Conversion of type II procollagen to collagen in Vitro: Removal of the carboxy-terminal extension is inhibited by several naturally occurring amino acids, polyamines, and structurally related compounds

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 215, No. 1, April 15, pp. 230-236, 1982

Conversion of Type II Procollagen to Collagen in vitro: Removal of the Carboxy-Terminal Extension Is Inhibited by Several Naturally Occurring Amino LASSE LXvish

Acids, RYHANEN;

of Llemmddogy,

Polyamines,

and Structurally

ELAINE

M. L. TAN, SIRPA JOUNI UITT03

Department

of Medicine,

Torrance, Received

September

UCLA

School

California

9, 1981, and in revised

Related

Compounds’

RANTALA-RYHANEN, of Medicine,

Ha&or-UCLA

AND

Medical

Center,

9050.9 form

November

3, 1981

Chick embryo sterna, which actively synthesize type II procollagen, were pulse-labeled with radioactive proline; protein synthesis was then inhibited by unlabeled proline and cycloheximide. After the inhibition of protein synthesis, several amino acids, polyamines, or structurally related compounds were added to the incubation medium. The conversion of procollagen, first to two intermediates, PC-collagen and pN-collagen, and then to collagen, was monitored by sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis. The addition of 50 mM fi-alanine, arginine, asparagine, glutamine, hydroxylysine, lysine, or ornithine, as well as agmatine, E-aminocaproic acid, S-2-aminoethylcysteine, cadaverine, canavanine, putrescine, or spermine clearly inhibited the removal of the carboxy-terminal extension and PC-collagen accumulated; the removal of the amino-terminal extension was not affected. The inhibition of the conversion was reversible and unaffected by fetal calf serum. The results suggest that the conversion of type II procollagen to collagen requires at least two separate proteinases for the removal of amino-terminal and carboxy-terminal extensions. The results further suggest that naturally occurring molecules may be used to modulate the rate of conversion of procollagen to collagen, and development of analogs of these compounds may provide the means to interfere with excessive deposition of collagen in diseases with tissue fibrosis. It is now well established that the interstitial collagens are first synthesized as precursor forms, procollagens, which con-

tain noncollagenous peptide sequences at both the amino-terminal and carboxy-terminal ends of the molecules. The extension peptides are removed from type I procollagen in the extracellular space by two separate enzymes, procollagen N-proteinase and procollagen C-proteinase,4 respectively (for recent reviews see (l-4)).

’ This study was supported in part by U. S. Public Health Service, National Institutes of Health Grants AM-28460 and GM-28333, and by a grant from March of Dimes-Birth Defects Foundation. A preliminary report of this work has been presented at the Western Regional Meeting of the American Federation for Clinical Research, Carmel, Calif., February 16-18, 1982 (Clin Res. 30:161A, 1982). s Recipient of a New Investigator Research Award AG-3172 from the National Institutes of Health. *Recipient of a Research Career Development Award 5-K04-AM-00897 from the National Institutes of Health. To whom all correspondence should be addressed. 0003-9861/82/050230-07$02.00/O Copyright All rights

0 1982 by Academic Press, Inc. of reproduction in any form reserved.

’ C-Terminal and N-terminal extensions refer to carboxy-terminal and amino-terminal extensions of procollagen; these extensions are cleaved by procollagen C-proteinase and N-proteinase, respectively. PC-collagen refers to a collagen precursor whose Nterminal extension has been removed and- pN-eollagen refers to a precursor whose C-terminal extension has been cleaved; pC-collagen and pN-collagen consist of PC-a and pN-a chains. 230

CONVERSION

OF TYPE

II PROCOLLAGEN

Recently, Leung et al. (5) reported that arginine, both L and D forms, as well as its analog, canavanine, inhibited the removal of the C-terminal extension from type I procollagen synthesized by chick embryo tendons. In the present study, we have examined the effects of a variety of amino acids, polyamines or their structural analogs on the conversion of type II procollagen to collagen using pulse-chase techniques. The results indicate that several naturally occurring amino acids and polyamines prevent the removal of the carboxy-terminal but not of the aminoterminal extension from type II procollagen. MATERIALS

AND

TO COLLAGEN

IN

CONTROL ISOMin

231

VITRO

LYS

180Min

METHODS

Materials. The following compounds were tested in pulse-chase experiments for their effectiveness in interfering with the conversion of type II procollagen to collagen: alanine (Sigma), @-alanine (Fisher), arginine (National Biochemical Corp.), asparagine (Nutritional Biochemical Corp.), glutamine (Nutritional Biochemical Corp.), glycine (Fisher), hydroxylysine (Sigma), isoleucine (Nutritional Biochemical Corp.), lysine (Sigma), ornithine (Sigma), phenylalanine (Nutritional Biochemical Corp.), threonine (Nutritional Biochemical Corp.), tryptophan (Nutritional Biochemical Corp.), valine (Nutritional Biochemical Corp.), S-2-aminoethylcysteine (Sigma), canavanine (Sigma), c-aminoeaproic acid (Sigma), agmatine (Sigma), cadaverine (Sigma), putrescine (Calbiochem), spermine (Sigma). [“C]Proline (NEC-235, sp act 287 mCi/mmol) was purchased from New England Nuclear. Fetal calf serum was obtained from Gibco and dialyzed against modified Krebs’ medium (6, 7). Fertilized eggs were obtained from Redwing Hatcheries, Los Angeles, California. Incubation coraditti. For pulse-chase experiments, five sterna from 17-day-old chick embryos were incubated in 1.0 ml of modified Krebs’ medium buffered with 30 mM Hepes,’ pH 7.6 (6,7), containing 10% dialyzed fetal calf serum, 13 mM D-glucose, 50 pg/ml ascorbic acid, and 50 g/ml fi-aminopropionitrile-HCl, unless otherwise stated. After a 30-min preincubation at 37”C, 3 &i of [“Clproline was added. Following another 30-min incubation, 50 ~1 of

5 Abbreviations used: Hepes, N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid; SDS, sodium dodecyl sulfate; Nas-EDTA, disodium ethylenediaminetetraacetic acid; NEM, N-ethylmaleimide; PMSF, phenylmethylsulfonyl fluoride.

20

40 20 40 MIGRATION (mm)

FIG. 1. Inhibition of conversion of type II PC-collagen to collagen by lysine and arginine. Seventeenday-old chick embryo sterna were pulse-labeled and chased, as described under Materials and Methods. In control samples, at the end of the pulse-labeling period (A), most of the [14C]proline-labeled collagenous polypeptides were [“C]Proa chains of type II procollagen. After a 1Wmin chase (B), a large fraction was recovered as [“C]ot chains. Addition of 50 mM arginine (C) or lysine (D) to the incubation at the end of the pulse-labeling period inhibited the removal of the C-terminal extension, and most of the [“lproline-labeled polypeptides at the end of the 180min chase were recovered as [i’C]pC-ol chains. The migration positions of Proa, PC-(Y, pN-a, and a chains of type II procollagen and collagen, identified on the basis of their susceptibility to bacterial collagenase, content of [“Clhydroxyproline, and comigration with purified type II procollagen and collagen polypeptides (10, 30), are indicated by the arrows.

the medium containing 11.0 mg/ml unlabeled proline was added, 5 min later, 50 ~1 of the medium containing 4.4 mg/ml cycloheximide was added; the delay in the addition of cycloheximide allowed chain completion of the ‘“C-labeled procollagen polypeptides prior to inhibition of protein synthesis (8, 9). Five minutes after the inhibition of protein synthesis, the test compound was added in 0.1 ml of the medium, and the incubation was continued for the time periods indicated under Results. The incubation was stopped by heating the samples at 100°C for 5 min in the presence of 3% SDS and the following final concentrations of protease inhibitors: 30 mM

232

RYHANEN

ET AL.

NazEDTA, 15 mM NEM, and 0.5 rn?d PMSF. The samples were homogenized and extracted, as described elsewhere (10). Aliquots of the SDS extract, containing 85.1 + 5.6% (mean + SD) of all the newly synthesized “C-protein were examined by SDS-polyacrylamide slab gel electrophoresis using 6% gels (11). The radioactive peptides were visualized by fluorography (12), and the bands were quantitated by scanning the fluorographs of the gels with an Ortec 4310 densitometer attached to a computerized Perkin-Elmer Sigma 1 integrator. After the addition of the test compounds, the pH of the incubation medium was measured. With compounds reported in this study, no significant change in the pH value was observed, except with S-2-aminoethylcysteine which lowered the pH to 6.95. Other methods. For the assay of r’“C]hydroxyproline, aliquots of the tissue homogenates were hydrolyzed in 6 M HCl for 24 h at 110°C. [“CjHydroxyproline was assayed by a specific radiochemical method (13). The total “C radioactivity was determined with a Beckman LS7500 liquid scintillation counter.

this study, the sterna were pulse-labeled for 30 min with [14C]proline, and the radioactive polypeptides were examined by polyacrylamide slab gel electrophoresis. The results indicated that at the end of the pulse, over 70% of the radioactivity in collagenous polypeptides migrated as procr chains of type II procollagen (Fig. 1 and Table I). If further incorporation of [14Clproline was inhibited and the incubation was continued for 60-180 min, a time-dependent conversion of [14C]Pro-a chains to [14C]a chains was noted in control samples; at 180 min, the conversion was over ‘75% complete (Table I). A major intermediate band, corresponding to [‘4C]pCcuchain was noted in all experiments (Fig. 1 and Table I). In addition, a minor intermediate band corresponding to [14C]pN-cy chain was also observed in gel scans (see Fig. l), but in most cases it was below the detection limit of the integrator. Therefore, the values for pN-cl! chains are not reported in Tables I and II. It was demonstrated that the inhibition of protein synthesis by the addition of cycloheximide and unlabeled proline was effective, in

RESULTS

Previous studies have demonstrated that chick embryo sterna synthesize type II procollagen, which has extension peptides at both ends of the molecule (14-1’7). In TABLE INCZORPORATION

I

OF [‘%)PROLINE, SYNTHESIS OF [“C]HM~ROXYPROLINE, AND CONVERSION OF PROCOLLAGEN TO COLLAGEN IN A PULSE-CHASE EXPERIMENT WITH AND WITHOUT LYSINE~

Sample

Time of chase (min)

Total r4C (X10-’ dpm)

[“C]Hydroxyproline (X10-’ dpm)

Relative distribution

(%)b

Procr

PC-cy

Control Control Control

0 60 180

16.5 14.9 14.0

7.3 6.2 6.9

72 18 0

28 24 24

0 58 76

Lysine Lysine Lysine Lysine Lysine

0 60 120 180 240

16.4 13.4 14.2 12.0 13.2

7.8 5.2 5.6 6.5 5.7

86 0 0 0 0

14 100 100 199 100

0 0 0 0 0

CY

D17-day-old chick embryo sterna, five per sample, were incubated in 1 ml of medium containing 3 pCi [“Cjproline, as indicated under Material and Methods. After a 30-min incubation, the incorporation of ‘“C was inhibited by the addition of unlabeled proline and cycloheximide, and the samples were chased with or without lysine (50 maa) for the time periods indicated. The incubations were terminated by the addition of protease inhibitors and SDS, and the samples were boiled and homogenized, as described in the text. The total incorporation of “C and the synthesis of [“Cjhydroxyproline were assayed, and aliquota of the SDS extract were processed for SDS-polyacrylamide slab gel electrophoresis. b The relative distributions of Proa, PC-cr, and a chains were calculated as percentage of total from the scans of fluorographs, similar to those shown in Fig. 1.

CONVERSION

OF TYPE II PROCOLLAGEN TABLE

TO COLLAGEN

233

IN VITRO

II

THE EFFECT OF FETAL CALF SERUM ON THE INHIBITION OF TYPE II PGCOLLAGEN CONVERSION TO COLLAGEN BY LYSINE AND REVERSIBILITY OF THE INHIBITION

Addition Experiment None None None None Lysine Lysine

of lysine (50 mbi)

FCS (10%)

Time of chase (min)

Relative distribution

(9%)

Procu

PC-L?

a

+ + + -

0 180 0 130 130 180

100 0 100 0 0 0

0 23 0 21 100 100

0 77 0 79 0 0

+

120

0

100

0

+

120 + 120

0

69

31

1”

Experiment 26 Lysine Lysine followed by fresh medium without lysine

a In experiment 1, the pulse-chase incubations with and without fetal calf serum were performed as described under Materials and Methods, and in Table I. The relative distributions of Proa, PC-cr, and a chains were determined as in Table I and Fig. 1. b In experiment 2, the sterna were pulse-labeled with [“C]proline for 30 min, protein synthesis was stopped by the addition of unlabeled proline and cycloheximide, and lysine was added in 50 mM concentration. After a 120-min chase period, one of the samples was processed for SDS-polyacrylamide slab gel electrophoresis; the medium of another incubate was removed, the sterna were rinsed, and the medium was replaced by fresh medium containing cycloheximide and unlabeled proline, but not lysine. After an additional 120-min incubation, the latter sample was prepared for gel electrophoresis.

that no significant changes during the chase period of O-240 min were found in the total i4C incorporation or synthesis of [‘4Cjhydroxyproline (Table I). When lysine or arginine (50 mM) was added to the incubation medium following the inhibition of protein synthesis, an inhibition of the removal of the C-terminal extension during the subsequent chase period was noted (Fig. 1 and Table I). The N-terminal extension, however, was removed and, as a result, polypeptides migrating in the position of PC-cu chains accumulated in the presence of these amino acids (Table I and Fig. 1). To examine the structural features of lysine and arginine, which are responsible for inhibition of the removal of the C-terminal extension, several other amino acids were tested. In addition to arginine and lysine, the following amino acids, in 50 mM concentration, clearly inhibited the removal of the C-terminal extension: asparagine, glutamine, hydroxylysine, and. or-

nithine. In addition, 100 mM 8-alanine was partially effective (Fig. 2). In contrast, alanine, glycine, isoleucine, phenylalanine, threonine, tryptophan, and valine had no effect in 50 mM concentration; none of these compounds were tested in higher concentrations. Several structural analogs of the effective amino acids were also tested. The naturally occurring polyamines, agmatine, putrescine, and cadaverine, derived by decarboxylation of arginine, ornithine, and lysine, respectively, also inhibited the removal of the carboxy-terminal extensions from type II procollagen when tested in 50 mM concentration. Furthermore, canavanine, S-2-aminoethylcysteine, and spermine were effective in the same concentration. Finally, +aminocaproic acid, a compound analogous to lysine but without the a-amino group, when tested in 50 mM concentration, inhibited the removal of the carboxy-terminal extensions. In further studies, two inhibitory com-

234

RYHtiEN

pounds with similar end groups but with different chain lengths, @-alanine and caminocaproic acid, were tested in different concentrations. e-Aminocaproic acid, consisting of a five-carbon chain with a terminal c-amino group (Fig. 3), inhibited the cleavage of the C-terminal extension in significantly lower molar concentrations than P-alanine which contains a terminal P-amino group (Fig. 2). In addition, lysine was compared with t-aminocaproic acid and cadaverine; the latter compounds have the same chain length as lysine, but are devoid of a-amino and a-carboxyl groups, respectively (Fig. 3). The results indicated that cadaverine is the most potent inhibitor of the four compounds tested in this experiment, causing a maximal inhibition already in 10 mM concentration. On the other hand, t-aminocaproic acid is less effective than Iysine (Fig. 2). Several control experiments were performed: First, it was important to demonstrate that the inhibitory compounds

0

20 40 CONCENTRATION (mM1

H

100

FIG. 2. Inhibition of conversion of type II PC-collagen to collagen by varying concentrations of cadaverine, lysine, e-aminocaproic acid, and 8-alanine. The pulse-chase experiments with these compounds were performed as described under Materials and Methods, and the relative amount of [l’ClpC-collagen at the end of the 180-min chase period was determined by scanning fluorographs of SDS-polyacrylamide gels. The relative distribution of pC-collagen is expressed as [‘pClpC-~ chains as percentage of the sum of [“CJPro-a, [“ClpC-cr, [“ClpN-a, and [“Cg chains. Symbols: 0 - - - 0, cadaverine; n n , lysine; 0 - - - 0, c-aminocaproic acid, 0 0, &alanine.

ET AL.

yH2

ARGININE

H,N-$-NH-CH,-CH,-C&-CH-COOH NH 7”2

LYSINE

&N-CH,-CH,-CH,-CH2-CH-COOH

CADAVERINE

H2N-CH,-CH2-CH,-CH,-CH,

7H2

E-AMINOCAPRO,C

@ALANINE

AC,D H2N-CH,-CH,-CH,-CH,-CH,-COOH

H2N-CH,-CH,-COOH

FIG. 3. Structures of some of the compounds inhibit the conversion of type II pC-collagen lagen.

which to col-

were not toxic to the cells, even though in the experiments described above, they were added to the incubation medium after the inhibition of protein synthesis. As a measure of tissue viability, the sterna were incubated with and without arginine (50 mM) for 3 h with [‘4C]proline. The total incorporation of 14C into protein and the synthesis of [14C]hydroxyproline were not significantly different in these two samples; the values in the presence of arginine were 80-85s of the controls. We have previously demonstrated that the conversion of type II procollagen to collagen in the system employed here is inhibited by the addition of Na2EDTA, and that the inhibition can be reversed by the addition of Ca2+ (10). To rule out the possibility that the inhibitory compounds tested here may act as chelators and deplete the tissue of calcium, in further control experiments the effects of arginine (50 mM) were noted in the presence of additional Ca2+ (50 InM). No difference between the Ca2+-enriched and control samples was noted. Since previous studies have suggested that some procollagen proteinases may be inhibited by serum (l&19), the conversion experiments in the presence of lysine were carried out without and with 10%” fetal calf serum. Serum had no effect on the conversion of procollagen to collagen: both the N-terminal and C-terminal extensions were removed similarly in samples with

CONVERSION

OF TYPE

II PROCOLLAGEN

and without serum (Table II, experiment 1). Also, 50 mM lysine inhibited the removal of the C-terminus regardless of the absence or presence of serum (Table II). Finally, the reversibility of the inhibition was tested by pulse-labeling sterna with [14C]proline for 30 min as described above; the protein synthesis was inhibited, and the samples incubated in the presence of lysine (50 MM) for 120 min. The medium was then replaced with fresh medium containing cycloheximide and unlabeled proline, but not lysine, and the incubation was continued for an additional 120 min. As expected, an accumulation of PC-~! chains was noted after the 120 min-chase in the presence of lysine (Table II, experiment 2). However, after the removal of lysine, the conversion proceeded and after a 120min additional incubation, a significant fraction of the accumulated pC-a chains had been converted to a chains (Table II). DISCUSSION

In the present study, we have demonstrated that several amino acids and structurally related compounds interfere with the conversion of type II procollagen to collagen in short-term organ cultures of chick embryo sterna in vitro. Specifically, fi-alanine, arginine, asparagine, glutamine, hydroxylysine, lysine, and ornithine, in concentrations varying from 10 to 100 mM, inhibit the removal of the Cterminal extension from type II procollagen. These observations extend the original findings by Leung et al. (5), indicating that arginine, both L and D forms, and its analog canavanine, inhibit the conversion of type I pC-collagen to collagen. We further demonstrate that several naturally occurring polyamines, such as agmatine, cadaverine, putrescine, and spermine, likewise inhibit the conversion of pC-collagen to collagen. In addition, a lysine analog, S-2-aminoethylcysteine, as well as canavanine, are inhibitory. Finally, c-aminocaproic acid, a well-known inhibitor of proteinases, such as plasmin, causes the accumulation of p&collagen in tissues. The common structural feature of all the inhibitory compounds is the presence of a free amino group. It is clear, however,

TO COLLAGEN

IN VITRO

235

that an a-amino group is not a sufficient structural feature to make a compound an inhibitor, since several amino acids, including alanine, glycine, isoleucine, leutine, phenylalanine, threonine, tryptophan, and valine, in 50 mM concentration, were not effective in our test system. On the other hand, asparagine and glutamine, which are amides of the corresponding dicarboxylic amino acids, were also effective in 50 mM concentration. Comparison between cadaverine, lysine, and t-aminocaproic acid indicated that cadaverine, which contains free amino groups at both ends of a five-carbon chain (Fig. 3), is the most potent inhibitor of the conversion, while t-aminocaproic acid, which has only one amino group, is the least effective of these three compounds. The chain length of the molecule may also be related to the effectiveness of the compound to serve as an inhibitor, since @-alanine is clearly less effective than c-aminocaproic acid. These two compounds possess similar end groups, but @alanine is two carbons shorter in length. It should be noted that the contribution of different structural features to the effectiveness of these compounds may be dependent on several factors, as for example, tissue penetration and charge properties of the molecule. The mechanisms of the inhibition of procollagen conversion by the compounds tested here are not clear at present. Leung et al. (5), on the basis of previous observations by Siegel (20), suggested that arginine might prevent the removal of the C-terminal extension by interfering with the aggregation of procollagen molecules. Alternatively, there could be a direct inhibition of the procollagen C-proteinase. In the present study, we have been able to inhibit the removal of the C-terminal extension with several compounds, including c-aminocaproic acid, a well-known proteinase inhibitor. These results thus suggest that the interference with conversion may he a result of direct inhibition of procollagen C-proteinase. In the case of type I collagen, it has been demonstrated that two separate proteinases, one for the removal of N-terminal extension and another one for the C-terminal extension, are required for the com-

236

RYH;6NEN

plete conversion of procollagen to collagen (l-5,19,21-27). The results presented here suggest that the conversion of type II procollagen to collagen likewise requires at least two separate proteinases, since only the removal of the C-terminal extension, but not that of the N-terminal one, was inhibited. It should be noted that under the tissue culture conditions used here, neither the procollagen N- nor C-proteinase is affected by the presence of fetal calf serum in the incubation medium. Although this observation suggests that the proteinases involved in the conversion of type II procollagen to collagen are not inhibited by serum, it should be interpreted with caution, since the tissue penetration of serum factors, such as az-macroglobulin, is not known. Accumulation of collagen is a major pathologic feature under several clinical conditions, including pulmonary fibrosis and liver cirrhosis, as well as various forms of dermal fibrosis, such as scleroderma, keloids, hypertrophic scars, and familial cutaneous collagenoma (4). No pharmacologic means to specifically reduce collagen accumulation in these conditions are currently available. The results of the present study suggest novel approaches to control the collagen deposition in pathologic fibrotic conditions. Potentially, administration of nontoxic compounds containing the appropriate structural features and available in a form which would allow achievement of effective tissue concentrations would lead to accumulation of PC-collagen. Since PC-collagen is not capable of forming fibers of ordinary tensile strength, these poorly crosslinked molecules are probably degraded by tissue collagenases at an accelerated rate (28). Similar approaches have been recently suggested by the use of synthetic peptides which interfere with the enzymatic conversion of procollagen to collagen (19, 29). REFERENCES 1. PROCKOP, D. J., KIVIRIKKO, K. I., TIJDERMAN, L., AND Guzaa~~, N. A. (1979) N. EngL J. &fed 301. 13-23.77-85. 2. BORNSTEIN, P., AND SAGE, H. (1980) Amu Rev. Biochem. 49,957-1003.

ET AL. 3. EYRE, D. R. (1980) Science 207,1315-1322. 4. Urrro, J., RYH~EN, L., AND TAN, E. M. L. (1981) in Progress in Diseases of the Skin (Fleischmajer, R., ed.), pp. 103-141, Grune % Stratton, New York. 5. LEUNG, M. K. K., FESSLER, L. I., GREENBERG, D. B., AND FESSLER, J. H. (1979) J. Bid Chm. 254,224-232. 6. KREBS, H. (1950) Biochim Biophvs. Acta 4,249257. 7. UITTO, J. (1970) B&him. Biophys. Acta 201,438445. 8. VUUST, J., AND PIEZ, K. A. (1972) J. Bid Chem 247, 856-862. 9. MILLER, E. J., WOODALL, D. L., AND VAIL, M. S. (1973) J. Bid Chem 248,1666-1671. 10. UITTO, J. (1977) Biochemistry 16.34213429. 11. KING, J., AND LAEMMLI, U. K. (1971) J. MoL Bid 62,465-477. 12. BONNER, M. W., AND LASKEY, R. A. (1974) Eur. J. Biochem 46,83-88. 1, JWA, K., AND PROCKOP, D. J. (1966) Ad IU. Biochem 15,77-83. 14. DEHM, P., AND PROCKOP, D. J. (1973) Eur. J. Biochem 36,159-X6. 15. M~ER, P. K., AND JAMHAWI, 0. (1974) B&him Biophys Acta 365,X%-168. 16. OLSEN, B. R., HOFFMANN, H.-P., AND PROCKOP, D. J. (1976) Arch Bidem BiophgK 175,341350. 17. U~rro, J., HOFFMANN, H.-P., AND PROCKOP, D. J. (1977) Arch. Biochem Biuphys 179,654-662. 18. NIJSGENS, B. V., GOEBELS, Y., SHINKAI, H., AND LAPI~~E, C. M. (1980) Bioch.em J. 191,699-706. 19. NJIEHA, F., MORIKAWA, T., TUDERMAN, L., AND PROCKOP, D. J. (1982) Biochemistry, in press. 20. SIEGEL, R. C. (1974) Pm Nat Ad Sk USA 71, 48264830. 21. KOHN, L. D., ISERSKY, C., ZUPNIK, J., LENEARS, A., LEE, G., AND LAPI~RE, C. M. (1974) Pm Nat Ad Sci USA 71,40-44. 22. FESSLER, L. I., MORRIS, N. P., AND FESSLER, J. H. (1975) Proc Nat. Acad. S& USA 72,4905-4909. 23. GOLDBERG, B., TAUBMAN, M. B., AND RADIN, A. (1975) CeU 4,45-50. 24. T~DERMAN, L., KIWRIKKO, K. I., AND PR~~KOP, D. J. (1978) Biocktim 17,2948-2954. 25. DUKSIN, D., DAVIDSON, J. M., AND BORNSTEIN, P. (1978) Arch. Biochem Biophya 186.326-332. 26. MORRIS, N. P., FESSLER, L. I., AND FESSLER, J. H. (1979) J. Bid Chem 254,11024-11032. 27. NUSGENS. B. V., AND LAP&E, C. M. (1979) Ad Biodmn 95.406-412. 28. VATER, C. A., HARRIS, E. D., AND SIEGEL, R. C. (1979) Bimhem J. 181,639-645. 29. MORIKAWA, T., T~DERMAN, L., AND PROCKOP, D. J. (1980) Biadmndtly 19,2646-2650. 30. Urm’o, J., ALLAN, R. E., AND POLAK, K. L. (1979) Eur. J. Bimhem S&97-103.