Chinese hamster lung cells synthesize and confine to the ... - PNAS

Proc. Nati. Acad. Sci. USA Vol. 77, No. 9, pp. 5206-5210, September 1980

Biochemistry

Chinese hamster lung cells synthesize and confine to the cellular domain a collagen composed solely of B chains (peptide analysis/immunofluorescent staining/cell adhesion)

MICHAEL A. HARALSON*, WILLIAM M. MITCHELL*, R. KENT RHODEStt, THOMAS F. KRESINAt*, RENATE GAYt, AND EDWARD J. MILLERt#

*fepa

ment of Pathology, Vanderbilt University, School of Medicine, Nashville, Tennessee 37232; and tDepartment of Biochemistry and *The Institute of Dental Research, University of Alabama Medical Center, Birmingham, Alabama 35294 Communicated by Sidney P. Colowick, July 20,1980

ABSTRACT The acid-soluble collagen extracted from cultured Chinese hamster lung (CHL) cell layers has been isolated after limited pepsin digestion and differential salt fractionation. Polyacrylamide gel electrophoresis of this material under denaturing conditions showed the presence of collagen chains with an apparent molecular mass of 120,000 daltons both before and after reduction, indicating the absence of interchain disulfide bonds in the native molecule. When chromatographed on CM-cellulose under denaturing conditions, the majority (>90%) of the CHL cell layer collagen chains eluted as relatively basic components slightly before the human a2(I) chain and coincident with the human B chain. In addition, the CM-cellulose elution profiles of the cyanogen bromide peptides derived from the human B chain and from the CHL cell ayer chain were essentially identical. Examination of CHL cells in culture by using affinity-purified antibody to human B chain revealed this colto localized in an extracellular matrix surrounding the laren cells. Furthermore, analysis of the culture medium indicated the absence of any comparable collagen chain. These data provide additional evidence for the existence of a molecular form of collagen composed solely of B chains and suggest that this molecular form o collagen has an unusual affinity for the cell layer in this system. The class of proteins collectively referred to as collagen represents various distinct gene products. The synthesis of these proteins is somewhat tissue specific and is altered in pathological conditions (1, 2). At present, at least nine genetically distinct chains have been described as the primary constituents of various collagen molecules. Four collagen chains-the al(I), a2(I), al(II), and al(III) chains-comprise the interstitial collagens (1). Recent evidence indicates that five additional unique collagen chains are present in collagen molecules in basement membrane-like structures and in basement membranes. These are the aA and aB chains (3-6) and aC chain (7) isolated from highly vascularized tissue, and two additional chains obtained from lens capsule (8-10) and human placenta (11-13). However, little is currently known about the mechanisms that control the differential expression of the collagen genes. This paucity of information reflects, at least in part, the limited number of systems available for studying the regulation and dissecting the genetics of mammalian procollagen bio-

been undertaken to evaluate the genetic type(s) of collagen produced by CHL cells. This report describes the collagen associated with CHL cells in culture.

EXPERIMENTAL PROCEDURES Materials. The sources of reagents for the biochemical procedures and for tissue culture have been detailed (5, 11, 14, 15). Pepsin (2585 units/mg) was obtained from Worthington, and [33H]proline (specific activity, 42 Ci/mmol; 12 ,uM; 1 Ci = 3.7 X 1010 becquerels) was purchased from Schwarz/Mann. The human B chain and human type I collagen were prepared as described (5, 16). The origin of the CHL cell line as well as the clone (HT1) used in these studies has been described (14, 17). Growth of Cells in Culture and Metabolic Labeling. CHL cells were maintained and grown in Dulbecco's modified Eagle's minimal essential medium supplemented with 10% fetal bovine serum as described (14, 15). For immunological studies, cells were grown in 15 X 60 mm dishes at 370C under a 10% C02/90% air atmosphere to t90% confluency. The medium was removed, the cell layer was washed with three 5-ml portions of cold phosphate-buffered saline, and the cells were allowed to dry at room temperature. For the production of radioactive cell-synthesized material, the cells were grown in 25 X 150 mm dishes to ;e90% confluency (-2 X 107 cells per dish), and then the medium was removed and replaced with Dulbecco's modified Eagle's medium (25 ml per dish) supplemented with ascorbic acid (50 ,g/ml) and [3H]proline (10 ,Ci/ml). After incubation for an additional 12 hr. the medium was removed and the cell layer was scraped into 0.5 M acetic acid (9 ml per dish). The suspension was stirred for 24 hr at 4VC, and the acid-insoluble material was removed by centrifugation at 10,000 X g for 15 min. The supernatant fraction was used as the starting material for all subsequent studies on the cell layer

collagen. Isolation and Purification of CHL Cell Layer Collagen. The acetic acid extract of the CHL cell layer was adjusted to 25% (wt/vol) (NH4)2SO4 and stirred for 24 hr. The suspension was clarified by centrifugation (20,000 X g for 45 min), and the

synthesis. This laboratory has recently reported that the HT1 clone of cultured Chinese hamster lung (CHL) cells devotes a significant proportion of its biosynthetic capacity to the synthesis of colUgenous material (14, 15), and the properties of this cell line suggest it to be a unique model system for dissecting the elements that regulate collagen gene expression in the mammalian cell. In order to characterize this system further, studies have

precipitate was dissolved in 15 ml of 0.5 M acetic acid. Human type I collagen (10 mg) was added as carrier, and the mixture was dialyzed for 20 hr against two changes of 1 liter of 0.5 M acetic acid. Pepsin was added to the dialyzed material to 100 lug/ml, and the mixture was stirred for 18 hr. The solution was then adjusted to 1.2 M in NaCl and stirred an additional 20 hr. and the insoluble material was collected by centrifugation (20,000 X g for 1 hr). The precipitate was then stirred in 1.0 M NaCl/50 mM Tris-HCl, pH 8.0, for 24 hr, and any remaining particulate material was removed by centrifugation at 10,000

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.

Abbreviation: CHL, Chinese hamster lung.

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Haralson et al.

X g for 15 min. The supernatant fraction was adjusted to 4.5 M in NaCi and stirred for 24 hr, and the insoluble material was collected by centrifugation at 20,000 X g for 1 hr. The precipitate was dissolved in 10 ml of 0.5 M acetic acid and dialyzed against two changes of 1 liter of 0.5 M acetic acid for 24 hr; the dialysate was lyophilized. All procedures described above were performed at 40C, and the typical yield of pepsin-resistant protein from CHL cell layers was approximately 3 X 106 cpm from 108 cells. NaDodSO4/Polyacrylamide Gel Electrophoresis of CHL Cell Collagen. Molecular weight analysis was performed essentially as described by Furthmayr and Timpl (18). Lyophilized preparations dissolved in sample buffer were heated for 15 min at 90C in either the presence or absence of 100 mM dithiothreitol as indicated in the text. After the addition of tracking dye and glycerol, the samples were applied to 0.6 X 10 cm 5% polyacrylamide gels and were electrophoresed at a constant current (8 mA per gel) until the tracking dye was -2 mm from the bottom of the gel. The distribution of radioactivity in the completed gels was determined in 2-mm segments, and the relative mobilities of the a, (3, and y components of human type I collagen were determined in parallel gels by described procedures (14). Preparation and Isolation of CHL Cell Layer Collagen Chains. Reduced and alkylated collagen chains from the CHL cell layer were prepared essentially as described (11). Briefly, the purified extract from the cell layer was dissolved in 5 M urea, the pH of the solution was adjusted to 8 with Tris base, and 2-mercaptoethanol was added to a final concentration of 0.1 M. After reduction for 4.5 hr at 250C, the material was alkylated for 10 min by the addition of sodium iodoacetate to a concentration of 0.2 M. The solution was then applied to a 1.2 X 125 cm column of agarose beads (Bio-Gel A-Sm, 200-400 mesh) equilibrated in 2 M guanidine-HCl and 50 mM Tris-HCl (pH 7.5), and the material was eluted at a flow rate of 10 ml/hr. The radioactive profile of the column eluent was determined by assaying aliquots (125 ,ul) of each 2.5-ml fraction in 10 ml of Aquasol. The fractions corresponding, in elution position, to the type I collagen a chains-the elution of which was simultaneously monitored at 226 nm-were combined, desalted on a 3 X 25 cm column of Bio-Gel P-2 (100-200 mesh) equilibrated in 0.1 M acetic acid, and lyophilized. CM-Cellulose Chromatography of Collagen Chains. CM-cellulose chromatography of the reduced and alkylated collagen chains recovered after agarose chromatography was performed as detailed (11). Samples were dissolved in buffer A (20 mM NaOAc, pH 4.8/1.0 M urea), heated at 42°C for 5 min, and applied to a 1.5 X 8 cm column of CM-cellulose equilibrated in buffer A. The column was then developed at 42°C by using a 300-ml linear salt gradient from 0 to 1.2 M NaCl in buffer A at a flow rate of 150 ml/hr, and 3.75-ml fractions were collected. Elution of the human al(I) and a2(I) chains was continuously monitored at 226 nm, and the elution of the radioactive CHL cell layer chains was determined by dissolving aliquots (0.5 ml) of each fraction in 10 ml of Aquasol and assaying in a refrigerated liquid scintillation counter. Preparation and CM-Cellulose Chromatography of CNBr-Derived Peptides. Purified CHL cell layer collagen chains obtained by CM-cellulose chromatography were desalted by gel filtration on Bio-Gel P-2 and lyophilized. Twenty milligrams of human B chain was added to the sample, the mixture was dissolved in 70% formic acid, and the chains were cleaved with CNBr as detailed (19). After incubation for 5 hr at 30°C, the reaction was terminated, and the mixture was lyophilized. The sample was then dissolved in buffer B (5 mM sodium citrate, pH 3.6/20 mM NaCI) and applied to a jacketed 0.9 X 7

Proc. Natl. Acad. Sci. USA 77 (1980)

5207

cm column of CM-cellulose equilibrated in buffer B. The column was then developed at 420C at a flow rate of 60 ml/hr by application of a 300-ml linear gradient from 20 to 180 mM NaCl in 5 mM sodium citrate, pH 3.6. Fractions (1 ml) were collected, and the elution of the peptides derived from the CHL cell layer chain and from the human B chain were determined as described in the preceding paragraph. Immunohistochemical Localization of Collagen Containing B Chains. Immunohistochemical staining of air-dried CHL cell layers was performed by using a specific antibody prepared against the human B chain as described (20, 21). Isolation and Purification of Collagen from CHL Cell Medium. The radioactive collagenous material secreted by CHL cells into the culture medium was isolated and purified in a manner identical to that described for the CHL cell layer collagen (see above). The typical yield of pepsin-resistant protein secreted by the cells into the culture medium was approximately 6 X 105 cpm from 108 cells.

RESULTS Isolation and Purification of CHL Cell Layer Collagen. The protocol was designed both to achieve a partial purification of the collagenous components and to ensure the recovery of all recognized genetic types of collagen. Because no radioactivity could be detected on the culture dishes after the cell layer had been scraped into acid and because no collagen could be solubilized by pepsin digestion of the acid-insoluble CHL cell layer fraction (data not shown), it was concluded that the results described in this report are an accurate assessment of the complete complement of collagen molecules synthesized by and associated with CHL cells in culture. NaDodSO4/Polyacrylamide Gel Electrophoresis of CHL Cell Layer Collagen. The CHL cell layer collagen was initially characterized with respect to chain composition, size of the constituent chains, and the presence of interchain disulfide bonds by electrophoresis on polyacrylamide gels. The pepsinresistant collagen obtained from the CHL cell layer was composed largely of components that after reduction, migrated slightly slower than the human al(I) chain and had an apparent molecular mass of approximately 120,000 daltons relative to human collagen chain standards (Fig. 1A Inset). Furthermore, this peak was present in the same amount and at the same relative mobility without reduction of the sample (Fig. 1B). This latter observation indicates that the native molecular form of the CHL cell layer collagen does not contain interchain disulfide bonds. Of the nine types of collagen chains described to date, only the B chain has an apparent Mr of 120,000 and is found in molecules lacking disulfide bonds (3, 5, 6, 19), suggesting that the CHL cell layer collagen contains this chain. Additionally, the profile of the material both before and after reduction suggests the virtual absence of any other type of collagen chain. CM-Cellulose Chromatography of CHL Cell Layer Collagen Chains. The CHL cell layer collagen was characterized further by CM-cellulose chromatography. The majority (>90%) of the chains derived from the CHL cell layer collagen eluted from the ion-exchange column as relatively basic components slightly preceding the elution of the human a2(I) chain (Fig. 2). A second component eluting slightly before the major component and composing less than 5% of the total radioactivity is thought to represent the molecular species of collagen secreted by CHL cells into the medium (see Fig. 5). Its presence in the cell layer likely reflects either adsorption from the medium or the existence of newly synthesized, but unsecreted, collagen. These results confirm the observation (Fig. 1) that the collagen found in CHL cell layers can be defined largely in terms of a


Biochemistry: Haralson et al.

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con'taining heated labeled material) molecular weight gels. The positions of migration of the al(I) and #,,3(I) components of human type I collagen are indicated by arrows. (Inset) Average mobility of the a (95,000), ,3 (190,000), andy (285,000) components as a function of size; arrow indicates position of migration of the major CHL cell layer collagen chain. Values presented have been corrected for scintillation counter background (41 cpm); the recovery of applied radioactivity was >80%.

single type of chain. In addition, the major CHL cell layer component elutes from CM-cellulose in approximately the same position as observed for the collagen B chain (4, 22), providing further support for the identity of the CHL cell layer component as B chain. CM-Cellulose Chromatography of CNBr Cleavage Products Derived from the CHL Cell Layer Chain. To provide further insight into the nature of the chain derived from the cell

layer collagen, its CNBr cleavage products were compared with the CNBr derived peptides of the human B chain. Not only were a similar number of peptides obtained, but also the ionexchange properties of the peptides generated from the human B chain and those of the chain derived from the CHL cell layer collagen were essentially identical (Fig. 3). The failure to detect peptide 8 in the CHL material may reflect either the low proline content of this peptide (19) or a species difference between human and hamster B chains. Immunohistochemical Studies. Additional evidence supporting the concept that the collagen found in CHL cell layers is composed of B chains and information about the localization of this material in culture was obtained by staining CHL cell layers with affinity-purified antibody specific for the B chain and molecules containing B chains (20, 21). The majority of the immunoreactive material synthesized by CHL cells was localized in an extracellular matrix surrounding the cells; only a small amount of intracellular staining was observed (Fig. 4). A similar staining pattern was also observed when the cells were less than 50% confluent; in agreement with this finding, collagen molecules composed of B chains have been isolated from the cellular layer of nonconfluent cultures (data not shown). Thus, the immunohistochemical results independently confirm the presence of molecules containing the collagen B chain in the CHL cell layer, demonstrate that the molecules are present largely in the form of an extracellular matrix, and indicate that the molecules are synthesized continually during the growth of CHL cells in monolayer culture. NaDodSO4/Polyacrylamide Gel Electrophoresis of Collagen in CHL Medium. In contrast to the collagen from the CHL cell layer, which contains pepsin-resistant chains that have an apparent Mr of 120,000 (Fig. 1), analysis of similar preparations of the collagen secreted by CHL cells into the culture medium reveals the presence of chains that have an apparent Mr of 83,000 (Fig. 5). These chains, which are not degradation products of B chains, represent a different type of collagen chain that has chemical and immunological properties quite distinct from those of the B chain. The data emphasize that little, if any, collagen composed of B chains is secreted into the culture medium. This finding and the immunohistochemical data indicating that molecules composed of B chains are deposited extracellularly lead us to conclude that the latter collagen has an unusual affinity for the cells in culture.

DISCUSSION The data presented here provide conclusive evidence that one 0.8 16 of the major molecular species of collagen synthesized by CHL cells in culture is a molecule composed solely of B chains. This 14 conclusion is based on the similar chemical features exhibited ~~~~~~~~~~~~~~~~~cq 0.6 cq 012 by the constituent chains of the CHL cell layer collagen and the ~~~ a~~~cl(I)l well-characterized human B chain. These include (i) the lack of interchain disulfide bonds, (Hi) an apparent molecular mass of 120,000 daltons, (iii) ion-exchange properties when chromatographed on CM-cellulose, and (iv) close similarity in the nature and number of CNBr cleavage products. These data, corroborate the results of a report (5) indicating that then, 2 collagen molecules having the molecular composition B3 are present in extracts of cartilage collagen. Furthermore, these results substantiate the concept that the aA and caB chains may 0 50 100 150 200 250 300 be used in the formation of more than one molecular species Effluent volume, ml of collagen (5, 7, 23, 24). FIG. 2. CM-Cellulose chromatography of CHL cell layer collagen Of additional interest are our observations that collagen chains. The sample (144,000 cpm of CHL cell layer chains and 10 mg composed of the B chains are apparently not secreted molecules chroA and buffer ml of 2 in of human type I chains) was dissolved medium by CHL cells. Indeed, this collagen culture the into matographed on CM-cellulose, and the elution of the radioactive CHL appears to be localized almost exclusively in an extracellular chains (a-0) and the human type I a chains (-) was followed. matrix surrounding the cells. At least two hypotheses can be Recovery of applied radioactivity was 85%. .1

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240 270 120 150 180 210 Effluent volume, ml FIG. 3. CM-Cellulose elution profile of CNBr cleavage products derived from the human B chain and the CHL cell layer chain. The sample (the peptides derived by CNBr cleavage of 55,000 cpm of the CHL cell layer chain and of 20 mg of the human B chain) was dissolved in 5 ml of buffer B and chromatographed on CM-cellulose, and the elution of the CHL cell layer chain peptides (@--) and the human B chain peptides (-) was determined. Recovery of applied radioactivity was 80%. 0

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advanced to explain the observation that molecules composed of B chains are found only in the cellular layer. First, retention of collagen composed of B chains within the cellular domain may be due to an inherent chemical property of this collagen that results in an unusually high affinity for the plasma membrane or the immediate cellular environment. In this location, it is likely that molecules composed of B chains serve as components of a specialized extracellular matrix, the continual synthesis or presence of which is necessary for adherence and spreading of the CHL cells on the plastic substratum (25). This proposal does not rule out the possible involvement of different collagens in cellular adhesion in other systems nor does it exclude a possible role for noncollagenous proteins. It has been

shown, for instance, that fibroblasts exhibit a high affinity for type I collagen (26) and that cultured fibroblasts grown in monolayers assemble a cell-surface protein meshwork apparently composed, at least in part, of type I collagen and fibronectin (27). In addition, it has been shown that preformed collagenous substrata containing one of several different types of collagen facilitate the attachment of a number of cell types in the presence of fibronectin (28). Our results, then, suggest that collagen composed of B chains is involved in the adhesion of CHL cells to the culture surface. Moreover, it has recently been observed that molecules composed of B chains are localized in mature hyaline cartilages within the lacunar spaces surrounding chondrocytes. Together with the data presented here, these observations strongly suggest that collagen molecules composed of B chains may be synthesized for a specialized function in the pericellular environment of several cell types. C 0

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FIG. 4. Immunohistochemical localization of collagen containing B chains. Air-dried CHL cell layers were incubated with an affinity-

purified antibody specific for the collagen B chain and molecules containing this chain. Then the cell layers were washed and incubated with fluorescein isothiocyanate-conjugated goat anti-rabbit IgG. The cell layers were washed again, embedded in phosphate-buffered glycerol, and observed by using a Leitz fluorescence microscope. (X230.)

5210

Biochemistry: Haralson et al.

An alternative explanation for the apparent localization of molecules composed of B chains within the cell layer of cultured CHL cells is the possible presence of a collagenase that specifically cleaves those molecules that are not incorporated into the extracellular matrix to give fragments that are rapidly degraded by other proteases. This possibility seems unlikely in view of the previously demonstrated resistance of molecules containing aA, aB, and aC chains to cleavage by mammalian collagenase (7). Nevertheless, further' experiments will be necessary to rigorously exclude this possibility. This work was originally undertaken to further characterize CHL cells as an in vtro model for investigating the molecular biology of collagen biosynthesis in the mammalian cell. The results indicate that these cells represent an appropriatemodel for evaluating the biosynthesis and regulation of gene expression for the collagen B chain and allow us to propose that the colla. gen B chain or its precursor(s) are present among the previously reported cell-free synthesized collagenous products directed by CHL cell components (14, 15). We are indebted to Donna Posey and Jennifer Spain for their aid in the preparation of this manuscript and to Ronald J. Gregory for his assistance with the photomicrograph. This work was supported by

National Institutes of Health Grants AM-18222, HL-11310, and DE02670. 1. Miller, E. J. (1976) Mol. Cell. Biochem. 13, 165-192. 2. Gay, S. & Miller, E. J. (1978) Collagen in the Physiology and Pathology of Connective Tissue (Fisher, New York). 3. Burgeson, R. E., El Adli, F. A., Kaitila, I. I. & Hollister, D. W. (1976) Proc. Natl. Acad. Sci. USA 73,2579-2583. 4. Chung, E., Rhodes, R. K. & Miller, E. J. (1976) Biochem. Biophys. Res. Commun. 71, 1167-1174. 5. Rhodes, R. K. & Miller, E. J. (1978) Biochemistry 17, 34423448. 6. Benz, H., Bachinger, H. P., Glanville, R. & Kuhn, K. (1978) Eur. J. Biochem. 92,563-567. 7. Sage, H. & Bornstein, P. (1979) Biochemistry 18,3815-3822.

Proc. Nati. Acad. Sci. USA 77 (1980) 8. Dehm, P. & Kefalides, N. (1978) J. Biol. Chem. 253, 66806686. 9. Gay, S. & Miller, E. J. (1979) Arch. Biochem. Biophys. 198, 370-378. 10. Dixit, S. N. & Kang, A. H. (1979) Biochemistry 18, 56865692. 11. Kresina, T. F. & Miller, E. J. (1979) Biochemtstry 18, 30893097. 12. Glanville, R. W., Rauter, A. & Fietzek, P. P. (1979) Eur. J. Biochem. 95, 383-389. 13. Sage, H., Woodbury, R. G. & Bornstein, P. (1979) J. Biol. Chem. 254,9893-9900. 14. Haralson, M. A., Frey, K. L. & Mitchell, W. M. (1978) Biochemistry 17, 864-868. 15. Haralson, M. A., Sonneborn, J. H. & Mitchell, W. M. (1978) J. Blol. Chem. 253, 5536-5542. 16. Chung, E'. & Miller, E. J. (1974) Science 183, 1200-1201. 17. Roufa, D. J. & Reed, S. J. (1975) Genetics 80,549-566. 18. Furthmayr, H. & Timpl, R. (1971) Anal. Biochem. 41, 510516. 19. Rhodes, R. K. & Miller, E. J. (1979) J. Blol.- Chem. 254, 12084-12087. 20. Sherman, M. I., Gay, R., Gay, S. & Miller, E. J. (1980) Dev. Biol. 74, 470-478. 21. Gay, R. E., Buckingham, R. B., Prince, R. K, Gay, S., Rodnan, G. P. & Miller, E. J. (1980) Arthritis and Rheum. 23,190-196. 22. Mayne, R., Vail, M. S. & Miller, E. J. (1978) Biochemistry 17,

446-452. 23. Jimenez, S. A., Yankowski, R. & Bashey, R. I. (1978) Biochem. Biophys. Res. Commun. 81,1298-1306. 24. Deyl, Z., Macek, K. & Adam, M. (1979) Blochem. Blophys. Res. Commun. 89,627-634. 25. Grinell, F. (1978) Int. Rev. Cytol. 53,65-144. 26.- Goldberg, B. (1979) Cell 16,265-275. 27. Bornstein, P. & Ash, J. F. (1977) Proc. Natl. Acad. Scl. USA 74, 2480-2484. 28. Kleinman, H. K., Murray, J. C., McGoodwin, E. B.-& Martin, G.

R. (1978) J. Invest. Dermatol. 71, 9-11.