine lipase has been recently obtained in a satisfactory state of purity. Aqueous extracts of porcine pancre-

177

PROTEINS OF EXOCRINE PANCREAS

atin are extracted with water. The extracts are fractionated by ammonium sulfate and acetone (143) as well as by selective absorption on tricalcium phosphate and aluminium hydroxide (16). The last step of the purification procedure is a zone electrophoresis on starch in a high potential gradient (16, 143). As shown in Fig. 14, an electrophoretically homogeneous protein

3530)

I

40

rnl

FIG.14. Electrophoretic homogeneity of porcine lipase (18). Starch columns (3.0 X 90 cm) equilibrated with 0.025 M acetate buffer, p H 5.25 (diagram on the left), or 0.025 M veronal buffer, pH 8.0 (diagram on the right). Potential gradient inside the column, 8 volts/cm; temperature, +l"C.Elution after 48 hr. Ordinates, per cent in 1ml eluate of the total lipase activity (solid line) and total proteins (dotted line) introduced into the column. The vertical dotted line gives the true origin, with due regard t o the electroosmotic flow. The figures along the peaks give the specific activity of some fractions. Specific activity of the sample introduced into the column, 3500. Abscissas, volume of eluate in milliliters.

is finally obtained after a 135-fold purification with a 20% yield (16). This protein is also homogeneous during chromatography on tricalcium phosphate. An additional proof of purity is given by the fact that the same maximal specific activity is reached, but not surpassed, when purification starts from porcine pancreatic juice (17). The diagram of Fig. 2 suggests that lipase could also be purified by chromatography on DEAF,-cellulose at pH 8.0. A series of other chromatographic techniques are not, siiitable for preparative purposes (144).

178

P. DESNUELLE AND M. ROVERY

Porcine pancreas and porcine pancreatic juice appear to contain a single protein endowed with lipolytic activity. As stated earlier, this protein corresponds to about 2.5 % of the total proteins of the juice. Its molecular activity (turnover number) is likely to be higher than 300,000 under the conditions of the test. Shortage of pure material has thus far prevented any investigation of its molecular properties. It is merely known to be quite soluble in water, to have an isoelectric point of 5.2 in 0.025 M acetate buffer, and to give a conventional protein spectrum. Lipase present in pancreatic juice is likely to be identical with the enzyme extracted from pancreatin. 2. Interactions with Insoluble Substrates and Inhibitors Porcine lipase exhibits a series of unusual properties (142). The most interesting one, directly connected with its structure, is certainly its specific interactions with emulsified esters. When an aqueous solution of lipase at pH 5 is mixed with an excess of a triolein emulsion and when the cream is separated by centrifugation, no lipase can be detected in the clear aqueous lower phase. All activity is in the cream. But it returns at once into water when the emulsion is broken (145). This simple experiment suggests that lipase is adsorbed at the oil/water interface of the emulsion and that this interface may be the normal site of its action. Such an assumption is confirmed by a series of experiments with substrates and inhibitors. a. Substrates. In the usual case of a water-soluble enzyme acting upon a water-soluble substrate in an homogeneous or “microheterogeneous” phase, a fully reversible equilibrium is assumed to exist between enzyme, substrate, and some kind of primary enzyme-substrate complex. This complex is further degraded at a given rate either directly or after an intramolecular “activation.” When substrate concentration is raised, the initial velocity of the reactJionincreases up to a point a t which all or almost all enzyme molecules are converted into the complex. Figure 15 shows that this classic picture does not hold for lipase. Pure lipase does not hydrolyze methyl butyrate and triacetin as long as the esters are dissolved in water. Conversely, lipolysis starts as soon as, by oversaturating the solution, some ester molecules begin to appear in an emulsified state. Thus, pure lipase hydrolyzes ester emulsions, not ester solutions (146). The same fact is demonstrated in a different way by the electrophoretic diagram of Fig. 16. When fractions isolated by zone electrophoresis of an impure lipase preparation are tested on ester emulsions, all the activities are found under a first peak which is the lipase peak. When the same fractions are tested on ester solutions, activities are found under another, wellseparated and more anionic peak (146, 147). The final proof that lipase is acting at the interface itself, and not inside the emulsified globules, is given by the following experiment: A coarser and

I

/+T-

0'1 O 2 0.30.40.5 O 6 d.7 08,i I6

Soluble

nsoluble I

5-

4-

20

3-

2I-

0 5s

1.0s (0.328M)1.5s

2 0s

/I

3 S

2 0 o

0.5s L 0.5s m

,-. A, , c JI.OS(0.153~) 1.5s , ' I.OS(0.153~) 1.5s

,

2,.0s 2.0s

,

I

FIG.15. Exclusive action of porcine lipase on emulsified esters (146). Ordinates: activity in per: cent of maximal activity on triolein emulsion. Abscissaa: lower axis, substrate amounts expressed in fractions of saturation for the solutions (on the left of the vertical dotted line) or in multiples of saturation for the emulsions (on the right of the line); upper axis, interfacial area expressed in lo6X cme in 100 ml. White circles, impure lipase containing some esterases. Black triangles, purified lipase. The substrate is triacetin on the left and methylbutyrate on the right.

180

1’. DESNUELLE AND M. ROVERY

a finer triolein emulsions are prepared. Initial velocities of the reaction induced by the same amount of lipase under the same conditions are plotted in each case against substrate “concentration.” When this concentration is expressed by the weight of the insoluble substrate in a given volume, two widely different curves are obtained. The velocity is much lower, for a

FIG. 16 Electrophoretic separation of the lipase and esterase activities of porcine pancreas (146, 147). Starch columns equilibrated with 0.025 M acetate buffer, p H 5.25. The activities of the fractions have been determined: ( a ) on emulsions of triolein and tributyriri (black circles), methyl oleate, methyl Iaurate, and p-nitrophenyllaurate (black triangles). ( b ) On solutions of methyl butyrate and p-nitrophenylacetate (crosses). White circles and dotted line, protein background. Figures along the first peak give the specific activity (lipase) of some fractions, determined against triolein emulsion. Ordinates and abscissas are the same as in Fig. 14.

givrii Wright, with the coarser emulsion than with the finer one. When

concentration is expressed by the interfacial area in a given volume (“interfacial concentration”), the curves obtained with both emulsions are almost identical. Furthermore, when different emulsions are prepared with the same weight of txiolein, the initial velocity of lipolysis depends upon the interfacial area of the emulsions. I n other words, a Michaelis curve of the ordinary shape can be drawn with lipase for a constant weight of insoluble substrate. It has been definitely established that this curve describes the gradual adsorption of lipase by an interface of increasing area. Then, a

PROTEINS O F EXOCRINE PANCREAS

181

Michaelis contant can be defined for lipase as being that interface which gives to the lipolytic reaction the half of its maximal velocity. This constant is independent of the amount of lipase used (148). b. Organophosphate Inhibitors. Porcine lipase is known to be unaffected by DFP and diethyl-p-nitrophenyl phosphate which inhibit most esterases by combining specifically with their active serine. I n fact, lipase is unaffected by organophosphate solutions. It is quickly and irreversibly inhibited by diethyl-p-nitrophenyl phosphate emulsions in a first-order reaction (149). To sum up, porcine lipase seems to be a very unusual protein which exhibits esterolytic activity when adsorbed a t an oil/water interface. Since no colipase has ever been discovered and since the enzyme itself can be inhibited by organophosphates, it may be postulated as a working hypothesis that lipase contains an esterolytic center built according to the same rules as are the centers of other esterases. The fact that lipase acts exclusively on emulsified esters and is exclusively inhibited by emulsified diethyl-p-nitrophenyl phosphate suggests further that the actual formation of this center is a consequence of interfacial adsorption. I n other words, lipase in solution, like chymotrypsinogen and trypsinogen, would he an inactive protein capable of conversion into an active enzyme by some limited changes in its tertiary structure. Instead of being induced by the splitting of a covalent bond, these changes would result in the case of lipase from interfacial adsorption (149).

J . Bovine Ribonuclease Bovine ribonuclease (150, 151) is a stable protein of relatively low molecular weight which contains no tryptophan. Extensive investigations have been carried out in recent years on its general structure and the origin of its enzymatic activity. However, the question of how many ribonucleases exist in the pancreas of beef and other species is still unsettled. When the crystalline enzyme or a sulfuric acid extract of bovine pancreas is submitted to partition (152) or ion-exchange chromatography on Amberlite IRC-50 (153), a major (ribonuclease A) and a minor (ribonuclease B) active peak are obtained. When phosphate or sucrose extracts of bovine pancreas are chromatographed on Amberlite IRC-50, three active peaks appear (154). Two of them are probably formed by ribonucleases A and B. Acid treatment of the third one converts it into the others and increases activity. Finally, chromatography of crystalline bovine ribonuclease on CM-cellulose in phosphate (155) as well as in univalent (156) buffers yields four active peaks. Recent investigations have been mostly performed on chromatographically homogeneous ribonuclease A.

182

P. DESNUELLE AND M. ROVERY

1. Amino Acid Arrangement and Location of the Disulfide Bridges

Amino acid arrangement and location of disulfide bridges in bovine ribonuclease have been completely elucidated in the last few years (157) by using a number of experimental approaches similar to those described by Sanger 10 years ago and employed by him for the determination of the covalent structure of insulin. This achievement is especially noteworthy since the single chain of ribonuclease contains 124 residues, that is to say, more than four times as much as the longer chain of insulin. Highly refined techniques were therefore required among which chromatography on cation-exchanger columns, instead of paper chromatography and electrophoresis, played a major role. Peptides were separated by manual chromatography and their amino acid composition was established by the use of an automatic amino acid analyzer. In a first series of experiments (158), performic acid-oxidized ribonuclease was digested by trypsin, chymotrypsin, or pepsin. The peptides arising during tryptic hydrolysis were taken as building stones of the whole sequence. Some of them could not be fully analyzed a t once, but all could be assembled together in the right order by taking advantage of “overlapping” sequences found in chymotryptic and peptic hydrolyzates. Then, the structures of the larger tryptic peptides were elucidated (159) and the complete sequence (160) was finally written as indicated in Fig. 17. It must be clearly realized that each shorter or larger peptide had to be obtained in pure form by chromatography and then analyzed. For the larger peptides, analysis required further breakdown and chromatographic fractionations. For the smaller ones, it required full elucidation of the structures by end-group methods and stepwise degradation by carboxypeptidase, aminopeptidase, and thiohydantoin cyclization. But the most striking point in the work is perhaps that yields were always determined, in order to see whether the sequence under investigation belonged to a single molecular entity, to a possible impurity, or to molecules derived from the principal components by some biosynthetic fluctuations around an average model. Yields were sometimes excellent. They could always be considered as satisfactory when unavoidable experimental losses were taken into account. All peptides were fully analyzed and found t o fit into the unique sequence of Fig. 17. To the best of our knowledge, therefore, the ribonuclease used appears to be a chemically homogeneous protein preparation, that is to say, a preparation in which all the molecules have the same covalent structure. This important observation suggests that the residue arrangement may be strictly determined even along a relatively large peptide chain and that, a t least in the case considered here, no fluctuation leading to a somewhat confusing microheterogeneity is taking place during biosynthesis of a given protein. Moreover, ribonuclease is the first enzyme for which full structural

F

-

-

rNH2

-.

Ala-Ala~Lys-Phe-Glu-ArqSer -Thr 5er-5er -Asp -His-Met-Glu-Ala-Ala

Thr

m

11 FIG.17. Amino acid arrangement and disulfide bridges in bovine ribonuclease (157). The arrows indicate the direction of the chain starting from the amino end. The heavy black links represent disulfide bridges.

184

P. DESNUELLE AND M. ROVEItY

information is available at the covalent level. The case of an enzyme is especially interesting since, as already stated, enzymatic activity is likely to be associated with the existence of a catalytic center. The results dewribed above prove that not only the nucleolytic center but also the entire ribonuclease molecule may have a predetermined structure. This does not mean of course that chemically different molecules cannot have the same activity, but that the residue arrangement may be strictly controlled well outside thc enter.^ Some years ago, Sanger also developed an elegant technique for locating the disulfide bridges of a protein. It includes partial degradation of the intact molecule, separation of the cystine peptides, and identification of the two cysteic acid peptides arising during performic acid oxidation of each cystine peptide. However, when insulin was submitted to degradation by enzymes and acid, more cystine peptides were formed than could be expected from the number of cystine residues. The reason for this anomaly is that disulfide interchanges are taking place during the degradation step leading to cystirie peptides. At least two types of reactions are involved, one inhibited by thiols in strong acid and the other catalyzed by the same thiols in neutral or alkaline solutions (161). In the last case, interchange is likely to be initiated by the well-known hydrolytic fission of disulfides. RiSSRl

+ OH- 2 RiS- + RISOH

As so011 as R& ailions are formed, the propagatioii of thc rcactioi: is induced hy tho following cyuilihris: 1tzSSlL II3SSRa

+ It&

+ It&

;2

RlSSjHL

2 RzSSR3

+ R,S-

+ It&,

etc.

At neutral or alkaline pH’s, interchanges are slowed down when the RSform is blocked by sulfhydryl reagents such as N-ethylmaleinimide or p chloromercuribenzoate. All disulfide bridges of insulin have been correctly located in this way (162). Unfortunately, the reaction of the RS- form drives the first equilibrium low-ard the right and consequently decreases the amount of the primitive disulfide bridges. Some bridges appear to be quite labile under these circumstances and their recovery is very low. This may lead to serious mistakes. Rovinr ribonurlease contains eight half-cystine residues which are Recent investigations at the National Hetlrt Institute, Bethesdtl, Maryland, and the Rockefeller Institute for Medical Research, New York, indicate that the sequence of ribonuclease involving residues 11 through 18 is incorrect as shown in Fig. 17. The correct sequence will be published shortly by both of the laboratories (personal communication from C. B. Anfinsen).

PHOTEINS OF EXOCRINE PANCREAS

185

numbered from I to VIII starting from the amino end of the chain. Two bridges (I-VI and IV-V) can be determined after degradation by subtilisin (163). The other two (II-VII) and III-VIII) are apparently destroyed or isomerized under these conditions. But they are found in 2 0 4 7 % yield after degradation by pepsin a t pH 2 followed by a short hydrolysis with trypsin and chymotrypsin (157). Pepsin seems to be the enzyme of choice for starting the degradation, since it is active a t slightly acidic pH’s, its specificity is broad, and it attacks proteins without requiring the help of a denaturing agent which may further labilize the bridges. A three-dimensional model of the ribonuclease molecule would be more significant than the two-dimensional representatiqn of Fig. 17. However, it may be noted that the ribonuclease structure is tightly coiled. Five parts may be roughly distinguished in the chain: two tails at both ends (NHZterminal tail, 25 residues; COOH-terminal tail, 14 residues) which are free of any S-S bridges, and three rings closed by S-S bridges. Eight residues are found in the smaller ring, as compared with six in the case of insulin, vasopressin, and oxytocin. The size of the larger rings (28 and 34 residues, respectively) is of the same order as in insulin (27 residues). 2. Correlation between Structure and Activity a. The Tertiary Structure. Complete elucidation of the covalcnt structure

of an active protein may appear as not very rewarding. No correlation can hr established immediately brtween structure and activity. This fact is not

srirprisirig in the casr of insulin and other hormones since the exact mode of action of this class of substances is still unknown. But catalysis induced by enzymes can sometimes be expressed in molecular terms and it is probably displayed by a restricted part of the molecule. Hence, the situation is more hopeful for enzymes than for any other active proteins thus far investigated. Enzymatic activity, however, is not merely associated with covalent structures, but chiefly with tertiary structure which is still more difficult to determine. The crucial role of tertiary structure is proved by the fact that denaturation brings about inactivation. Even with proteins which may be reversibly denatured, such as chymotrypsin and trypsin, activity is lost as long as denaturation persists. Ribonuclease appeared for a while to be an exception, since it was still active in 8 M urea. But it was shown later that phosphate ions, a t a concentration as low as 0.003 M , and polyphosphates induced in urea-denatured ribonuclease spectral changes usually associated with refolding (164). It could then be assumed that ribonucleic acid, the actual substrate, was also able to refold the denatured form and prevent inactivation in this way. In other words, even in ribonuclease, the active center is probably not built by adjacent residues in a tail or a ring, but by some residues correctly located in space by the superimposed

186

P. DESNUELLE AND M. ROVERY

tertiary structure. About 15-41 % of the chain is present in an a-helical configuration in the native enzyme (165, 166). Most of this configuration is lost when the protein is denatured. Precise studies have been carried out in order to correlate ribonuclease inactivation and denaturation. The rate of ribonucleic acid hydrolysis normally increases with temperature up to 50-55°C. Its slower increase and later its decrease at higher temperatures suggest that the enzyme is inactivated afterward (167). Temperatures of 50-55°C correspond exactly t o the range in which, as shown by intrinsic viscosity and optical rotation determinations, the protein starts to unfold (168). All the observations concerning the opposite phenomenon, that is to say, the activation of an inactive precursor (see above), are also consistent with the view that active centers are dependent on tertiary structure. It must be recognized that the role played by tertiary structure complicates to a great extent the already heavy task of protein chemists. However, the full elucidation of the covalent structure of an enzyme is of fundamental importance for many reasons. It represents the first and compulsory step toward a better understanding of the tertiary structure. It allows also a closer chemical approach to the problem of enzyme inactivation and activation. The only condition is to keep in mind that, however specific and local at first sight, any covalent change may exert long-range actions on the tertiary structure. Several examples are found in the older literature of enzymes which are inactivated as soon as some of their “unreactive” groups begin to be involved in a chemical reaction. When unreactive groups are forced to react, the tertiary structure of the protein is likely to be modified and the inactivation may be attributed to this modification (169). b. The NH2-Terminal Sequence. When bovine ribonuclease A is incubated with crystalline subtilisin a t 3°C and p H 8.0, a single bond is split without impairing activity (170-173). Chromatography of the digestion mixture shows the presence of about 10% of the original enzyme and 70% of another active protein (RNase-S) moving more slowly on Amberlite IRC50. This latter is certainly different from ribonuclease since it contains an additional NHz-terminal serine residue, is quickly inactivated by trypsin, and does not display the characteristic activity in 8 M urea. But it has the same specific activity in water against a series of specific substrates, the same amino acid composition, and the same heat stability. It cannot be fractionated by dialysis against water, chromatography, and ammonium sulfate fractionation. Thus, the simplest idea is to suppose that a peptide bond has been split in a ring and that ribonuclease has been converted into a new active protein with two open chains bound by S-S bridges. However, when the digestion mixture is precipitated by trichloroacetic acid or is dialyzed against 8 M urea, a protein (S-protein) and a peptide (S-peptide) are separated. The peptide contains 20 residues which cor-

PROTEINS OF EXOCRINE PANCREAS

187

respond exactly to the first 20 residues of the NHz-terminal sequence of ribonuclease. Both parts are inactive when taken separately. But their mixture in equimolar amounts (RNase-S’) is almost as active as the original enzyme. It appears therefore that subtilisin splits in quite a specific way the twentieth bond linking an alanine and a serine residue in the KHzterminal tail of ribonuclease and that the liberated peptide remains tightly bound to the rest of the molecule. This latter, containing 104 residues, is not active by itself, but it is activated by its close association with the peptide. Although no rational interpretation can be presented for the time being, these results deserve the utmost attention. Why does subtilisin, known for its broad specificity, split a single bond in ribonuclease under a given set of conditions? Why does this splitting actually inactivate the molecule? Why does trypsin become able to attack the chain after this primary splitting? Why is the S-peptide so tightly bound to the S-protein in a way reminiscent of the association between neurophysin and the two hormonal peptides vasopressin and oxytocin (174)? How finally can inactive S-protein containing 104 residues become an active ribonuclease through its association with the S-peptide? An experimental approach to the last two problems is to modify the S-peptide groups and to determine the influence of these modifications on the strength and activity of the association. Several derivatives of S-peptide (la-hydroxy ; lc-, 7ediguanidino; la-, 16-, 7etriguanidino; la-acetamido-1 e, 7e-diguanidino; la-, le-, 7e-triacetamido; tetramethyl ester; sulfonium or sulfone derivatives of methionine on position 17) have been prepared. Their ability to associate with S-protein and to form with it an active enzyme have been investigated (175-177). Two possible explanations of the activating effect of the peptide may be considered. First, the peptide may contain a part of the active center and the association of peptide-protein may be so intimate that the whole organization of the center is retained even after the splitting of the twentieth bond. Second, all parts of the center may be present in the rest of the protein and its configuration, by itself labile, may be stabilized or even reformed by the peptide. c. The COOH-Terminal Sequence. Ribonuclease is very quickly inactivated by pepsin. Inactivation runs parallel with the appearance of the COOH-terminal tetrapeptide Asp.Ala.Ser.Va1 (178, 179). Pepsin action is probably more complex than the specific splitting induced by subtilisin. Nevertheless, an inactive preparation with the same sedimentation constant as ribonuclease and having no additional NHz-terminal residue can be obtained in high yield by chromatography of the peptic digest. It seems therefore quite possible that one or several of the four last residues of the chain have something to do with activity. d. Speci$ic Reactions with Iodo- or Bromoacetate. Ribonuclease is easily

188

P. DESNUELLE AND M. ROVERY

inactivated by iodoacetate (180) and bromoacetate (181).The first interest of this observation is to show that reagents thought to be specific for protein SH groups and even used sometimes for defining the sulfhydryl character of some enzymes may act on and inactivate enzymes devoid of SH groups. Many investigations carried out on the so-called sulfhydryl enzymes with inappropriate techniques and unspecific reagents are of little significance. The very unusual pH-dependence of ribonuclease inactivation by iodoacetate led to a careful study of the groups involved at various pH’s (180). The technique was to identify the carboxymethylated residues by chromatography after hydrolysis of the modified protein. It was shown in this way that lysine is mostly involved a t higher pH’s and methionine a t acidic pH’s. After reaction at pH 5.5-6.0, a chromatographically homogeneous and inactive protein was isolated which differed from riboriucdease only by carboxymethylation of a single histidine residue (180). By using C14-labeled bromoacetate, it was confirmed that total inactivation was brought about by alkylation of a single histidine residue arid it was shown that this residue is the one hundred and ninteerith one in the peptide chain (182). This beautiful correlation between alkylation of a single group and inactivation can be compared with the one existing between phosphorylation of one serine residue and inactivation of DFP-sensitive esterases. It raises the question of the unusually high reactivity of the group concerned and the role played by this group in the catalytic center. The crucial importance of the tridimensional environment on the reactivity of protein groups is again stressed by the fact that the histidine residue is alkylated a t a normal rate in denatured ribonuclease and that, in contrast, the reactivity of the methionine residues is enhanced by denaturation (183). A similar situation is met with subtilisin-modified ribonuclease. In the inactive, presumably denatured S-protein methionine residues are easily alkylated a t pH 6.0 by iodoacetate and the resulting derivative can no longer bind the S-peptide. But, alkylation of the presumably native association Sprotein S-peptide yields large amounts of carboxymethylhistidine (184). I n other words, the time-honored concept according to which denaturation increases group reactivity by unfolding the chain(s) is valid for “ordinary” groups. There exists some groups involved in the active center aiid some bonds involved in the activation of precursors to which a special coiling or folding of the native chain gives a very high reactivity. This reactivity actually decreases when the native configuration is lost. It is also interesting to note that the one hundred and ninteenth histidine residue and the Phe-Asp bond split by pepsin (see above) are located very close to each other in the COOH-terminal tail of ribonuclease. Inactivation occurs readily when this part of the molecule is modified.

+

PROTEINS O F MXOCHINE PANCREAS

189

e. Reduction and Reoxidation. It has been known for several years that the four disulfide bridges of ribonuclease are split by reductive agents to give an inactive product and that the extent of inactivation roughly parallels the extent of reduction as measured by the number of SH groups. Thioglycolic acid has now been shown to contain polythioglycolides formed by polymerization of thioesters. These polymers are able to react with the protein amino groups (thiolation) and thus to alter significantly the results due to reduction alone. Freshly distilled thioglycolic acid or mercaptoethanol must therefore be used (185). New technical refinements have been described for the full reduction of ribonuclease by mercaptoethanol (186). They include treatment of the protein at p H 8.6 in 8 M urea and elimination of the low molecular weight compounds by passage through Sephadex G-25 equilibrated with 0.1 M acetic acid. The SH groups are fairly stable in dilute acetic acid solution. They may be s t a b b e d further by specific alkylation under well-selected conditions or reaction with p-chloromercuribenzoate. In this latter case, SH groups may be liberated again at will and the reduced protein again obtained in a pure state by mercaptoethanol treatment and chromatography on Sephadex. It had already been pointed out 2 years ago that reduced ribonuclease regains some activity when exposed to air at p H 8.0 (187). An 80-100% reactivation has now been achieved by avoiding thiolation and surface denaturation by air bubbling. (185, 186). This reactivation is very interesting since random combination of eight SH groups to form four disulfide bridges may be expected to give 105 isomers (188). Thus, the question arises whether all the possible isomers of reoxidized ribonuclease have the same activity, or whether the original isomeric form is almost entirely regained during oxidation. The latter assumption seems to be substantiated by a comparative study of native and reoxidized ribonuclease. No difference can be detected in the chromatographic behavior of both substances, fingerprinting of cystine peptides formed by subtilisin degradation, isoionic point, optical rotation, intrinsic viscosity, immunological properties (189), and geometry of the molecules as revealed by X-ray diffraction (190). Some minor changes, however, are noted between the ultraviolet spectra of' both proteins and between their ability to give crystals of high quality. Final proof of the identity of disulfide bridges in native and reoxidized ribonuclease will probably be given by a closer study of the cystine peptides arising during pept]ic degradation (157) of both proteins. The results already obtained suggest that the primary structure, that is to say, the amino acid arrangement along the ribonuclease chain, fully determines a unique mode of attachment of half-cystine residues, as well as a unique helical coiling and folding of the chain. The necessary information for the

190

P. DESNUELLE AND M. ROVERY

determination of these structures seems to be contained in the first 104 residues] since reoxidized S-protein is activated by S-peptide (191).

K . Sheep and Mouse Ribonucleases As already pointed out earlier, it is important to purify the same enzyme or the same precursor from several species in order to compare the general structure of the molecules, the structures of their active centers, or the mode of activation of the precursors. Chromatography of sulfuric acid extracts of sheep pancreas on CM-cellulose equilibrated with 0.01 M phosphate buffer, pH 6.0 gives an initial active peak near the solvent front and then four other peaks when a concentration and pH gradient is applied to the column (155). These four peaks may be further purified under the same conditions and they behave in exactly the same way during repeated chromatography. Therefore they probably represent four different sheep ribonucleases which have been numbered I, 11, 111, and IV. The behavior of the first peak is more complex. When passed through a DEAE-column, it gives rise to one major, one minor, and two very small peaks. But, after passage through Amberlite IRC-50, very little active material is found at the original position on CM-cellulose. Most of the activity migrates as ribonucleases I, 11, and 111. The first peak is perhaps formed by an association of the already known ribonucleases with some acidic compound which becomes dissociated on Amberlite. Evidence is available in the case of crystalline beef (156) and mouse ribonucleases (154) that this compound may be a polynucleotide. All sheep ribonucleases I to IV have nearly the same specific activity and the same isoionic point which is very similar to the value found for beef ribonucleases A and B. The sequences in beef ribonuclease A and sheep ribonuclease I11 have been compared by the fingerprint technique (192). Only thrce specific differences are seen: substitution of a threonine by a serine a t position 3, substitution of a lysine by a glutamic acid a t position 37, and a still undetermined modification probably located between positions 99 and 104. Sulfuric acid, phosphate] and sucrose extracts of mouse and beef pancreas have also been compared chromatographically on Amberlite IRC-50 (154). For both species, the sulfuric acid extracts give two peaks which correspond to ribonucleases Aand B. When prepared from supernatant free of zymogen granules and microsomes, phosphate and sucrose preparations give a third peak, (10-15 % of the total activity in the case of mouse; 1% in the case of beef) which can be converted into the previous ones by acid treatment. This treatment induces a two- to three-fold increase in activity and the dissociation of the acidic compound already referred to above. The chemical structure of mouse ribonuclease(s) is still unknown.

PROTEINS OF EXOCRINE PANCREAS

19L

REFERENCES 1. Keller, P. J., Cohen, E., and Neurath, H. (1958).J . B i d . Chem. 233, 344. 2. Jorpes, J. E.,and Mutt, V. (1955). Acta Chem. Scand. 9, 1040. 3. Mutt, V.,and Werner, B. (1954).Scand. J. Clin. & Lab. Invest. 6 , 228. 4. Keller, P. J., Cohen, E., and Neurath, H. (1959). J. B i d . Chem. 234, 311. 5. Morris, A. J., and Dickman, S. R. (1960).J . B i d . Chem. 236, 1404. 6. Anfinsen, C. B., and Redfield, R. R. (1956).Advances i n Protein Chem. 11. 1. 7. Neurath, H. (1957).Advances i n Protein Chem. 12, 379. 8. Hirs, C. H. W. (1953). J. B i d . Chem. W6, 93. 9. Lewis, U.J., Williams, D. E., and Brink, N. G. (1956).J . Biol. Chem. 222, 705. 10. Lewis, U.J., and Thiele, E. H. (1957). J . Am. Chem. SOC.79, 755. 11. Weil, L.,and Telka, M. (1957).Arch. Biochem. Biophys. 71,204. 12. Grant, N. H., and Robbins, K. C. (1957).Arch. Biochem. Biophys. 66, 396. 13. Marchis-Mouren, G.(1959).Sc. D. Thesis, University of Marseille. 14. Marchis-Mouren, G., Charles, M., Ben Abdeljlil, A., and Desnuelle, P. (1961). Biochim. el Biophys. Acta 60,186. 15. Fisher, E.H.,and Bernfeld, P. (1948).HeZv. Chim. Acta, 32, 1831. 16. Marchis-Mouren, G.,Sarda, L., and Desnuelle, P. (1959). Arch. Biochem. Biophys. 83. 309. 17. Marchis-Mouren, G.,Sarda, L., and Desnuelle, P. (1960). Biochim. et Biophys. Acta 41, 358. 18. Moore, B. W., and Lee, R. H. (1960). J. B i d . Chem. 236, 1359. 19. Smith, B.W., and Roe, J. H. (1949).J . B i d . Chem. 179, 53. 20. Hokin, L.E.(1951). Biochem. J . 60,216. 21. Dickman, S. R.,Aroskar, J. P., and Kropf, R. B. (1956). Biochim. et Biophys. Acta 21, 539. 22. Reboud, J. P., Ben Abdeljlil, A., and Desnuelle, P. Unpublished experiments. 23. Fisher, E. H., and Bernfeld, P. (1948).Helv. Chem. Acta 31, 1834. 24. Palade, G.E.,and Siekevitz, P. (1956).J. Biophys. Biochem. Cytol. 2 , 171. 25. Siekevitz, P., and Palade, G. E. (1958).J . Biophys. Biochem. Cytol. 4, 309. 26. Siekevitz, P.,and Palade, G. E. (1958).f. Biophys. Biochem. Cytol. 4, 203. 27. Palade, G.E.(1959). In “Subcellular Particles” (T. Hayashi, ed.), p. 64.Ronald Press, New York. 28. Heidenhain, R. (1875). Arch. ges. Physiol. Pjluger’s 10, 557. 29. Weiss, S.B., ACE,G., and Lipman, F. (1958).Proc. Natl. Acad. Sci. U.S . 44,189. 30. Siekevitz, P., and Palade, G. E. (1958).J . Biophys. Biochem. Cytol. 4, 557. 31. Siekevitz, P.,and Palade, G. E. (1959).Federation Proc. 18, 324. 32. Siekevitz, P., and Palade, G. E. (1960).J . Biophys. Biochem. Cytol. 7 , 619. 33. Hirs, C.H.W., Moore, S., and Stein, W. H. (1953).J. B i d . Chem. 2C0, 493. 34. Hokin, L. E.(1955).Biochim. et Biophys. Acta 18,379. 35. Cohen, E.,and Keller, P. J. (1960).Federation Proc. 19, 345. 36. Keller, P. J., and Cohen, E. (1961).J. Biol. Chenz. 236, 1407. 37. Greene, L.J. (1960).Federation Proc. 19, 132. 38. Marshall, J. M. (1954). Exptl. Cell Research 6, 240. 39. Van Lancker, J. L., and Holtzer, R. L. (1959). J. B i d . Chem. 234, 2359. 40. Dickman, S.R.,and Morrill, G. A. (1959).Ann. N. Y.Acad. Sci. 81, 585. 41. Laskowski, M., and Laskowski, M., Jr. (1954). Advances i n Protein Chem. 9,203. 42. Northrop, J. H., Kunitz, M., and Herriott, R. (1948). “Crystalline Enzymes,” 2nd ed., Columbia Univ. Press, New York.

192

P. DESNUELLE AND M. ROVERY

43. Desnuelle, P., and Rovery, M. (1956). Proc. Intern. Conyr. Biochern. 3rd Congr., Brussels, 1966, p. 78. 44. Desnuelle, P., and Rovery, M. (1957). In “Symposium on Protein Structure” (A. Neuberger, ed.), p. 155. Methuen, London. 45. Dixon, G. H., Neurath, H., and Pechkre, J. F. (1958).Ann. Rev. Biochem. 27,489. 46. Keith, C. K., Kazenko, A., and Laskowski, M. (1947). J. B i d . Chem. 170, 227. 47. Keller, P. J., Cohen, E., and Neurath, H. (1956). J. Bzol. Chem. 223,457. 48. Keller, P. J., Cohen, E., and Neurath, H. (1958). J . Biol. Chem. 230, 905. 49. Folk, J. E. (1956). J. A m . Chem. SOC.78, 3541. 50. Folk, J. E., and Gladner, J. A. (1958). J. B i d . Chem. 231, 379. 51. Gladner, J. A., and Folk, J. E. (1958). J . Biol. Chem. 231, 393. 52. Pechkre, J. F., Dixon, G. H., Maybury, R. H., and Neurath, H. (1958). J. Biol. Chem. 233. 1364. 53. Folk, J. E., Piee, K. A., Carroll, W. R., and Gladner, J. A. (1960). J . Biol.Chem. 236, 2272. 54. Miller, K. D. (1958). Federation Proc. 17, 276. 55. Alkjaersig, N., Fletcher, A. P., and Sherry, S. (1958). J . Biol. Chem. 233, 81. 56. Yamashina, I. (1956).Arkiv. Kemi 9,225. 57. Yamashina, I. (1956). Biochim. et Biophys. Acta 20,433. 58. Grossman, M. I., Greengard, H., and Ivy, A. C. (1942). A m . J. Physiol. 138, 676. 59. Grossman, M. I., Greengard, H., and Ivy, A. C. (1944). A m . J . Physiol. 144.38. 60. Desnuelle, P., Reboud, J. P., and Ben Abdeljlil, A. (1961). Ciba Foundation Symposium Exocrine Pancreas (in press). 61. Junqueira, L. C. U., Hirsch, G. C., and Rothschild, H. A. (1955). Biochem. J . 61, 275. 62. Keller, P. J., Cohen, E., and Neurath, H. (1961). J. Biol. Chem. 236, 1404. 63. Jacobsen, C. F. (1947). Compt. rend. trav. lab. Carlsberg, Sbr. chim. 26, 325. 64. Desnuelle, P. (1960). I n “The Enzymes” (P. D. Boyer, H. Lardy, and K. Myrback, eds.), 2nd ed., Vol. IV, p. 93. Academic Press, New York. 65. Rovery, M., Poilroux, M., Yoshida, A., and Desnuelle, P. (1957). Biochim. el Biophys. Acla 23.608. 66. Bettelheim, F. R. (1955).J . Biol. Chem. 212,235. 67. Rovery, M., Gabeloteau, C., de Vernejoul, P., Guidoni, A., and Desnuelle, P. (1959). Biochim. et Biophys. Acta 32,256. 68. Pechhre, J. F., Dixon, G. H. Maybury, R. H., and Neurath, H. (1959). Biochim. el Biophys. Acta 31,259. 69. Meedom, B. (1956). Acta Chem. Scand. 10, 150. 70. Meedom, B. (1956).Acta Chem. Scand. 10, 881. 71. Davie, E. W., arid Neurath, H. (1955). J . Biol. Chem. 212, 515. 72. Desnuelle, P., and Fabre, C. (1955).Biochim. el Biophys. Acta 18, 49. 73. Neurath, H., Rupley, J. A., and Dreyer, W. J. (1956). Arch. Biochem. Biophys. 66, 243. 74. PechBre, J. F., and Neurath, H. (1957). J . Biol.Chem. 229, 389. 75. Rovery, M., and Desnuelle, P. Unpublished experiments. 76. Hartley, B. S. (1960). Ann. Rev. Biochem. 20, 45. 77. Neurath, H., and Dixon, G. H. (1957). Federation Proc. 16,791. 78. Meedom, B. (1958). Biochim. et Biophys. Acta 30, 429. 79. Rovery, M., Dinh Van Hoang, and Desnuelle, P. Unpublished experiments. 80. Cecil, R., and McPhee, J. R. (1955). Biochem. J . 60,496. 81. Bailey, J. L., and Cole, R. D. (1959). J . Biol. Chem. 234, 1733. 82. Swan, J. N. (1957). Nature 180, 643.

PIEOTEINS O F EXOCRINE PANCREAS

193

83. Spackman, D. H., Stein, W. H., and Moore, S. (1958). Anal. Chem. SO, 1190. 84. Wilcox, P. E., Cohen, E., and Tan, W. (1957). J . Biol. Chem. 228,999. 85. Rovery, M., Charles, M., Guy, O., Guidoni, A., and Desnuelle, P. (1960). Bull. S O C . chim. biol. 42, 1235. 86. Rovery, M., Guy, O., Maroux, S., and Desnuelle, P. Unpublished experiments. 87. Kaasel, B., and Laskowski, M. (1961). J. Biol. Chem. 236,1996. 88. Smith, E. L., Brown, D. M., and Laskowski, M. (1951). J. Biol. Chem. 191, 639. 89. Oosterbaan, R. A., Kunst, P., Van Rotterdam, J., and Cohen, J. A. (1958). Biochim. et Biophys. Acta 27, 556. 90. Hartley, B. S. (1958). Biochem. J. 70, 4P. 91. Hartley, 13. S. (1958). Proc. Intern. Congr. Biochem., 4lh Congr., Vienna, Vol. 8, p. 87. V2. Hartley, B. S. (1959). J. Cellular Comp. Physiol. 64, 203. 93. Hartley, B. S. (1961). Proc. Intern. Congr. Biochem., bth Congr., Moscoiu (in press). 94. S h m , F., Keil, B., Holeyiiovskf, V., Meloun, B., Mikeb, O., and VanBbek, J. (1958). Collection Czechoslov. Chem. Commun. 23, 985. 95. Keil, B., and Sbrm, F. (1959). Collection Czechoslou. Chem. Commun. 24, 1558. 96. VanBEek, J., Keil, B., Meloun, B., and Sbrm, F. (1959). Collection Czechoslov. Chem. Commun. 24. 3148. 97. Meloun, B., Holeyiiovskf, V., VanGek, J., Keil, B., and S6rm, F. (1959). Colteclion Czechoslov. Cham.. Clom,mun 24.3002. 98. VanBbek, J., Meloun, B., Kostka, V., Keil, B., and Sbrm, F. (1960). Biochim. et Biophys. Acta 97, 169. 99. Tom&Bek, V., Holeybovskf, V., Mikeg, O., and Sbrm, F. (1960). Biochim. et Biophys. Acta 38. 570. 100. Meloun, B., VanBbek, J., Prusik, Z., Keil, B., and S6rm, F. (1960). Collection Czechoslov. Chem. Commun. 26, 571. 101. Keil, B., Sbrm, I?., HoleyRovskf, V., Kostka, V., Meloun, B., Mikeb, O . , Torn&Bek, V., and VanhEek, J. (1959). Collection Czechoslov. Chem. Commun. 24, 3491. 102. Kassel, B., and Laskowski, M. (1960). Federation Proc. 19,332. 103. Kassel, B., and Laskowski, M. (1957). Abstr. A m . Chem. SOC.1Slst Meeting, 6812. 104. Kassel, B. (1959). Federation Proc. 18, 257. 105. Rovery, M., Guy, O., and Desnuelle, P. (1960). Biochim. et Biophys. A d a 42,554. 106. Guy, 0. Unpublished experiments. 107. Gabeloteau, C., and Desnuelle, P. (1957). Arch. Biochem. Biophys. 69,475. 108. Gabeloteau, C., and Desnuelle, P. (1960). Biochim. et Biophys. A d a 42, 230. 109. Hofmann, T. (1960). Biochem. J. 74, 41P. 110. Hofmann, T. (1960). Biochem. J . 76, 26P. 111. Wootton, J. F., and Hess, G. P. (1958). Biochim. et Biophys. Acta 29, 435. 112. Viswanatha, T., Wong, R. C., and Liener, I. E. (1958). Biochim. et Biophys. A d a 29, 174. 113. Liener, I. E., and Viswanatha, T. (1959). Biochim. el Biophys. Acta 36. 250. 114. Tallan, H. H. (1958). Biochim. et Biophys. Acta 27, 407. 115. Liener, I. E. (1960). Arch. Biochem. Biophys. 88, 216. 116. Maroux, S., Rovery, M., and Desnuelle, P. Unpublished experiments. 117. Harris, J. I. (1959). Biochem. J. 71, 434. 118. Ravin, H. A., Bernstein, P., and Seligman, A. M. (1954). J. Biol. Chem. 208, 1. 119. Hofstee, B. H. J. (1957). Biochim. el Biophys. Acta 24, 211. 120. Martin, C., Golubow, J., and Axelrod, A. E. (1958). Biochim. et Biophys. Acta 27, 430.

194

P. DESNUELLE AND M. ROVERY

121. Martin, C., Golubow, J., and Axelrod, A. E. (1959). J. Biol. Chem. 234, 1718. 122. Inagami, T., and Sturtevant, J. M. (1960). J . B i d . Chem. 236, 1019. 123. Viswanatha, T., Lawson, W. B., and Witkop, B. (1960). Biochim. et Biophys. Acta 40,216. 124. Green, N. M., and Neurath, H. (1954). I n “The Proteins” (K. Bailey and H. Neurath, eds.), Vol. 11, Part B, p. 1057. Academic Press, New York. 125. Neurath, H., and Dreyer, W. J. (1955). Discussions Faraday SOC.No. 20, 32. 126. Charles, M., Rovery, M., Maroux, S., and Desnuelle, P. Unpublished experiments. 127. Anson, M. L. (1935). Science 81,467. 128. Neurath, H. (1960). I n “The Enzymes” (P. D. Boyer, H. Lardy, and K. Myrback, eds.), 2nd ed., Vol. IV, p. 11. Academic Press, New York. 129. Neurath, H., Brown, J. R., Greenshields, R. N., Keller, P. J., and Yamasaki, M. (1961). Federation Proc. 20, 220. 130. Thompson, E. 0. P. (1953). Biochim. et Biophys. Acta 10. 633. 131. Ando, T., Fujioka, H., and Kawanishi, Y. (1959). Biochim. et Biophys. Acta 34, 296. 132. Vallee, B. L., Rupley, J. A., Coombs, T. L., and Neurath, H. (1958). J. A m . Chem. Soc. 80,4750. 133. Vallee, B. L., Rupley, J. A., Coombs, T. L., and Neurath, H. (1960). J . B i d . Chem. 236, 64. 134. Coleman, J. E., and Vallee, B. L. (1960). J. Biol. Chem. 236, 390. 135. Vallee, B. L., Coombs, T. L., and Hoch, F. L. (1960). J. B i d . Chem. 236, PC45. 136. Coleman, J. E., and Vallee, B. L. (1961): Federation Proc. 20, 220. 137. Folk, J. E., and Gladner, J. A. (1960). J. Biol. Chem. 236, 60. 138. Waldschmidt-Leitz, E., Ziegler, F., Schaffner, A., and Weil, L. (1931). 2.physiol. Chem. Hoppe Seyler’s 197, 219. 139. Weil, L., Seibles, T. S., and Telka, M. (1959). Arch. Biochem. Biophys. 79, 44. 140. Folk, J. E., and Gladner, J. A. (1961). Biochim. et Biophys. Acta 48, 139. 141. Smith, E. L., and Stockell, A. (1954). J. B i d . Chem. 207, 501. 142. Desnuelle, P. Advances i n Enzymol. 23,129. 143. Sarda, L., Marchis-Mouren, G., Constantin, M. J., and Desnuelle, P. (1957). Biochim. el Biophys. Acta 23, 264. 144. Marchis-Mouren, G., Constantin, M. J., and Desnuelle, P. (1958). Bull. SOC. chim. biol. 40, 2019. 145. Sarda, L., Marchis-Mouren, G., and Desnuelle, P. (1957). Biochim. el Biophys. Acta 24, 425. 146. Sarda, L., and Desnuelle, P. (1958). Biochim. et Biophys. Acta 30, 513. 147. Sarda, L., PasBro, L., and Desnuelle, P. Unpublished experiments. 148. Sarda, L., Benzonana, G., and Desnuelle, P. Unpublished experiments. 149. Desnuelle, P., Sarda, L., and Ailhaud, G. (1960). Biochim. et Biophys. Acta 37, 570. 150. Laskowski, M. (1951). In “The Enzymes” (J. B. Sumner and K. Myrback, eds.), Vol. I, Part 2, p. 956. Academic Press, New York. 151. Anfinsen, C. B., and White, F. H., Jr. (1961). In “The Enzymes” (P. D. Boyer, H. Lardy, and K. Myrback, eds.), Vol. V, p. 95. Academic Press, New York. 152. Martin, A. J. P., and Porter, R. R. (1951). Biochem. J . 49, 215. 153. Hirs, C. H. W., Moore, S., and Stein, W. H. (1953). J. Biol. Chem. 200. 493. 154. Dickman, S. R., Morrill, G. A., and Trupin, K. M. (1960). J. B i d . Chem. 236,169. 155. Iqvist, S. E. G., and Anfinsen, C. B. (1959). J. Biol. Chem. 234, 1112. 156. Taborsky, G. (1959). J. B i d . Chem. 234, 2652.

PROTEINS O F EXOCRINE PANCREAS

195

157. Spackman, D. H., Stein, W. H., andMoore, S. (1960). J. Biol. Chem. 236. 648. 158. Hirs, C. H. W., Moore, S., and Stein, W. H. (1958). I n “Symposium on Protein Structure” (A. Neuberger, ed.), p. 211. Methuen, London. 159. Him, C. H. W. (1960). J. B i d . Chem. 236, 625. 160. Hirs, C. H. W., Moore, S., and Stein, W. H. (1960). J. B i d . Chem. 236, 633. 161. Ryle, A. P., and Sanger, F. (1955). Biochem. J. 60, 535. 162. Ryle, A. P., Sanger, F., Smith, L. F., and Kitai, R. (1955). Biochem. J. 60, 541. 163. Ryle, A. P., and Anfinsen, C. B. (1957). Biochim. et Biophys. Acta 24, 633. 164. Anfinsen, C. B.( 1957). Federation PTOC.16, 783. 165. Harrington, W. F., and Schellman, J. (1956). Compt. rend. trav. lab. Carlsberg SBr. chim. 30,21. 166. Yang, J. T., and Doty, P. (1957). J. A m . Chem. SOC.79, 761. 167. Kalnitsky, G., and Resnick, H. (1959). J . Biol. Chem. 234, 1714. 168. Weber, R. E., and Tanford, C. (1959). J. A m . Chem. SOC.81, 3255. 169. Desnuelle, P., and Rovery, M. (1949). Biochim. et Biophys. Acta 3, 2G. 170. Richards, F. M. (1955). Compt. rend. trav. lab. Carlsberg Skr. chim. 29, 329. 171. Richards, F. M. (1958). Proc. Natl. Acad. Sci. U . S . 44, 162. 172. Richards, F. M. (1958). Federation Proc. 17, 296. 173. Richards, F. M., and Vithayathil, P. J. (1959). J . Biol. Chem. 234, 1459. 174. Acher, R., Chauvet, J., and Olivry, G. (1956). Biochim. et Biophys. Acta 22,421. 175. Vithayathil, P. J., and Richards, F. M. (1960). J. Biol. Chem. 236, 1029. 176. Vithayathil, P. J., and Richards, F. M. (1960). J. BioE. Chem. 296,2343. 177. Vithayathil, P. J., and Richards, F. M. (1961).J. Biol. Chem. 236, 1380. 178. Anfinsen, C. B. (1955). Biochim. et Biophys. Acta 17, 593. 179. Anfinsen, C. B. (1956). J. Biol. Chem. 221, 405. 180. Gundlach, H. G., Stein, W. H., and Moore, S. (1959). J . Biol. Chem. 234, 1754. 181. Barnard, E. A., and Stein, W. D. (1959). J. Mol. Biol. 1, 339. 182. Stein, W. D., and Barnard, E. A. (1959). J. Mol. Biol. 1,350. 183. Stark, G. R., Stein, W. H., and Moore, S. (1961). J . Biol. Chem. 236, 438. 1%. Vithayathil, P. J., and Richards, F. M. (1961). J. Bio2. Chem. 236, 1386. 185. White, F. H., Jr. (1960). J. Biol. Chem. 236, 383. 186. Anfinsen, C. B., and Haber, E. (1961). J. Biol. Chem. 236, 1361. 187. White, F. H., Jr., and Anfinsen, C. B. (1959). Ann. N. Y. Acad. Sci. 81, 515. 188. Kauzmann, W. (1959). “Sulfur Proteins” (R. Benesch et al., eds.), p. 93. Academic Press, New York. 189. White, F. H., Jr. (1961). J. Biol.Chem. 236, 1353. 190. Bello, J., Harker, D., and De Jarnette, E. (1961). J . B i d . Chem. 236, 1358. 191. Haber, E., and Anfinsen, C. B. (1961). J. Biol. Chem. 236, 422. 192. Anfinsen, C. B., Aqvist, S. E. G., Cooke, J. P., and Jonsson, B. (1959). J. Biol. Chem. 234. 1118.