Cell Surface Enzymes: Effects on Mitotic Activity and Cell Adhesion

Cell Surface Enzymes: Effects on Mitotic Activity and Cell Adhesion H. BRUCE BOSMANN Department of Pharmacology and Toxicology, University of Rochester Cancer Center, University of Rochester School of Medicine and Dentistry, Rochester, New York I. Introduction . . . . . . . . A. Purpose of This Article. . . . . . B. The Cell Plasma Membrane. . . . . 11. Cell Surface Enzymes and Mitosis . . . . A. Cell Surface Enzymes . . . . . . B. Sublethal Autolysis . . . . . . C. Cell Surface Proteases . . . . . . D. Cell Surface Proteases and Mitosis . . . E. Conclusions . . . . . . . . 111. Cell Surface Enzymes and Cell Adhesion . . A. Cell Adhesion . . . . . . . B. Complementary Macromolecule Hypothesis . C. Cell Surface Charge and Complementary RNA Mechanism . . . . . . . . D. Probable Events in Cell Adhesion . . . E. Cell Surface Enzymes and Adhesion . . . F. Conclusion . . . . . . . . IV. Summary . . . . . . . . . References . . . . . . . . .

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I. Introduction In the last decade, and in particular of late, two interrelated concepts concerned with the cell plasma membrane have emerged. The first is that enzymes and substrates located on the external surface of the plasma membrane of the cell mediate a variety of cellular interactions, including cell-cell adhesion, cell-cell recognition, and cell-cell communication. The second concept is that cell surface enzymes or, more accurately, enzymes active at the external cell surface, mediate genomic events, in particular, cellular mitosis.

A. PURPOSE OF THIS ARTICLE This article is concerned exclusively with the above two concepts, as indicated in the title. Although they are relatively new, several reviews have already appeared. The purpose of this report is to present these concepts and critically examine the evidence for, and implica1

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tions of, these ideas. No attempt is made to be exhaustive; only references deemed significant have been included, and they may be consulted for the other important work that contributed to these theories. B. THE CELL PLASMAMEMBRANE Although many people are familiar with the Danielli-Davson model of the plasma membrane (Danielli and Davson, 1934) and with the fluid mosaic model of Singer and Nicolson (1972), currently in vogue, many intervening models are less well known, such as the micellar model (Lucy, 1968), the subunit model (Green et d., 1967), and the unit membrane model (Robertson, 1966). It should be emphasized that each of these models, including the present one, is just that-a model. The fluid mosaic model, in which cellular membranes are visualized as two-dimensional solutions of oriented globular proteins and lipids, is based on thermodynamic considerations and does not explain all the properties of the various cell membranes (Singer and Nicolson, 1972). Furthermore, although it has been known for a long time that lipid and protein are major membrane components, it has become increasingly clear that complex proteins and lipids and glycoconjugates (glycoproteins, glycolipids, and perhaps glycosaminoglycans) play a pivotal role in the cell membrane (Cook and Stoddard, 1973). Also, although membrane biogenesis is far from clearly understood, current thinking points to similarities among plasma membranes, organelle membranes, and the rough and smooth endoplasmic reticulum with respect to the terminal N-acetylneuraminic acid residues on their surfaces (Gersten et al., 1974), and the plasma membrane is now recognized to contain many enzyme activities. Finally, the plasma membrane of nucleated mammalian cells rapidly turns over its components (Warren and Glick, 1968) and is constantly subjected to capping, phase transition, and rearrangement, as predicted by the fluid mosaic model (Singer and Nicolson, 1972). 11. Cell Surface Enzymes and Mitosis As it becomes clearer that mitosis is in itself a complex event, it becomes evident that the factors influencing mitosis (or, as they are sometimes referred to, growth control factors) are even more complex. Various cases can b e made for a host of growth control factors being involved in influencing mitosis-insulin or insulin-like activity ( Dulak and Temin, 1973; Oka and Topper, 1974; Baseman et aE., 1974), environmental p H (Eagle, 1973), amino acids (Ley and Tobey, 1970; Short et al., 1972; Brunner, 1973; Paul, 1973; Pardee, 1974), microtubules

CELL SURFACE ENZYMES

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(Edelman, 1976), dimerized RNase ( Bartholeyns and Baudhuin, 1976), glucocorticoids ( Thrash and Cunningham, 1973), “serum factors” (Holley and Kiernan, 1971; Fan et al., 1973; Leffert, 1974), cyclic nucleotides (Burk, 1968; Grimm and Frank, 1973; Willingham e t al., 1973; Eker, 1974; Hovi et al., 1974; Nesbittet al., 1976), various ions or concentrations thereof (Rubin, 1973; Rubin and Koide, 1976; Whitfield et al., 1974), folic acid (Balk et al., 1973), polyamines (Pohjanpelto, 1973), disaccharides ( Rheinwald and Green, 1974), linoleic acid ( Holley et al., 1974), phytohemagglutinin in lymphocytes (Romeo et al., 1973; Ruddon et al., 1974), dextran sulfate (Scholnick e t al., 1973; Goto et al., 1973; Clarke et al., 1976), bacterial lipopolysaccharides (Greaves and Janossy, 1972), trypsinized concanavalin A (Trowbridge and Hilborn, 1974), cytosine arabinoside (Hawtry et al., 1973; Yoshikura, 1974);periodate ( Novogrodsky and Katchalski, 1972; Parker et al., 1974; Kent and Pogo, 1974), bromodeoxyuridine (Grady and North, 1974; Meuth and Green, 1974), dimethyl sulfoxide (Kisch et al., 1973; Borenfreund et al., 1974), and many others. I n this article we limit the discussion to the role of cell surface or cell surface active enzymes and mitosis. It should be noted, however, that evidence for cell surface control of division and DNA synthesis is not at all limited to classic mammalian models. One of the most elegant examples of cell surface control of macronuclear DNA synthesis, involving the large ciliate Stentor, was presented by de Terra (1975). Using grafting and microsurgery technique, he demonstrated that nuclei of Stentor associated with distinct regions of the cell surface can b e made asynchronous with regard to DNA synthesis even though they share a common endoplasm. These results can be interpreted to mean that information controlling DNA synthesis in Stentor is associated with the cell plasma membrane and not the cytoplasm, and indeed that specific regions of the cell surface are so involved. A.

CELL SURFACEENZYMES

The entire notion that cell surface enzymes can influence mitosis assumes surface membrane-to-genome communication. As a corollary to this, one must also assume that a system exists within the cytoplasm for such communication, that is, cellular molecules or components capable of transferring information from the cell external surface to the cell nucleus. It is also important to realize that a cell surface active enzyme need not be present at the cell surface at all times; it is conceivable that it migrates there during a critical period or is even present extracellularly as an enzyme released from the cell itself or from another cell.

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H. BRUCE B O S M A ”

The presence of an enzyme on the external surface of a plasma membrane is difficult to establish. Even purification of plasma membranes (absolute purification from nucleated cells is at present impossible) and identification of enzymes in that plasma membrane fraction is fraught with problems: (1)The enzyme activity, or lack thereof, in vivo may bear no relationship to the activity in the purified membrane. (2) In most instances, because of the “inside-out” problem, one cannot be sure the enzyme is located on the membrane’s external surface. (3) In vitro assay usually employs optimum cofactors, substrate and enzyme concentrations, a situation which may or may not obtain in viuo. Therefore many assay methods for external surface enzymes rely on the intact cell assay method which involves many assumptions, such as that the substrate does not enter the cell, that there is no substrate breakdown, that cells with disrupted membranes do not exist in the assay, and so on. These problems are considered in the following discussion. B. SUBLETHALAUTOLYSIS This article concentrates on lytic enzyme activity at the cell surface, which can be thought of as having one of three sources: (1)enzymes originating within the cell (the lysosome?), which move to the cell surface but not out of the cell; (2) enzymes which after their biogenesis are continually associated with the external surface of the cell and are “uncapped,” activated, or expressed only at a certain point in the cell mitotic cycle; or (3) enzymes secreted from the cell, which alter the cell surface from their position in the environment. The first alternative is actually a corollary of the general phenomenon “sublethal autolysis” (Weiss and Mayhew, 1967a,b) in which enzymes, presumably of lysosomal origin, constantly modify the cell’s external surface. It should be noted that increased cell surfacedegrading enzymes may continually degrade the plasma membrane, affecting surface properties such as antigenicity, adhesiveness, membrane transport, membrane architecture, and rearrangement. The hypothesis is not restricted to proteases but may include glycosidases (Bosmann et al., 1974b), lipases, and other potentially lytic enzymes. The hypothesis is unproven, and how the enzymes are released from the lysosome and function correctly at the cell surface periphery is unknown.

C. CELL SURFACEPROTEASES As pointed out in Section I,A, given the present procedures for purifying plasma membranes, it is essentially impossible to prove directly

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that enzymes are present at the external periphery of nucleated mammalian cells. In the enucleated erythrocyte, however, strong evidence has been presented that the plasma membrane is rich in proteases. Using the Anson (1938) colorimetric method, Morrison and Neurath (1953) found that proteases were present in the erythrocyte, and Moore et al. (1970), using the same method, isolated a proteinase active at p H 7.4 from human erythrocyte plasma membranes. Bernacki and Bosmann (1972), using 3H-acetylated hemoglobin as a substrate, isolated and purified two proteinases, one with optimum activity at pH 3.4 and one at p H 7.4, from 0.1% Triton X-100 extracts of pure human erythrocyte plasma membranes. The pH 3.4 optimum proteinase was 75 times as active as the p H 7.4 optimum proteinase. Other evidence for proteinases on the surface of mammalian cells is indirect and depends for validity mainly on the assumption of a lack of protein substrate entry into the cell, a lack of lysed cells in the assay preparation, and a lack of enzyme secretion. It should be emphasized that the measurement of proteinase activity at the cell surface never gives a true representation of how many enzyme molecules are present at this surface nor the rate of turnover or autolysis of these proteases. Protease activity at the cell surface in nucleated mammalian cells and proteases secreted from the cell are discussed in Section II,D.

D. CELL SURFACEPROTEASESAND MITOSIS A potentially important observation was made in 1970 by two independent groups of investigators who demonstrated that mild protease treatment of density-inhibited normal cells temporarily released these cells from so-called contact inhibition. Sefton and Rubin (1970) and Burger (1970) showed that trypsin treatment of contact-inhibited cultured fibroblasts released these cells in such a way that they underwent another round of division. The implications of this finding were potentially great, since they were interpreted to mean that a molecule on the external cell surface was altered (probably a peptide linkage was broken) by the trypsin, and that this alteration caused the nucleus to respond and the cell to undergo division. Furthermore, since as early as 1969 elevated hydrolase levels have been found in oncogenically transformed cells ( Bosmann, 1969a) and elevated protease levels have been found in a variety of oncogenic and tumor cells (Bosmann, 1972a; Bosmann et al., 1974b), the idea that cell surface proteases may function in the control of both normal and abnormal growth has aroused much interest. Another exciting series of reports appeared in which tumor cells were shown to produce a specific protease (Un-

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keless et al., 1974) which activated serum plasminogen (Quigley et al., 1974), and this activation was thought to regulate tumor cell growth specifically (Ossowski et al., 1974). On further examination, however, these hypotheses and the data turned out to be more complicated than they had at first appeared. The hypotheses and complications, as well as the implications for future work, are considered in the following discussion.

1. Exogenously Added Proteases and Mitosis As stated above, Burger (1970) and Sefton and Rubin (1970) found

that exogenously added trypsin or pronase stimulated growthinhibited fibroblasts in culture to undergo a subsequent round of mitosis. Although this finding was not reproducible in cell line 3T3, a mouse embryo fibroblast continuous line culture (Glynn et al., 1973), the phenomenon of protease stimulation of mitosis was confirmed in chick embryo fibroblasts (CEFs) by Vaheri et al. (1974), and Blumberg and Robbins (1975) have indicated that trypsin, collagenase, plasma, a-chymotrypsin, and thrombin, when added to culture medium at concentrations of 0.08-2.2 pg/ml induced cell division in CEFs. Thus it seems fair to conclude that proteases added to the medium of growing but density-inhibited cells do indeed induce mitosis. Two important questions remain: (1) Is the protease effect specific or would, as many people believe, any perturbation of the cell surface cause a similar effect? (2) What is the sequence of events accompanying and continuing after the interaction of the protease at the cell surface to initiate division, i.e., what is the nature of the “receptor” protein on the surface that is cleaved and what is the nature of the secondary stimulus for division? Several proposals have been made in answering the second question, including alteration in lectin binding, decreased intracellular CAMP, increased uridine transport, and so on (see, for example, Noonan and Burger, 1973), and it seems certain that, whatever the initiating event is, other mediators (see this section) are also involved in the complex process of surface-togenome communication.

2 . In Vivo Proteases at the Cell Surface and Division Although it is generally conceded that much can be learned from adding exogenous material to in viuo or in uitro experimental models, one usually turns to making correlations in unperturbed in vivo or in vitro models to prove hypotheses. The key here, however, is the word correlations, because in most instances only correlations can be made

and whether the correlation is causal or casual cannot be discerned.

CELL SURFACE ENZYMES

7

This is now especially true in the study of normal versus neoplastic cells; where once the search was for differences between “normal” and “tumor” tissue, we now have enough differences to fill several volumes-the crucial problem is to identify the important differences. The surface protease and cellular mitosis hypothesis has held up remarkably well in comparisons of neoplastic, or unrestrained, growth .with normal growth, as well as in the comparative situation with aging cells (Section 11,D,5). As stated, elevated hydrolase levels in transformed cells were observed as early as 1969 (Bosmann, 1969a). I n 1972, three groups reported finding elevated hydrolase levels in transformed cells (Bosmann, 1972a; Schnebli, 1972; Kazakovh et al., 1972), and Unkeless e t al. (1973), and Ossowski et al. (1973) originally claimed that oncogenically transformed, but not normal, cells induced fibrinolytic activity in the culture serum (see Section II,D,4). Furthermore, using the Rous sarcoma-CEF system, Rousassociated virus (RAV, a virus that infects but does not oncongenically transform), and temperature-sensitive Rous mutants it was possible to demonstrate that the elevations in protease levels were actually a function of oncogenic transformation and not merely virus infection (Bosmann et al., 1974b). Finally, it was demonstrated (Bosmann and Hall, 1974) that human tumor tissue contained higher levels of proteolytic enzyme activity than normal adjacent tissue. Thus it seems well established that tumor and transformed cells contain elevated proteases. Recent experiments (Spataro et al., 1976) have established that little or no proteolytic activity is secreted into the media of SchmidtRuppin Rous sarcoma virus-transformed CEFs (SR-RSV-CEFs), Rous-associated virus-infected CEFs (RAV-CEFs), or normal C E F cells grown in serumless medium. However, when normal, transformed, and infected cells maintained in serumless medium were incubated with 3H-acetylated hemoglobin, a significant proteolysis of the hemoglobin (a sixfold increase compared to that in normal C E F cells) was found only in the SR-RSV-CEF cells. A sensitive fluorescent assay for peptides confirmed the greatly increased levels of cell surface-associated proteolytic activity in the SR-RSV-CEF cells at p H 7.6. Thus for the first time convincing evidence for cell surface neutral protease has been demonstrated, and this activity is elevated sixfold in oncogenic virus-transformed cells compared to normal control cells (Spataro e t al., 1976). It has been demonstrated in chemically synchronized murine leukemic cells that the release of a cell surface neutral pH enzyme occurs primarily in the G,-M phase of the mitotic cycle (Bosmann, 1974a).

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Thus activity of this surface active protease ( H . B. Bosmann, unpublished observation), as well as its release (Bosmann, 1974a),occurred primarily when these leukemic cells were preparing for division and during division. Whether the secretion of the protease stimulated other cells to divide, or whether each cell released its own growth stimulus in the form of a neutral protease, is not known. The above experimental data indicate that (1) oncogenically transformed cells probably have elevated levels of proteases compared to their normal counterparts, (2) some of this neutral protease activity is probably associated with the external cell surface and is active there, and ( 3 )this activity seems to be most active in the G,-M phase of the mitotic cycle as the cell prepares for division. The data do not demonstrate that the neutral protease induced, stimulated, or effected cell division.

3. Protease Inhibitors and Cell Division The circumstantial evidence described above leads to the suggestion that a neutral protease may be responsible for stimulating, activating, or participating in inducing cell division. If so, it seems logical that protease inhibitors can inhibit cell division. Indeed, protease inhibitors, such as N-a-tosyl-L-lysyl chloromethane, N-a-tosyl-Larginyl methyl ester, and N-a-tosyl-L-phenylalanyl chloromethane, have been shown to inhibit selectively the growth and morphology of oncogenically transformed cells (Schnebli and Burger, 1972; Prival, 1972; Goetz et al., 1972; Hozumi et al., 1972; Troll et al., 1970). Unfortunately, most of the agents used seem to have general toxic effects and are alkylating agents which can penetrate into the cell and cause mitotic arrest (Chou et al., 1974; Schnebli and Haemmerli, 1974). However, the question of selectivity still remains (i.e., the inhibitors were more toxic against oncogenic cells), and more conclusive experiments with bead-immobilized inhibitors or less generally toxic inhibitors need to be performed before this potentially fruitful idea is discarded.

4. Tumor-Associated Fibrinolysis In 1925, Fisher compared the growth of normal and malignant

tissue on plasma clots and found that malignant tissue lysed the clot whereas normal tissue did not. This observation was neglected until Reich and his associates (1974) used it as a basis for determining that only tumor cells lyse fibrin, and that the cells secrete a protease which converts plasminogen (a serum component) to plasmin, which then hydrolyzes the fibrin. The reports of Reich's group stimulated much

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9

interest in protease activity as it relates to plasmin and fibrin. However, several laboratories have reported that fibrinolysis is not restricted to oncogenic cells. Chen and Buchanan (1975) observed that oncogenically transformed cells produce fibrinolytic activity in the absence of plasminogen or serum, and that plasminogen is not necessary for maintenance of the transformed properties of RSV-CEF cells. Thus it seems that the fibrinolysis concept of Reich (1974) may be but a subset of the general concept regarding neutral surface active protease described in Section II,C.

5. Proteases and Mitosis in Aging Cells WI-38 cells, which divide a given number of times and then cease to grow (i.e., die), have been used extensively as an in vitro model of aging. However, since these cells initially (during passages 1 to 36) are under little growth restraint (i-e.,they continuously grow and divide) but during later passages (37 to 50) growth and division are severely restricted, they make an excellent model for the study of normal growth controls. Thus it is of importance to this article that, when WI-38 cells are in early passages and are dividing, they have a normal level of neutral protease (Bosmann et al., 1976). I n later passages when the cells are senescent, the neutral protease activity decreases and goes to zero detectable activity when the cells die, even though WI-38 lysosomal cell enzymes are constant in number or increasing at this time (Bosmann et al., 1976). Thus when young cells need a stimulus for division, the neutral protease is present, but when the cells are dying or dead, and presumably no mitotic stimulus is present, the neutral protease is absent. Therefore WI-38 cells provide another correlation between protease activity and cellular mitosis.

E. CONCLUSIONS The following conclusions can be made about proteases and mitosis. 1. Proteases, particularly those active at neutral pH, can stimulate mammalian cells to undergo mitosis. Whether or not this is a specific effect and how it is mediated are unknown. 2. Protease levels, especially at the cell surface, seem to follow the growth patterns of cells, i.e., levels are elevated during uncontrolled growth and depressed during lack of growth. 3. Inhibition of cell growth by traditional protease inhibition seems unlikely to proceed through the inhibition of protease action, since most of these inhibitors directly inhibit DNA synthesis.

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4. The specific tumor protease that activates serum plasminogen does not seem to be specific to tumor cells, nor does it in all situations seem to require plasminogen. Thus this hypothesis may be a subset of conclusion 1 above. 5. Proteases at the cell surface and mitosis offer a virtually unlimited area for exploration, since conditions for surface expression of the proteases are unknown and factors regulating mitosis seem to be extremely complex. Since growth control is important in a normal situation, in neoplasia, and in aging, delineation of the role of surface proteases (or other hydrolases) in this control is needed. It is likely that proteases at the cell surface in some way mediate growth effects if only by the constant remodeling of the cell surface known to occur during plasma membrane turnover and biogenesis. 111. Cell Surface Enzymes and Cell Adhesion

As complex as cell division is, cell adhesion seems to be equally complex. The two mechanisms may indeed be related, since cellcell contact possibly mediates growth control via mechanisms similar to those discussed in Section II,D for cell division. There are many ways of classifying factors mediating adhesion (and as we will see in Section III,A, adhesion is the result of many complex events). This article is concerned only with cell-cell adhesion although, with the advent of artificial organs and the use of dialysis units, it can readily be appreciated that cell adhesion to materials such as those used in artificial organ components or dialysis tubing is an important area for research.

A. CELL ADHESION Cell adhesion is probably not a single mechanism. Two distinct concepts have been proposed for adhesion, even though it may be found that true cell-cell adhesion requires both mechanisms. The first is the “glue hypothesis,” a complementary macromolecule hypothesis wherein adhesion is mediated by macromolecules which can be solubilized and secreted at the surface. Adding these isolated macromolecules (“glue molecules”) to a cell suspension similar to the one from which they were derived immediately causes aggregation. The second adhesion mechanism is the “recognition,” or “lockand-key,’’ hypothesis. Solubilization of the macromolecules postulated to work in this mechanism is usually difficult, and adding the “lock” and “key” to a given cell suspension which has the potential to aggregate does not cause aggregation. This report is concerned with

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one case of the second hypothesis: complementary enzyme-substrate molecules on cell surfaces mediating adhesion. B.

COMPLEMENTARY MACROMOLECULE HYPOTHESIS

It is appropriate to consider examples of the glue hypothesis with respect to the complementary macromolecule model. The glue hypothesis differs only in the fact that perhaps only one molecule is the glue substance and that the substance can be solubilized and causes aggregation when added to the cell suspension. The complementary macromolecule theory (Tyler, 1946, Weiss, 1974) states that molecules-usually glycoproteins-complementary to each other and present on the surface of different cells, bind at the time of cell contact in an antigen-antibody type of reaction, and cellular adhesion results. These macromolecules do not necessarily differ chemically. The possibility of such a mechanism is seen in the fertilizin-antifertilizinreaction of adhesive components isolated from sea urchin eggs and sperm. Fertilizin, a glycoprotein isolated from sea urchin eggs, agglutinates sea urchin sperm. Many studies have been made on this system (for a review, see Metz, 1967). Similar glycoprotein components can be isolated from mating types of the yeast Hansenula (Crandall et d., 1974) and the ciliate Blepharisma (Miyake and Beyer, 1974). When such components are added to suspensions of the opposite mating type, cell agglutination occurs (a glue-type reaction). Agglutinating components can be isolated from the medium of Chlamydomonas gametes and added to the opposite mating type to cause agglutination (Wiese, 1965). These components (gamones or isoagglutinins) were found to be flagellar membrane fragments which bud off the surface as a normal process (McLean et al., 1974). Treatment of sponges with calcium- and magnesium-free sea water dissociates the cells and releases a glycoprotein factor into the medium. When the factor is added to dissociated cells in the presence of calcium or magnesium, the cells aggregate (Moscona, 1968).Similar cell-aggregating factors have been isolated from embryonic chick neural retina cells and brain cells (Garber and Moscona, 1972a,b). Two main types of reactions exemplify complementary molecule interactions. One is the antigen-antibody reaction itself, and the other is lectin binding. Although antibodies are not known to be present on cell types other than those involved in the immune response, it would not be improbable to have an interaction similar to the binding of lectin to oligosaccharides. However, lectin binding itself is relatively nonspecific except for terminal monosaccharides and can easily be reversed by free oligo- or monosaccharides. Cell adhesion is not known

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to be suspectible to dissolution by free oligo- or monosaccharides. A reversible mechanism which allows for changes in adhesiveness such as those observed in developing embryonic systems would have to be operable. I n addition, antigen-antibody complexes are believed to be relatively permanent, although Mitler and Nussenzweig (1975) showed that antigen-antibody aggregates can be solubilized by complement. A mechanism for the dissolution of adhesive bonds in the complementary molecule model may involve the destruction of both the adhesive bond and the molecules involved b y hydrolytic enzymes. Such a mechanism may operate in sea urchin eggs, which during fertilization release a proteolytic enzyme (Vacquier et al., 1972a) which subsequently prevents polyspermy by detaching supernumerary sperm (Vacquier et al., 197213). Proteases were also found on the surface of mammalian cells (Spataro et al., 1976). It is possible that complementary molecules react with each other through the formation of disulfide bridges; proteins may interact by proper alignbond. Specificity ment of -SH groups, with the resulting -S-Swould be gained through complementary configuration of the proteins. The -SH groups may also mediate nonspecific adhesion of cells to inert substances such as glass and polystyrene (Grinnel and Srere, 1971; Grinnel et al., 1972). Rao (1969, 1973) demonstrated that sulfhydryl groups may function in certain morphogenetic processes in the chick embryo. George and Rao (1975)showed that the aggregation of chick embryonic liver and kidney cells could be inhibited in the presence of carboxypyridine disulfide, which is assumed to bind to -SH groups. Blockage of sulfhydryl groups by this method may hinder steric alignment of complementary molecules. C. CELL SURFACECHARGEAND COMPLEMENTARY RNA MECHANISM Much discussion has centered on the question of whether or not cell-cell recognition, adhesion, and aggregation involve a charge at the cell surface. Since like cells seek out like cells, it seems unlikely that such a simple mechanism is possible; the net negative charge on the surface of like cells would tend to repel rather than attract. Charge coupling may, however, be important after initial cell contact has been made, with negative surface groups coupling with positive groups deeper in the membrane. Consideration of charge coupling gave rise to the concept of a complementary RNA mechanism, attributable primarily to L. Weiss and E. Mayhew. In the complementary RNA mechanism, single-stranded RNA on the cell surface aligns with a complementary sequence of bases, also

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in a single strand, on another cell surface. This results in the formation of a hydrogen bond and adhesion of the cells (Mayhew, 1974a and b) Weiss and Mayhew (1966) found that RNA “footprints” were left on glass after mammalian cells were detached with RNase. RNase treatment of whole cells affected the electrophoretic mobility of several cell types, indicating a possible peripheral or external localization of RNA (Weiss and Mayhew, 1967a,b;). It is interesting to note that the electrophoretic mobility of erythrocytes was not affected by RNase (Weiss and Mayhew, 1967a,b). This cell type is not, and should not be, adhesive, since it must flow freely for circulation. Kolodny (1971)presented evidence for the transfer of RNA between mammalian cells, but the mechanism ofthis transfer was not determined. Synthetic polynucleotides have been shown to bind to the cell surface of Erlich ascites cells (Mayhew, 197413). A major problem in the study of RNA in membranes results from the apparent ubiquity of RNA in the cytoplasm, and breakage of cells during certain types of experiments may result in RNA binding to the plasma membrane (DePierre and Karnovsky, 1973). In spite of this, evidence is accumulating for the existence of RNA in or on plasma membranes (Davidson and Shapot, 1970; Emmelot and Bos, 1972). Its function may be similar to that suggested by Mayhew, 1974a) for cell adhesion. However, it could function in nonribosomal protein synthesis, as demonstrated by Strominger and his co-workers (Petit et al., 1968; Roberts et al., 1968) for bacterial cell wall synthesis.

D. PROBABLE EVENTSIN CELL ADHESION The two mechanisms presented briefly above, as well as the hypothesis detailed in Section III,E, are only a sampling ofthe many possible mechanisms for cell-cell adhesion. Furthermore, several of the proposed mechanisms may operate in concert. For cell-cell adhesion to occur, the following conditions are necessary and sufficient. 1. The cells must be in close proximity to one another (a thermodynamic consideration). 2. The cells must recognize each other and prepare to adhere (this follows from “sorting out” experiments and denotes a high degree of specificity). 3. Correct cell-cell alignment must occur (probably mechanical or a function of condition 2 above). Corollary: Correct membrane configuration alignment must occur (perhaps conformation changes or “ uncapping”).

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4. An initial bonding force must be initiated (if condition 2 has not already occurred). 5. “Permanent” bonding forces must be secured (these should be reversible in the short term, e.g., rounding up at mitosis, and in the long term, e.g., dissociation). 6. Cells must be able to restrict condition 2 above to prevent possible but incorrect recognition and bonding. Corollary: In neoplasia, condition 6 is probably lacking. Thus, simply put, for cell-cell adhesion to occur a high degree of specificity is required and the reaction must be reversible. The latter point illustrates the consideration that the event of cell adhesion need not be a static one, i.e., it may consist ofcontinual formation and breakage of linking bonds.

E. CELL SURFACE ENZYMESAND ADHESION In 1970 S. Roseman, of Johns Hopkins University, made a rather startling proposal (Roseman, 1970) that has had a profound influence on the understanding of cell adhesion. Although the hypothesis was stated in terms of glycosyltransferases and acceptors on the cell surface (and is considered in these terms in the following discussion), it can be stated in general terms as follows. An enzyme on one cell would interact with a substrate on another cell to form an activated intermediate or high-energy enzyme-substrate complex. The energy of this complex would hold the cells together, and completion of the reaction would then cause the cells to dissociate. If one assumes a dual substrate reaction involving substrate X anchored to the periphery of cell 1, a soluble substrate Y, and an enzyme Z anchored to cell 2, the reaction would be: 22

x 1 + Y F=[Xl+ Y

+ Z2I==X’1+

Y‘ + 22

where X’1 and Y’ are the products of the reaction, and the enzyme on cell 2 (Z2) remains unchanged. It is the energy of the complex in the brackets in the reaction that would supply the energy to hold cells 1 and 2 together. It should be noted that this energy could mediate cell-cell recognition or adhesion and, because it has enzyme and substrate components, it carries a high degree of specificity. Completion of the reaction provides for reversibility.

1. Glycosyltransferases The hypothesis that the enzyme-substrate ectoenzyme system mediates cell-cell interactions was developed by Roseman and his

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colleague S. Roth (for a comprehensive review see Shur and Roth, 1975) utilizing glycosyltransferases, primarily glycoprotein glycosyltransferases, as the models for both the theory and experimental data. The glycoprotein glycosyltransferases function according to the following reaction:

Glyconucleotide protein + monosacacceptor charide

2':ge

.

[

.

Activated intermediate

1

Glycoprotein acceptor

I

Terminal monosaccharide

+ nucleotide

where Me2+is a divalent metal cation. Several points should be made about this reaction, some of which even workers in the field seem to ignore:

1. The reaction is a bisubstrate reaction; Michaelis-Menten kinetics therefore do not apply, and the Cleland model must be used. 2. The reaction is usually, although not always, catalyzed by a divalent metal cation (Me2+),which means that it involves a simultaneous four-body hit if the metal is not carried by the enzyme or one of the substrates. 3. The reaction seems to be specific for all components; that is, it requires the correct glycoprotein acceptor even for recognition of the amino acid and oligosaccharide primary structure, correct nucleotide monosaccharide, correct divalent cation, and correct enzyme. 4. Other factors, such as lipids, may also be cofactors in this complex reaction. For a long time glycoprotein glycosyltransferases were thought to

be localized exclusively in the smooth endoplasmic reticulum or Golgi apparatus of cells (Cooket al., 1965; Neutra and Leblond, 1966). Later, however, they were found in mitochondria1 membranes (Bosmann and Martin, 1969; Bosmann, 1971a) and other organelles. Interestingly, glycoprotein glycosyltransferases were identified in plasma membranes by fractionation procedures as early as 1968 (Hagopian et al., 1968; Bosmann, 1969b). However, since it is impossible to obtain pure plasma membrane preparations, identification of cell external surface glycosyltransferases has proceeded using whole-cell assays.

16

H. BRUCE BOSMANN

2. Cell Surface Glycoprotein Glycos yltransferase Ectoenx yme Systems The rather cumbersome title of this section denotes that the enzymes are glycosyltransferases and that both the enzyme and the acceptor molecule are located on the cell surface. In the last 6 years about 60 reports have been published indicating the presence of these glycosyltransferase ectoenzymes in whole-cell preparations; some of these are mentioned here, and others by implication in Section III,E,3. It should be emphasized that none of these reports indicates definitely that the ectoenzyme systems play a role in cell-cell interaction. In whole-cell preparations, glycosyltransferases have been found on the surfaces of embryonic neural retina cells (Roth et al., 1971), transformed mouse fibroblasts (Bosmann, 1972b; Datta, 1974; Roth and White, 1972; Webb and Roth, 1974; Roth et al., 1974; Patt and Grimes, 1974), human blood platelets (Bosmann, 1971b, 1972c; Barber and Jamieson, 1971; Jamieson et al., 1971), intestinal cells (Weiser, 1973a,b), kidney tubular cells (Kirschbaum and Bosmann, 1973a,b), embryonic liver cells (Arnold et al., 1973), RSV-CEFs (Bosmann et al., 1974a; Morgan and Bosmann, 1974; Spataro et al., 1975), high and low metastasis melanomas (Bosmann et al., 1973), and rat dermal fibroblasts (Lloyd and Cook, 1974). The above paragraph mentions only a few of the reports in which glycosyltransferase ectoenzyme systems have been identified chemically; certainly many more could be referenced. It should be pointed out that, in the case of kidney tubular transferases (Kirschbaum and Bosmann, 1973b), folic acid and lysolecithin (Kirschbaum and Bosmann, 1973c) have been shown to accelerate transferase activity. The folic acid effect has been shown to affect substrate concentration (Geren and Ebner, 1974), while the lysolecithin effect has been reported extensively by others. In addition to the biochemical evidence in the above-mentioned studies, Porter and Bernacki (1975) showed conclusive autoradiograph evidence for the existence of glycosyltransferase ectoenzyme systems. In spite of the overwhelming evidence for cell surface glycosyltransferase ectoenzyme systems, Deppert et al. (1974) and Keenan and Morrk (1975) questioned the existence of the transferases on the basis of the methodology used in determining their presence. The questions revolved around hydrolysis of the substrate by surfaceactive hydrolases, broken cells in assay preparations, and lack of a function for the transferases. Answers to these questions already existed in the literature, and several investigators (see, for example, Shur and Roth, 1975) have picked up the gauntlet and successfully de-

17

CELL SURFACE ENZYMES

fended the existence of external surface glycosyltransferases. It seems safe to conclude, at this time, that cell surface glycoprotein glycosyltransferase ectoenzyme systems exist. An interesting corollary to the objection of a lack of function of hydrolases at the cell surface is that they may provide additional or renewed acceptor molecules at the cell surface (Bosmann, 1 9 7 2 ~ )In . the accompanying schematic model, the enzyme-substrate reaction would constantly undergo renewal and would remain in a dynamic rather than a static state.

acceptor

+

nucleotide monosac charide

Cell 2 dYwsY1

wansferase

.

Nucleotide monosaccharide

I

Cell 1 - _ Cell 2 acceptor transferase

I

Me2+ No adhesion

I

Cell 1 acceptor '

Adhesion Glywsidase sublethal autolysis

Cell 1 acceptor

I

11

+

Cell 2 transferase

Monosaccharide

+ nucleotide + Me*' No adhesion

No adhesion

I n this scheme the glycosidase or hydrolase could make cell 1 available for another round of formation of activated intermediate. If one envisions large numbers of these reactions occurring constantly, it is easy to perceive how a dynamic cell-cell interaction may occur. In experiments with chemically synchronized murine leukemic cells L5178Y it was found that the surface glycosyltransferase systems expressed maximal activity during the S phase of the mitotic growth cycle (Bosmann, 1974b). This is consistent with cells rounding up at mitosis and having the least cell-cell contact during the M period and the most at the GI-S interface, the S phase, and the S-G, interface (Bosmann, 197413).

3. Cell Surface Glycoprotein Glycosyltransferase Ectoenxyme Systems and Adhesion While there seems to be no proof that glycosyltransferase ectoen-

zyme systems play a role in cell-cell interactions, several correlations can be made, one of which is as follows.

18

H. BRUCE BOSMANN

Glycosyltransferase ectoenzyme systems that transfer galactose, glucose, N-acetylglucosamine, N-acetylneuraminic acid, mannose, and fucose have been detected on vegetative cells and gametes of Chlamydomonas moewusii. Gametes have higher levels of activity of the transferase ectoenzyme systems than do morphologically identical vegetative cells, as determined by the transfer of monosaccharide to endogenous cell surface acceptors. When and - gametes are mixed, there is a significant increase in activity of the transferase ectoenzyme systems. No such enhancement of activity occurs when + and - vegetative cells are mixed. Flagellar membrane vesicles obtained from and - gametes show high transferase ectoenzyme system activity on a per milligram of protein basis and also demonstrate enhanced activity on mixing (McLean and Bosmann, 1975). A mixture of and - vesicles from vegetative cells and of sexually incompatible gametes did not show enhanced transferase activity (Bosmann and McLean, 1975). Therefore glycosyltransferases and acceptors seem to be located on the flagellar membrane and appear to have a function particularly related to gametic cells. The mechanism of cellular adhesion or recognition proposed by Roseman (1970), involving glycosyltransferases and acceptors, is strongly supported by data for the mating reaction in Chlamydomonas (McLean and Bosmann, 1975). Other, similar correlations have been made for growth, adhesion, and recognition between cells but, until an enzyme-substrate complex is isolated and shown unequivocally to be involved in cell-cell interaction, the role of these ectoenzyme systems will be open to speculation.

+

+

+

F. CONCLUSION From the above discussion, we can conclude the following.

1. Cell adhesion is a complex event, probably encompassing a variety of cellular or biochemical processes for its completion. 2. Glycoprotein glycosyltransferases and their acceptors, the socalled glycoprotein glycosyltransferase ectoenzyme system, probably are present at the external surface of cell membranes and are enzymically active there. 3. No direct proof exists that these ectoenzymes mediate cell-cell adhesion. Various lines of correlative evidence suggest roles for the ectoenzyme in cell recognition, hemostasis, and so on, but the evidence is only suggestive. 4. It is difficult to reconcile how a three- or four-body collision (or many of them) would be possible in three-dimensional space, especially when at least two of the bodies are firmly anchored to cells, in

CELL SURFACE ENZYMES

19

order to bring about formation of the enzyme-substrate complex necessary for this theory to be viable. 5 . It is evident that, although much of the enzyme-substrate work in its relation to cell interactions has been performed on glycoprotein glycosyltransferase ectoenzyme systems, any enzyme-substrate system, if present, may function in a similar or even more efficient manner thermodynamically. 6. The many studies cited above should provide a strong stimulus to investigators to prove that glycosyltransferase ectoenzyme systems indeed mediate some form of cell-cell interaction.

IV. Summary The plasma membrane has come a long way from being thought of simply as an inert barrier which functions to define inside from outside for a cell. Now we know not only that the external surface of the cell communicates with its environment but also that it may be important in determining cell events such as mitosis and cell adhesion. This knowledge, as well as a realization of the role of the cell periphery in immunological processes and its potential role in neoplasia, has brought this once neglected organelle to the forefront of biochemical and molecular biological research. It is a good time, however, not only to continue research on membranes to try to determine definitively their biological functions, but also to take a step backward and assess the data and approaches used in studying the plasma membrane, so that we may determine whether we are simply making correlations which may or may not have relevance to the real world or whether we are truly proving hypotheses about the function of the plasma membrane. REFERENCES Anson, M. L. (1938).J . Gen. Physiol. 22, 79. Arnold, D., Hommel, E., and Risse, H. J . (1973).Biochem. Biophys. Res. Comrnun. 54, 100. Balk, S. D., Whitfield, J. F., Youdale, T., and Braun, A. C. (1973).Proc. Natl. Acad. Sci. U.S.A. 70,675. Barber, A. S., and Jamieson, G . A. (1971).Biochirn. Biophys. Acta 252, 533. Bartholeyns, J., and Baudhuin, P. (1976).Proc. Natl. Acad. Sci. U.S.A. 73, 573. Baseman, J . B., Paolinia, D., Jr., and Amos, H. (1974).J.Cell Biol. 60, 54. Bernacki, R. J., and Bosmann, H . B. (1972).J.Membr. Bid. 7, 1. Blumberg, P. M . , and Robbins, P. W. (1975).Cell 6 , 137. Borenfreund, E., Steinglass, M., Komgold, G., and Bendick, A. (1974).Ann.N . Y. Acad. Sci. 243, 164. Bosmann, H. B. (1969a). Exp. Cell Res. 54,217. Bosmann, H. B. (1969b). Life Sci. 8, 737.

20

H. BRUCE BOSMANN

Bosmann, H. B. (1971a).Nature (London),New Biol. 234,54. Bosmann, H . B. (1971b). Biochem. Biophys. Res. Commun. 43, 1118. Bosmann, H. B. (1972a). Biochim. Biophys. Acta 264,339. Bosmann, H . B. (1972b). Biochem. Biophys. Res. Commun. 48,523. Bosmann, H . B. ( 1 9 7 2 ~ )Biochim. . Biophys. Acta 279,456. Bosmann, H . B. (1974a).Nature (London) 249, 144. Bosmann, H . B. (1974b). Biochim. Biophys. Acta 339,438. Bosmann, H . B., and Hall, T. C. (1974). Proc. Natl. Acad. Sci. U.SA. 71, 1833. Bosmann, H. B., and McLean, R. J. (1975).Biochem. Biophys. Res. Commun. 63,323. Bosmann, H . B., and Martin, S. S. (1969).Science 164, 190. Bosmann, H. B., Bieber, G. F., Brown, A. E., Case, K. R., Gersten, D. M., Kimmerer, T. W., and Lione, A. (1973). Nature (London) 246,487. Bosmann, H . B., Case, K. R., and Morgan, H. R. (1974a).Exp. Cell Res. 83, 15. Bosmann, H. B., Lockwood, T., and Morgan, H. A. (1974b). Exp. Cell Res. 83,25. Bosmann, H . B., Gutheil, R. E., Jr., and Case, K. R. (1976). Nature (London) 261, 499. Brunner, M. (1973).Cancer Res, 33,29. Burger, M. M. (1970).Nature (London) 227, 170. Burk, R. R. (1968).Nature (London) 219, 1272. Chen, L. B., and Buchanan, J. M. (1975). Proc. Natl. Acad. Sci. U.S.A. 72, 1132. Chou, I., Black, P. H., and Roblin, R. 0. (1974). Nature (London) 250,739. Clarke, G . D., Shearer, M., and Ryan, R. J. (1976). Nature. (London) 252, 501. Cook, G. M. W., and Stoddard, R. W. (1973).“Surface Carbohydrates of the Eukaryotic Cell.” Academic Press, New York. Cook, G. M. W., Laico, M. T., and Eylar, E. H. (1965).Proc. Natl. Acad. Sci. U S A . 54, 247. Crandall, M., Lawrence, L. M., and Saunders, R. M. (1974).Proc. Natl. Acad. Sci. U S A . 71,26. Danielli, J . F., and Davson, H. (1934).J . Cell. Comp. Physiol. 5,495. Datta, P. (1974).Biochemistry 13,3987. Davidson, S., and Shapot, V. S. (1970).Fed. Proc., Fed. Am. SOC.Exp. Biol. 6,34(r. DePierre, J. W., and Kamovsky, M. L. (1973).1.Cell Biol. 56,275. Deppert, W., Werchau, H., and Walter, C. (1974).Proc. Natl.Acad. Sci. U.S.A. 71,3068. deTerra, N . (1975).Nature (London) 258,300. Dulak, N . C., and Temin, H. M. (1973).J.Cell. Physiol. 81, 153. Eagle, H. (1973).J.Cell. Physiol. 82, 1. Edelman, G. M. (1976).Science 192,218. Eker, P . (1974).J.Cell Sci. 16, 301. Emmelot, P., and Bos, C. J. (1972).J.Membr. Biol. 9, 83. Fan, K., Fisher, K. M., and Edlin, G. (1973).Exp. Cell Res. 82, 111. Fisher, A. (1925).Arch. Mikrosk. Anat. Entwicklungsmech. 104,210. Garber, B. B., and Moscona, A. A. (1972a).Deu. Biol. 27,217. Garber, B. B., and Moscona, A. A. (1972b). Deu. Biol. 27,235. George, J. V., and Rao, K. V. (1975).J . Cell. Physiol. 85, 547. Geren, L. W., and Ebner, K. E. (1974). Biochem. Biophys. Res. Commun. 59, 14. Gersten, D. M., Kimmerer, T. W., and Bosmann, H. B. (1974).J.Cell Biol. 60,764. Glynn, R. D., Thrash, C. R., and Cunningham, D. D. (1973). Proc. Natl. Acad. Sci. U S A . 70,2676. Goetz, I. E., Weinstein, C., and Roberts, E. (1972).Cancer Res. 32,2469. Goto, M . , Kataoka, Y., Kimura, T., Goto, K., and Sato, H. (1973).Exp. Cell Res. 82,367. Grady, L. J., and North, A. B. (1974).Exp. Cell Res. 87, 120.

CELL SURFACE ENZYMES

21

Greaves, M., and Janossy, G. (1972). Transplant. Reu. 11,87. Green, D. E., Allman, D. W., Buchmann, E., Baum, A., Kopaczyk, K., Korman, E. F., Lipton, S., MacLennan, D. H., McConnell, D. G., Perdue, J. F., Rieske, J. S., and Tzagoloff, A . (1967).Arch. Biochem. Biophys. 119,312. Grimm, J., and Frank, W. (1973).Eur. J. Biochem. 40,555. Grinnel, F., and Srere, P. A. (1971).J.Cell B i d . 51, 146 (abstr.). Grinnel, F., Milan, M., and Srere, P. A. (1972).Arch. Biochem. Biophys. 153, 193. Hagopian, A., Bosmann, H. B., and Eylar, E. H. (1968).Arch.Biochem. Biophys. 128, 387. Hawtry, A. D., Scott-Burden, T., Jones, P. and Robertson, G. (1973). Biochem. Biophys. Res. Commun. 51, 1282. Holley, R. W., and Kiernan, J. A. (1971). Growth Control Cell Cult., Ciba Found. Symp., 1970 p. 3. Holley, R. W., Baldwin, J. H., and Kiernan, J. A. (1974).Proc. Natl. Acad. Sci. U S A . 71, 3976. Hovi, T., Keski-Oja, J., and Vaheri, A. (1974).Cell 2, 235. Hozumi, M., Ogawa, M., Sugimura, T., Takeuchi, T., and Umezawa, H. (1972).Cancer Res. 32, 1725. Jamieson, G. A., Urban, C. L., and Barber, A. J. (1971).Nature (London),New B i d . 234, 5. Kazakovi, 0. V., Orekhovich, C. V., Purchot, L., and Schreck, J. M. (1972).J . Biol. Chem. 247,4224. Keenan, T., and MorrB, D. J. (1975). FEBS Lett. 55, 7. Kent, J. L., and Pogo, B. G . (1974). Biochem. Biophys. Res. Commun. 56, 161. Kirschbaum, B. B., and Bosmann, H. B. (1973a).Nephron 12,211. Kirschbaum, B. B., and Bosmann, H. B. (1973b). Biochem. Biophys. Res. Commun. 50, 510. Kirschbaum, B. B., and Bosmann, H . B. ( 1 9 7 3 ~ )FEBS . Lett. 34, 129, Kisch, A. L., Kelley, R. O., Crissman, H., and Panton, L. (1973).J. Cell Biol. 57,38. Kolodny, G. M. (1971). E x p . Cell Res. 65, 313. Leffert, H. L. (1974).J . Cell Biol. 62,767. Ley, K. D., and Tobey, R. A. (1970).J . Cell Biol. 47,453. Lloyd, C. W., and Cook, G. M. W. (1974).J. Cell Sci. 15, 575. Lucy, J. A. (1968).Br. Med. Bull. 24, 127. McLean, R. J., and Bosmann, H. B. (1975).Proc. Natl. Acad. Sci. U.S.A. 72,310. McLean, R. J., Laurendi, C. S., and Brown, R. M. (1974).Proc. Natl. Acad. Sci. U.S.A. 71, 2610. Mayhew, E. (1974a).J.Theor. Biol. 47,483. Mayhew, E. (197413).E x p . Cell Res. 86,87. Metz, C. B. (1967).In “Fertilization” (C. B. Metz and A. Monroy, eds.), Vol. 1, p. 163. Academic Press, New York. Meuth, M., and Green, H. (1974).Cell 2, 109. Mitler, G. W., and Nussenzweig, V. (1975).Proc. Natl. Acad. Sci. U . S A . 72,418. Miyake, A., and Beyer, J. (1974). Science 185,621. Moore, G. L., Kocholaty, W. F., Cooper, D. A., Gray, J. L., and Robinson, S. L. (1970). Biochim. Biophys. Acta 212, 126. Morgan, H. R., and Bosmann, H. B. (1974).Proc. Soc. E x p . B i d . Med. 146, 1146. Morrison, W. L., and Neurath, H. (1953).J . Biol. Chem. 200,39. Moscona, A. A. (1968).Deu. Biol. 18,250. Nesbitt, J. A., Anderson, W. B., Miller, Z., Pastan, I., Russell, T. R., and Godspodarowicz, D. (1976).J. Biol. Chem. 251,2344.

22

H. BRUCE B O S M A ”

Neutra, M., and Leblond, C. P. (1966).J.Cell Biol. 30, 137. Noonan, K. D., and Burger, M. M. (1973).E x p . Cell Res. 80,405. Novogrodsky, A., and Katchalski, E. (1972).Proc. Natl. Acad. Sci. U . S A . 69,3207. Oka, T., and Topper, Y.J. (1974). Proc. Natl. Acad. Sci. U.S.A. 71, 1630. Ossowski, L., Unkeless, J. C., Tobia, A., Quigley, J. P., Rifkin, D. B., and Reich, E. (1973).J.E x p . Med. 137, 112. Ossowski, L., Quigley, J. P., and Reich, E. (1974).J.Biol. Chem. 249,4312. Pardee, A. B. (1974). Proc. Natl. Acad. Sci. U.S.A. 71, 1286. Parker, J . W., O’Brien, R. L., Steiner, J., and Paolilli, P. (1974).E x p . Cell Res. 83,220. Patt, L. M. and Grimes, W. J. (1974).J.B i d . Chem. 249,4157. Paul, D. (1973).Biochem. Biophys. Res. Commun. 53,745. Petit, J. F., Strominger, J. L., and Soll, D. (1968).J.B i d . Chem. 243,757. Pohjanpelto, P. (1973).E x p . Cell Res. 80, 137. Porter, C. W., and Bernacki, R. J. (1975). Nature (London) 256,648. Prival, J. T. (1972).Ph.D. dissertation, pp. 120-135. Massachusetts Institute of Technology, Cambridge. Quigley, J . P., Ossowski, L., and Reich, E. (1974).J . B i d . Chem. 249,4306. Rao, K. V. (1969).Wilhelm Roux’ Arch. Entwicklungsmech. Org. 163, 161. Rao, K. V. (1973).Wilhelm Roux’ Arch. 173,22. Reich, E. (1974).In “Control of Proliferation in Animal Cells” (B. Clarkson and R. Baserga, eds.), pp. 351-355. Cold Spring Harbor Lab., Cold Spring Harbor, New York. Rheinwald, J. G., and Green, H. (1974). Cell 2,287. Roberts, W. S. L., Petit, J. F., and Strominger, J. L. (1968).J.B i d . Chem. 243,768. Robertson, J. D. (1966).Biomol. Organ., Ciba Found. Symp. 1965 pp. 357-417. Romeo, D., Zabucchi, G., and Rossi, F. (1973). Nature (London),New Biol. 243, 111. Roseman, S. (1970). Chem. Phys. Lipids 5,270. Roth, S . , and White, D. (1972). Proc. Natl. Acad. Sci. U S A . 69,485. Roth, S . , McGuire, E. J., and Roseman, S. ( 1 9 7 1 ) ~Cell . Biol. 51, 536. Roth, S., Patterson, A., and White, D. (1974).J . Supramol. Struct. 2, 1. Rubin, H. (1973).J . Cell. Physiol. 82,231. Rubin, H., and Koide, T. (1976). Proc. Natl. Acad. Sci. U S A . 73, 168. Ruddon, R. W., Weisenthal, L. M., Lundeen, D. E., Bessler, W., and Goldstein, I. J. (1974).Proc. Natl. Acad. Sci. U S A . 71, 1848. Schnebli, H . P. (1972). Schweiz, Med. Wochenschr. 102, 1191. Schnebli, H. P., and Burger, M. M. (1972). Proc. Natl. Acad. Sci. U.S.A. 69, 3825. Schnebli, H. P., and Haemrnerli, G. (1974).Nature (London)248, 150. Scholnick, P., Lang, P., and Racher, E. (1973).J. Biol. Chem. 248, 5175. Sefton, B. M., and Rubin, H. (1970).Nature (London)227,843. Short, J., Brown, R. F., Husakovh, A., Gilbertson, J. R., Zemel, R., and Lieberman, I. (1972).J . Biol. Chem. 247, 1757. Shur, B. D., and Roth, S. (1975). Biochim. Biophys. Acta 415,473. Singer, S . J . , and Nicolson, G. L. (1972).Science 175, 720. Spataro, A. C., Morgan, H. R., and Bosmann, H. B. (1975).Proc. SOC.E x p . Biol. Med. 149,486. Spataro, A. C., Morgan, H. R., and Bosmann, H. B. (1976).J . Cell Sci. 21,407. Thrash, C. R., and Cunningham, D. D. (1973).Nature (London) 227,843. Troll, W., Klassen, A., and Janoff, A. (1970). Science 169, 1211. Trowbridge, I. S., and Hilborn, D. A. (1974).Nature (London) 250,304. Tyler, A. (1946).Growth 10,7. Unkeless, J . C., Tobia, A., Ossowski, L., Quigley, J. P., Rifkin, J . B., and Reich, E. (1973).J.E x p . Med. 137, 85.

C E L L SURFACE ENZYMES

23

Unkeless, J. C., Dano, K., Kellerman, G. H., and Reich, E. (1974).J.B i d . Chem. 249, 4295. Vacquier, V. D., Epel, D., and Douglas, L. A. (1972a).Nature (London)237,34. Vacquier, V. D., Tegner, M. J., and Epel, D. (1972b). Nature (London) 240,352. Vaheri, A. E., Ruoslahti, E., and Hovi, T. (1974). I n “Control of Proliferation in Animal Cells” (B. Clarkson and R. Baserga, eds.), p. 305. Cold Spring Harbor Lab., Cold Spring Harbor, New York. Warren, L., and Click, M. C. (1968).J. Cell Biol. 37,729. Webb, G. C., and Roth, S. (1974).J. Cell Biol. 63, 796. Weiser, M. M. (1973a).J.Biol. Chem. 248, 2536. Weiser, M. M. (1973b).J.Biol. Chem. 248,2542. Weiss, L., and Mayhew, E. (1966).J.Cell. Physiol. 68,345. Weiss, L., and Mayhew, E. (1967a).J. Cell. Physiol. 69,381. Weiss, L., and Mayhew, E. (1967b).N . Engl. /. Med. 267,1354. Weiss, P. (1947).Yale J . Biol, Med. 19, 235. Whitfield, J. F., MacManus, J. P., Boynton, A. L., Gillan, D. J., and Isaacs, R. J. (1974). J . Cell. Physiol. 84, 445. Wiese, L. (1965).J. Phycol. 1,46. Willingham, M. E., Johnson, G. S., and Pastan, I. (1973). Biochem. Biophys. Res. Commun. 48,743. Yoshikura, H. (1974).Exp. Cell Res. 85, 123.