The polyribosomal synthesis of collagen

BIOCHIMICA ET BIOPYSICA ACTA

411

BBA 95534

T H E POLYRIBOSOMAL S Y N T H E S I S OF COLLAGEN

G E O R G M A N N E R * , R O B E R T H. I ~ R E T S I N G E R * * , B E R N A R D S. G O U L D AND ALEXANDER RICH

Department o/ Biology, Massachusetts Institute o/ Technology, Cambridge, 2VIass. (U.S.A.) (Received J u l y 25th, 1966)

SUMMARY

The formation of collagen, on polyribosomes has been studied. Collagen was identified its characteristic content of hydroxyproline, which is derived metabolically b y from proline. [14C]Proline was injected into chick embryos and nascent collagen was measured in sucrose density gradients by the isolation of protein-bound [14C]hydroxyproline. The results indicate that collagen is synthesized on very large polysomes. Ribonuclease, puromycin and dodecyl sulfate studies indicate that the nascent peptide chains already contain hydroxyproline suggesting that hydroxylation occurs prior to complete peptide chain assembly. There is suggestive evidence that the large polysomes m a y result from the aggregation of nascent collagen peptide chains.

INTRODUCTION

Collagen is the major fibrous protein in the vertebrate world and in mammals constitutes over one quarter of the total body protein. It is responsible for m a n y of the unique properties of connective tissue and it has been studied extensively. The collagen fibers of connective tissue are formed b y an association of tropocollagen molecules which are approximately 2800 A in length and 15 • in diameter. Tropocollagen has a molecular weight somewhat over 300 ooo and consists of three polypeptide chains which are wound around each other in a triple helical manner. Less is known of the genetics and biosynthesis of collagen than of its chemistry. Within the past few years it has become generally recognized that protein synthesis occurs on clusters of ribosomes which are assembled on a messenger RNA coded for the particular protein 1-4. In a number of instances, there appears to be a relation between the size of the protein molecule and the size of the polyribosomal or polysomal cluster involved in the synthesis of the protein. For example, the hemoglobin polypeptide chains have a molecular weight of about 17 ooo and are synthesized on clusters of 5~6 ribosomes a. The enzyme fl-galactosidase has an asymmetric unit with a molecular weight of about 135 ooo and is synthesized on a poly* P r e s e n t address: D e p a r t m e n t of Surgery, Albert Einsteirt School of Medicine, N e w York, N.Y. ** P r e s e n t address: I n s t i t u t de Biologie Moldculaire, Geneva, Switzerland.

Bioehim. Biophys. Acta, 134 (i967) 4 i i - 4 2 9

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G. MANNER eta].

somal cluster containing about 5° ribosomes 5-v. The messenger RNA for collagen has as yet not been identified. It is not known whether the information for the assembly of the three chains is contained in separate messengers or whether it is contained in a very large polycistronic messenger molecule. There is some evidenceS, 'a which suggests that collagen peptide chains may be composed of subunits. This implies that even smaller messenger-RNA molecules would be found than those coding for the individual ~ polypeptide chains which have a molecular weight near Ioo ooo. The experiments described here were initiated with the aim of identifying the polyribosomal structure associated with collagen synthesis in order to obtain some indirect evidence about the size of the messenger RNA and some information about the probable nature of the assembly of the maeromoleeule. Collagen is unique among proteins in that it contains substantial amounts of hydroxyproline which is formed by the hydroxylation of prolinO °. Accordingly, collagen was identified on the polysomes of chick embryos by the presence of proteinbound hydroxyproline. The major results of this research show that the hydroxyproline-containing polypeptide chains attached to polysomal clusters have all the characteristics associated with nascent or growing polypeptide chains. Furthermore, they are found predominantly with a fraction of very large polyribosomes. Parts of this work have already been reported briefly n.

METHODS

lncubatio~ o/ chick embryos and analysis o~ polysomal /ractions by density-gradient centri/ugation Square openings 1.5 2.o cm on a side were cut into fertilized eggs on the second to fourth day of incubation after removal of 2 ml of egg white through a pinhole at the sharp end. The openings were sealed with masking tape and the eggs reineubated until use. After S--9 days of incubation, the seal was removed and 2.5 IO ffC of 14C-labeled amino acid dissolved in o.o5-o.1 ml of physiological saline was injected into a surface blood vessel of the chorio-allantoie membrane using a fine needle. Incubation was continued for an additional specified time, usually 2o-3o rain. The embryo was then removed from the shell and washed repeatedly in ice-cold RS buffer containing o.oI M KC1, o.ool 5 M MgCI~ and o.oi 3I Tris HCI (pH 7.4-7.6). The brain was removed because its high lipid content appeared to interfere with subsequent operations. The remainder of the embryo was homogenized with o.5 1.o ml of ice-cold RS buffer in a cold Dounee tissue grinder by xo-I2 strokes of the loosely fitting pestle. Heavy particles were removed by eentrifugation at i o o o o ×g for 15 rain. Lipid membrane material was dissolved by adding sodium deoxycholate to the supernatant to a final concentration of o.5 °,'o.The supernatant was then layered over 25-28 ml of a linear sucrose gradient (15 3o % w/w) having the same ionic composition as the homogenization medium. Centrifugation was carried out near o ° for various time periods but most frequently for ~2o rain at 25 ooo rev./min in the SW 25 swinging-bucket rotor of the Spinco L preparative ultracentrifuge. After centrifugation the plastic centrifuge tube was punctured and serial fractions were collected. The absorbance at 26o mt, of the fractions was assayed continuously in a Gilford spectrophotometer. 13iochim. 13iophys. .4eta, 134 (~067) 41l 429

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Fig. I, C h r o m a t o g r a p h i c s e p a r a t i o n of p r o l i n e a n d h y d r o x y p r o l i n e , o. 5 m g of b o v i n e s e r u m a l b u m i n h a d a d d e d to it o. I m g each of c a r r i e r L-proline a n d L - h y d r o x y p r o l i n e . The m i x t u r e w a s t h e n h y d r o l y z e d as in METHODS. A f t e r r e m o v a l of t h e HC1, t h e h y d r o l y s a t e w a s a p p l i e d a t t h e o r i g i n as a i 2 - c m wide b a n d . To one p o r t i o n , l a b e l e d A, were a d d e d r a d i o a c t i v e p r o l i n e a n d h y d r o x y p r o l i n e . N o n - r a d i o a c t i v e p r o l i n e a n d h y d r o x y p r o l i n e , a b o u t o.o 5 nlg each, were s p o t t e d as m a r k e r s on each side, B, of t h e sanlple. The c h r o m a t o g r a p h y p r o c e e d e d 2o h as d e s c r i b e d in METHODS. The s t r i p A w a s c u t o u t a n d c o u n t e d in a V a n g u a r d s t r i p c o u n t e r as s h o w n in (A). The r e m a i n d e r of t h e c h r o m a t o g r a m w a s s p r a y e d w i t h o. 5 % n i n h y d r i n in acetone, s h o w n in (B). The p o s i t i o n s of t h e circled m a r k e r s p o t s c o r r e s p o n d to t h e p e a k s in t h e c o u n t e r t r a c i n g . P r o l i n e fornls a pale y e l l o w c o m p l e x w i t h n i n h y d r i n a n d h y d r o x y p r o l i n e a y e l l o w - g r e e n one, t h u s t h e circled s p o t s do n o t s h o w up v e r y s t r o n g l y in t h i s p h o t o g r a p h of t h e c h r o m a t o g r a m . (C) A s a m p l e c h r o m a t o g r a m from a f r a c t i o n of t h e sucrose d e n s i t y g r a d i e n t . The m a t e r i a l w a s p r e p a r e d as a b o v e w i t h c a r r i e r p r o l i n e a n d h y d r o x y p r o l i n e , b u t t h e r a d i o a c t i v i t y w a s o b t a i n e d from t h e samp le. The s t r i p w a s c u t i n t o s e v e n s e g m e n t s , e l u t e d a n d c o u n t e d as d e s c r i b e d in METHODS. T h e r e is a good s e p a r a t i o n b e t w e e n p r o l i n e a n d h y d r o x y p r o l i n e .

14iochim. Biophys. Acta, 134 (1967) 411 429

414

G. MANNER et aI.

Isolation o~ protein labeled with proline and hydroxyproline and measurement o~ the radioactivity U n l a b e l e d chick e m b r y o h o m o g e n a t e (0.5 mg) was a d d e d to each fraction as carrier protein. The t o t a l p r o t e i n in the i n d i v i d u a l fractions was p r e c i p i t a t e d with cold t r i c h l o r o a c e t i c acid a d d e d to give a final c o n c e n t r a t i o n of 5 %. I t was s e p a r a t e d b y low-speed centrifugation, w a s h e d 2 3 times with cold 5 o~ ,.o trichloroacetie acid a n d twice w i t h cold e t h a n o l - e t h e r (3:1, v/v). The fractions were t h e n air-dried a n d h y d r o l y z e d in sealed t u b e s w i t h 6 MHC1 for 3 h at 135 ° or 18 h at IiO °. The h y d r o l y s a t e was dried, dissolved in o.I ml of distilled w a t e r a n d s t r e a k e d on W h a t m a n 3 MM paper. C h r o m a t o g r a p h y was carried out using w a t e r - s a t u r a t e d p h e n o l - o cresol ( I : I , v/'v) in an a t m o s p h e r e of 0.3 O//o a m m o n i a . The proline and h v d r o x y proline were e l u t e d from the p a p e r w i t h water, dried a n d c o u n t e d on a NuclearChicago l o w - b a c k g r o u n d gas-flow counter. The effectiveness of the c h r o m a t o g r a p h i c s e p a r a t i o n is shown in Fig. I. W h e n the t o t a l u p t a k e of g e n e r a l l y 14C-labeled amino acids was to be measured, o.3 mg of bovine serum a l b u m i n was a d d e d to each fraction as carrier after which t h e y were p r e c i p i t a t e d w i t h 5 o/ ,.o trichloroacetic acid. The p r e c i p i t a t e s were collected on Millipore filters a n d the r a d i o a c t i v i t y d e t e r m i n e d on a l o w - b a c k g r o u n d counter.

Enzyme and detergent treatment T r e a t m e n t w i t h p a n c r e a t i c ribonuclease was carried out in RS buffer at o ° for 15 min prior to g r a d i e n t centrifugation. The c o n c e n t r a t i o n used was I / , g / m l . T r e a t m e n t w i t h collagenase was p e r f o r m e d at 20 ° in RS buffer with CaC12 a d d e d to a c o n c e n t r a t i o n of o . o i M. 5O-lOO/*g eollagenase per ml was used a n d i n c u b a t i o n was for 3o rain prior to g r a d i e n t centrifugation. The sodium d o d e c y l sulfate t r e a t m e n t s were carried out at 25 ° b y g e n t l y stirring a solution containing o.I M NaC1, o.oo5 M Tris-HC1 (pH 7.0) a n d 0.50//o sodium dodecvl~ sulfate. The solution was t h e n d i a l y s e d against w a t e r at 2 ° for 2 h a n d then centrifuged on a 5 20 o~, sucrose grad i e n t r'.

Materials The 14C-labeled a m i n o acids, proline a n d tyrosine, with specific activities of 33-I8offC:'/,mole were p u r c h a s e d from Nuclear-Chicago Corporation a n d New E n g l a n d Nuclear, Inc. l~C-labeled algal h y d r o l y s a t e , used as a general a m i n o acid label, was o b t a i n e d from New E n g l a n d Nuclear, Inc. I t s specific a c t i v i t y was o.8 mC/mg. Before use, the solutions were e v a p o r a t e d to dryness and redissolved in o. 9 ° o NaC1 solution. F e r t i l i z e d eggs from white leghorn chickens were o b t a i n e d locally. T h e y were s t o r e d at I 3 - I 5 °. I n c u b a t i o n was i n i t i a t e d b y p u t t i n g t h e m in a 37 ° i n c u b a t o r . Crystalline p u r o m y c i n was a gift from Dr. J. M. RUECSECCEa of the Lederle L a b o r a t o r i e s , Pearl River, New York. Collagenase was a gift from Dr. J. FRANZBLAU, B o s t o n U n i v e r s i t y School of Medicine. I t h a d a specific a c t i v i t y of IOO Seifter-Gallop units per m g a n d h a d no d e t e c t a b l e non-specific p r o t e o l y t i c activity. P a n c r e a t i c ribonuclease (3 × crystallized) was p u r c h a s e d from W o r t h i n g t o n Biochemicals, inc.

Biochim. Biophys. Acta, i34 (1967) 411-429

POLYRIBOSOMAL SYNTHESIS OF COLLAGEN

415

RESULTS

Ribosomes characteristically sediment as a sharply defined peak which can be detected b y their absorption at 26o m#. Chick embryo ribosomes have a sedimentation constant near 75 S. In contrast to this, polyribosomes or polysomes sediment in a broadly distributed band covering a wide range of sedimentation constants. This arises because of the varied numbers of ribosomes held together by messenger RNA. This distribution is clearly evident in Fig. 2a which shows the results of a sucrose gradient analysis of the ribosome-containing extract from a chick embryo injected with l~C-labeled algal hydrolysate. The 75-S single ribosomes show up clearly in the absorbance tracing. In the distribution shown in Fig. 2a, the absorbance m a x i m u m occurs in the region of polysomes containing 5-6 ribosomes. Sedimenting more rapidly than this is a broad, continuous distribution of ultravioletabsorbing material extending down to the bottom of the gradient. This distribution resembles that seen in a mammalian cell, such as HeLa cells, where very large polysomes ( > 50 ribosomes) are found near the bottom of the gradient 13. It differs sharply from the narrow distribution of polysomes seen in the reticulocyte2, 3 where hemoglobin is essentially the only product of protein synthesis and it is associated with polysomes containing 4-7 ribosomes. The radioactivity from the general amino acid label in Fig. 2a does not show a peak in the single ribosome region, but rather in the polysome region. It is important to notice that the gelatinous pellet at the bottom of the tube also contains radioactivity. The pellet contains very large polysomes. Gentle treatment with ribonuclease characteristically (Fig. 2b) breaks up the polysomes, transferring both the ultraviolet-absorbing material and the radioactivity into the region of single ribosomes. Significantly, it reduces the radioactivity found in the pellet to

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Fig. 2. Sucrose density-gradient analysis for a 9-day-old chick e m b r y o injected with 5 g C 14Clabeled algal h y d r o l y s a t e and incubated f u r t h e r for 3o Inin. The e m b r y o w a s homogenized as in METHODS, and 1. 5 ml of the io ooo × g s u p e r n a t a n t was placed on top of the sucrose density gradient (15-3o (~o, w / w in RSB buffer). The centrifugation w a s carried o u t at 2 ° and centrifugation time is lO 5 Inin. The material is sedimenting to the left. The a r r o w on the base line to the r i g h t r e p r e s e n t s the last fraction in all gradients. Absorbance at 260 In/~ is a solid line and c o u n t s / m i n are the open circles. (a) General amino acid label. (b) An equal aliquot of (a) was t r e a t e d w i t h I/~ g of ribonuclease per Inl for 15 min at 2 ° before layering it on the sucrose gradient.

]3iochim. ]3iophys. Acla, I34 (1967) 4 i i - 4 2 9

416

G. MANNER et al.

about one-third of its value in the untreated material, but does not reduce it to zero. However, all of the radioactivity from the non-pelleted polysomes is released by ribonuclease. It should be noted that the peak of radioactivity in lqg. 2a occurs in a region of larger polysomes rather than at the peak of absorbance. This implies that the radioactivity per ribosome is greater with larger polysomes. This m a y be readily explained if the nascent or growing polypeptide chains are, on the average, longer for larger polysomes or longer messenger RNA molecules. In order to identify the polysomal sites specifically involved in collagen formation, chick embryos were incubated for 2o min after iniection of 5/lC of IlaC~proline and the distribution of acid-precipitable [14Clproline and [14Clhydroxyproline was then determined on the sucrose gradients and compared in ribonucleasetreated and untreated aliquots. The results are presented in lqg. 3a. It can be seen that the distribution of trichloroacetic acid-precipitable [14Clproline is essentially identical with the distribution of the general amino acid label shown in Fig. 2a. The polysomes rather than the single ribosomes appear to be active in protein synthesis and there is relatively more radioactivity associated with the larger polysomes. However, the distribution of trichloroacetic acid-precipitable [~*C~hydroxyproline is strikingly different. As can be seen in Fig. 3 a, significant amounts of Ft4Ci]hydroxy proline are found almost exclusively in the gelatinou~ pellet, indicating that all or nearly all of the collagensynthesizing polysomes must be sufficiently large to sediment within the I2o-min centrifugation period. Ribonuclease treatment of the homogenate before gradient eentrifugation (Fig. 3b), again results in a shift of most of the ultraviolet-absorbing material as well as the radioactivity from the polysomes into the single ribosome fraction. At the same time, the size of the pellet and its radioactivity are reduced in a manner analogous to that seen when the ~¢C-labeled amino acid mixture was used. The data in Figs. 2 and 3 are representative of a large number of experiments. There is some variation with different embryos both in the total amount of polysomal material extracted and the relative distribution of the ribosomes and polysomes.

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Fig. 3- Sucrose d e n s i t y - g r a d i e n t a n a l y s i s of h o m o g e n i z e d chick e m b r y o injected w i t h 5 /~C F14Cjproline a n d allowed to i n c u b a t e for 2o rain. A b s o r p t i o n (solid line) a n d r a d i o a c t i v i t y (open circles) are p l o t t e d w i t h o t h e r c o n d i t i o n s as described in Fig. 2. (a) S a m p l e after i2o m i n of c e n t r i f u g a t i o n . (b) E q u a l a l i q u o t of (a} b u t after t r e a t m e n t w i t h ribonuclease as in Fig. 2 (b).

Biochim. lCiophys. Acla, I34 (1967) 411 429

POLYRIBOSOMAL SYNTHESIS OF COLLAGEN

417

In typical experiments, a gradient would have 5 absorbance units at 26o m# under the monomer ribosome peak, about 15 in the polysome region and about 1.5 in the pellet after it has been resuspended. After a 2o-min incubation with ~4C-labeled amino acids, about 9 ° % of the trichloroacetic acid-precipitable radioactivity is found in the top 3 ml of the gradient. This indicates that no more than IO (I() is associated with nascent protein chains attached to ribosomes.

Characteristics o/the rapidly sedimenting material It is evident that from the point of view of collagen formation the material which is pelletable in a I2o-min eentrifugation is of primary interest because it contains the greatest concentration of hydroxyproline radioactivity. It is equally evident that it becomes important to establish whether the material is in fact collagenlike and whether there are other nascent proteins in this pellet fraction. Tile following experiments were performed to answer these questions. In most cases a larger percentage of the proline than of the hydroxyproline bound to the pelletable material was removed b y ribonuclease treatment. This results in a higher hydroxyproline:proline radioactivity ratio in the ribonuclease-treated pellet as compared with the untreated pellets. This ratio normally varies from 1:6 to I : I O in pellets from untreated homogenates which increases to 1:4 in pellets from ribonuclease-treated aliquots. This would make it appear that at least some of the polysomes involved in collagen formation are for some reason le~s sensitive to ribonuclease than those synthesizing other proteins.

Collagenase experiments Experiments were carried out to ascertain whether the hydroxyproline radioactivity in the pellet was indeed in collagen molecules. For this purpose resuspended pellet material was treated with highly purified collagenase which was free of nonspecific proteases 14. After a 2-h centrifugation, as in Fig. 3, a pellet was resuspended in the usual RS buffer solution and then divided into four aliquots. One served as a control which was kept at o °. Another aliquot was treated with collagenase then cooled to o ° and exposed to ribonuclease treatment in the usual way. A third sample was treated with ribonuclease, while the fourth was treated with collagenase, then all the samples were resubjected to gradient centrifugation and the newly formed pellets were examined. The results are shown in Fig. 4It is apparent that collagenase alone removes virtually all the bound [14C~hydroxyproline from the pelletable fraction. Ribonuclease, when used alone, liberated 55-6o o/ of both [14C]proline and [14C]hydroxyproline. When it is used after collagenase treatment, it removes half of the remaining El~C]proline. Collagen normally has a ratio of proline to hydroxyproline of about 1.2 : I - i . 4 : I. Collagenase treatment liberated 15o counts/min of proline. This gives a ratio of about 1.9:1 which is slightly higher than that observed for adult chick collagen. In view of the high specificity of collagenase the results strongly support the interpretation that collagen polypeptides are present in the pelletable material. The slightly high ratio. of proline to hydroxyproline might suggest that the nascent collagen peptides attached to ribosomes have not been fully hydroxylated. However, further experiments will be Biochim. Biophys. Acts, 134 (t967) 41i 420

418

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necessary to establish this point with certainty. The Ca 2+ treatment necessary for the activation of collagenase was found to precipitate the ribosomes. Thus it was not possible to determine whether collagenase treatment resulted in the liberation of ribosomes or polysomes from the pelletable material.

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i Fig. 4. T h e p e l l e t m a t e r i a l o b t a i n e d as in Fig. za was r e s u s p e n d e d in t{S buffe r a n d t r e a t e d a s d e s c r i b e d u n d e r METHODS. F o u r e q u a l a l i q u o t s h a d e i t h e r no t r e a t m e n t (control), c ol l a ge na s e , r i b o n u e l e a s e or b o t h of t h e l a t t e r . F o l l o w i n g this, t h e m a t e r i a l w a s a g a i n p e l l e t e d in a s uc ros e g r a d i e n t , a n d t h e r a d i o a c t i v i t y in the new p e l l e t s was m e a s u r e d .

Adsorption control An experiment was carried out in order to establish whether the radioactivity found associated with the pelletable fractions actually constituted particle-bound nascent polypeptides rather than non-specifically adsorbed protein. A supernatant containing the polysomes from a non-radioactive chick embryo homogenate obtained after centrifuging at IO ooo ×g was mixed with a solution of radioactive protein supernatant. The latter was prepared by a I5o-min centrifugation at IOO o o o × g of the homogenate obtained from chick embryos incubated overnight with IoffC of [14C]proline. The material was subjected to sucrose gradient centrifugation with the results shown in Fig. 5- Under these conditions, no radioactivity was found associated with the non-pelleted ribosomal fractions and only traces with the pellet. It can therefore be concluded that the radioactivity associated with the particulate fractions is not bound by non-specific adsorption.

Non-collagenous protein in the pelletable polysomes The hydroxyproline :proline ratio in the pellet is low for collagen. In addition, collagenase removes only part of the [14C]proline from the pellet and all of the hydroxvproline. This suggests that non-collagenous proteins as well as collagen m a y be formed by the polysomes in the pelletable fraction. This was confirmed by experiments in which []14C]tyrosine was injected into chick embryos. After homogenization and gradient centrifugation in the usual manner, the analysis showed that Biochim. Biophys. =icla, ~34 (t967) 4 ~I 429

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Fig. 5. A d d i t i o n of r a d i o a c t i v e p r o t e i n to n o n - r a d i o a c t i v e p o l y s o m e s . A n o n - r a d i o a c t i v e e m b r y o h o m o g e n a t e w a s p r e p a r e d as in METHODS. H y d r o x y p r o l i n e - a n d proline-labeled s u p e r n a t a n t p r o t e i n was p r e p a r e d as described in t h e t e x t . T h i s r a d i o a c t i v e s u p e r n a t a n t p r o t e i n w a s c o m b i n e d w i t h t h e n o n - r a d i o a c t i v e h o m o g e n a t e a n d c e n t r i f u g e d 120 rain t h r o u g h a 15-3 ° °/o s u c r o s e gradient. T h e poor resolution of t h e r i b o s o m e p e a k is p r o b a b l y d u e to t h e large a m o u n t of m a t e r i a l l a y e r e d on t h e gradient.

radioactivity was incorporated not only in the polysome fractions distributed through the gradient but also in the pellet-forming fraction. For example, in one experiment the I14Cltyrosine in the polysome peptide bound peak had 380 counts/min while the pellet after a i2o-min centrifugation had 73o counts/min. Since collagen contains only traces of tyrosine this shows that other rapidly labeled proteins are present in the pellet.

Kinetics o/ labeling It is known from previous worklS, 16 that after the administration of labeled proline to collagen-forming systems, a lag of several minutes occurs during which the proline but not the hydroxyproline in the newly-formed collagen is radioactive. The significance of this lag period has been interpreted differently by different workers as indicating hydroxylation of proline before or after the assembly of the collagen polypeptide chain. However, in either case, it is clear that the lag period represents the time necessary for the hydroxylation of proline. Accordingly, it was of interest to determine whether this same lag is reflected in the pellet fraction by varyinL• the incubation time after the injection of E14Clproline. A 9o-see incubation with ~a4C~proline showed that even after this brief incubation time significant proline radioactivity had been incorporated in the pelletable material as well as in the slowersedimenting polysome fractions. However, no hydroxyproline-associated radioactivity appeared in this short time period (Fig. 6a). In a 2o-min incubation carried out simultaneously under identical conditions, the radioactivity of the pellet-bound hydroxyproline amounted to almost 2 1 % of the pellet-bound proline (Fig. 6b). The kinetics of the labeling of the pellet thus appears to be similar to that seen in previous experiments on collagen formation 15. Biochim. Biophys..4cta, 134 (1967) 411-429

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Fig. 6. Two e m b r y o s were each i n j e c t e d w i t h 5 ,uC [14CJproline a n d were t h e n a l l ow e d to i n c u b a t e for 9o sec a n d 20 rain r e s p e c t i v e l y . H o m o g e n a t e s were t r e a t e d as in Fig. 2, a n d a i c e - r a i n sucrose g r a d i e n t c e n t r i f u g a t i o n was c a r r i e d out. (a) 9e-see i n c u b a t i o n ha s no h y d r o x y p r o l i n e in pellet. (b) 2o-nfin i n c u b a t i o n h a d h y d r o x y p r o l i n e r a d i o a c t i v i t y a t eo ° o of p r o l i n e r a d i o a c t i v i t y .

Shorter cenlri/u.<,ation time The rapid sedimentation of the collagen-forming material results in the accumulation of nearly all of the particle-bound [l'~Cihydroxyproline in the pellet after a 2-h gradient centrifugation. Shorter centrifugation times were therefore used in an attempt to characterize better the sedimentation properties of the collagenforming material. Fig. 7 shows the results of three independent experiments, A-C, where 20 min of centrifugation was used. The absorbance profile shows that the

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Biochi~L lqiophys. Acta, 134 (z967) 4 r i - 4 2 9

421

POLYRIBOSOMAL SYNTHESIS OF COLLAGEN

polysome peak has not quite separated from the top of the gradient. Radioactive proline follows the absorbance profile, but radioactive hydroxyproline appears to move with a rapidly sedimenting peak near the leading polysome edge. However, it is not a sharply defined peak and some hydroxyproline radioactivity has already accumulated in the pellet after only 20 rain of centrifugation.

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Fig. 8 shows the results of gradient analyses using 55-rain centrifugation. In Fig. 8a the polysome material is just separating from the non-particulate material at the top of the gradient. The distribution of proline radioactivity indicates that the bulk of the proline follows the absorbance curve while hydroxyproline radioactivity has a different distribution with a quite diffuse peak sedimenting around 400 S. By this time considerable amounts of I14C]proline and E14C]hydroxy proline have accumulated in the pellet. The results of ribonuclease treatment of the homogenate prior to a 55-rain centrifugation (Fig. 8b) are comparable to those obtained with longer centrifugation times. The proline and hydroxyproline radioactivities from the polysome fractions are almost completely shifted towards the more slowly sedimenting part of the gradient and single ribosomes can now be seen. About 40-5 ° o/., of both [z4C]proline and [14C]hydroxyproline remains associated with the pelletable material while the remainder is broken down into slower sedimenting particles.

Absorption spectra o/ the pellet Since the bulk of the particle-bound hydroxyproline is in the pellet some properties of the resuspended pellet have been investigated. On the basis of the Biochim. Biophys. Acta, 134 (1967) 411-429

6. MANNER et al.

422

experiments previously described it is clear that the pellet contains nascent protein, including collagen chains. The absorption spectrum of resuspended pellet material was examined, and compared to the spectrum of other ribosomal fractions. Fig. 9 shows the ultraviolet absorption spectrum of the material from the experiment in Fig. 3. Curve A is the spectrum of Fraction 8, Fig. 3 a. The ratio of absorbance at 26o m F to that at 280 mF is i.O which is charactelistic of ribosomes. Curve B represents the spectrum of the pellet from Fig. 3a, resuspended in o. 7 ml of RS buffer. The ratio A260 m,/A2s 0,,,, is identical to that seen in Curve A, suggesting that it is the ribosomes that are predominant in absorbing in the ultraviolet region. Curve C shows the results with the resuspended pellet from Fig. 3b following ribonuclease treatment. The A260 m,/A2so ,n, ratio is now I . I :I, which indicates that a large proportion of the ribosomes have been freed from pelletable material by ribonuclease. The pellet, however, still contains nucleic acid, presumably ribosomal material, since the m a x i m u m absorption is still at 20o mF, rather than 28o m F.

Effect o~ deoxycholate concentration Deoxycholate is generally used to solubilize the lipid of the endoplasmic reticulum and release the polysomes. In order to insure that sufficiently high concentrations of deoxycholate were being used to remove membrane material, ex-

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F i g . 9. S p e c t r a of p o l y s o m e s a n d p e l l e t s . S p e c t r u m A is f r o m F r a c t i o n 8 of t h e g r a d i e n t s h o w n in F i g . 3 a. S p e c t r m n t3 is f r o m t h e p e l l e t of F i g . 3 a r e s u s p e n d e d in 0. 7 m l of R S b u f f e r . S p e c t r u m C is t h e r e s u s p e n d e d p e l l e t of F i g . 3 b d i s s o l v e d in I.O m l of R S b u f f e r . T h i s is a f t e r r i b o n u c l e a s e t r e a t n l e n t of a n a l i q u o t of F i g . 3a. S p e c t r a A a n d B h a v e a A260 m/u//1280 m/* r a t i o of I : 6; s p e c t r u m C h a s a r a t i o of 1.1 : [. Fig. i o . E f f e c t of d e o x y c h o l a t e t r e a t m e n t o n p o l y s o m e p r e p a r a t i o n s . S i x c h i c k e m b r y o s w e r e i n j e c t e d w i t h a b o u t 8 / , C e a c h of L [14C]proline a n d a l l o w e d to i n c u b a t e 3° r a i n b e f o r e b e i n g h o m o g e n i z e d t o g e t h e r . T h e ~o o o o ~ g s u p e r n a t a n t w a s d i v i d e d i n t o e q u a l a l i q u o t s . E a c h a l i q u o t w a s b r o u g h t t o a d i f f e r e n t d e o x y c h o l a t e c o n c e n t r a t i o n o f o % t o 2.0 % b e f o r e u l t r a c e n t r i f u g a t i o n t h r o u g h a ~ 5 - 3 o % s n c r o s e g r a d i e n t f o r IZO m i n . E a c h p e l l e t w a s d i s s o l v e d in i . o m l ] i S b u f f e r ; and its spectrum was recorded before the sample was analyzed for proline and hydroxyproline radioactivity. The plotted data represents an average of two such experiments.

Biochim. Biophys. ~qcta, 134 ( i 9 6 7 ) 4 1 1 - 4 2 9

POLYRIBOSOI~AL SYNTHESIS OF COLLAGEN

423

periments were carried out to determine the absorbance and radioactivity of pellets obtained after treatment of the homogenate with varying concentrations of deoxycholate. Equal aliquots of homogenate were treated with concentrations varying from 0 to 2 %; after 120 min centrifugation through a 15-30 % sucrose gradient the resulting pellet was analyzed. The results are presented in Fig. I0. These show slight differences in absorbance at 26o m# of pellets derived from homogenates treated with from o.2 to 2.o o/,o deoxycholate. The radioactivity found in [14C]proline and hvdroxyproline does decrease with increased deoxycholate concentration; however, there is only a 4 ° o/ decrease between o.2 and 2,o o/ o deoxvcholate and the effect clearly has largely leveled off at I °/o deoxycholate. The greatest change is observed between untreated aliquots and those treated with as low as o.2 o7 ,'o deoxycholate. This is in agreement with the fact that treatment with o.2 °/o deoxycholate is usually sufficient for dissolving the membranes of the endoplasmic reticulum ~7.

Release o/ ribosomes [rom pellet by ribonuclease A reasonable conclusion that could be drawn from the above is that very large polysomes accumulate in the pellet which forms after a i20-min centrifugation. A characteristic feature of polysomes is their marked sensitivity to low levels of ribonuclease even at low temperature and their disaggregation into single ribosomes with the retention of the attached nascent polypeptide chains. This was demonstrated for the collagen system b y resuspending a pellet from a I20-min centrifugation in 2 ml of RS buffer solution and treating it with 2/~g ribonuclease per ml at 2 ° for 20 min. After removal of pelletable material by centrifugation at I0 000 × g, the supernatant was layered over a sucrose gradient and then centrifuged at high speed. The results shown in Fig. I I indicate that ribonuclease treatment of the isolated pellet produces single 75-S ribosomes from the pelletable material. The single ribosome fraction also contains the bulk of the radioactive proline and hydroxyproline released by the ribonuclease treatment. This supports the conclusion that the pellet contains nascent protein chains attached to ribosomes. It is apparent that the distribution of radioactive hydroxyproline is diffuse and that some of it is still attached to slightly larger polysomes containing more than one ribosome.

Identi/ication o/nascent polyp@tide chains in sodium dodecylsul/ale gradient When ribosomes with nascent polypeptide chains are treated with the detergent sodium dodecylsulfate the ribosomal protein is denatured but the nascent polypeptide chain remains attached to transfer RNA and it can be identified through the characteristic sedimentation of transfer RNA near 4 S (ref. 12). This technique was applied to the collagen-synthesizing polysomes in order to establish whether the hydroxyproline-containing polypeptides are indeed attached to ribosomes as peptidylsRNA. The pellet obtained from chick-embryo homogenate by gradient centrifugation for 120 min was resuspended, treated with sodium dodecylsulfate and then subiected to density gradient centrifugation for 22 h. The results are presented in Fig. 12. The tracing of the absorbance at 260 rot, shows two m a x i m a near the bottom of the gradient corresponding to the I8-S and 28-S components of ribosomal RNA, and a peak close to the top of the gradient corresponding to a sedimentation constant of

Biochim. Biophys. Acta, 134 (1967) 4I~-429

424

G. MANNER

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Fig. 11. Sucrose d e n s i t y - g r a d i e n t a n a l y s i s of r i b o n u c l e a s e t r e a t e d pellet. T h e p e l l e t o b t a i n e d a f t e r a i 2 o - m i n c e n t r i f u g a t i o n of t h e h o m o g e n a t e from t h r e e e m b r y o s i n j e c t e d as in Fig. 3 w a s res u s p e n d e d b y g e n t l e h o m o g e n i z a t i o n a n d t r e a t e d w i t h r i b o n u c l e a s e (2 # g / m l ) for 35 rain at. 2 °. G r o s s p a r t i c u l a t e m a t e r i a l was r e m o v e d b y c e n t r i f u g a t i o n a t l o ooo .v g for 15 rain. This clarified s o l u t i o n was t h e n p l a c e d on a 15-3o % sucrose g r a d i e n t in R S t)uffer a n d c e n t r i f u g e d for 27o rain a t 54 ooo.< g. The large p e a k c o r r e s p o n d s to single r i b o s o m e s a n d s m a l l e r a b s o r b a n c e p e a k s c o r r e s p o n d to d o u b l e a n d t r i p l e ribosomes. Fig. 12. C e n t r i f u g a t i o n of s o d i u m d o d e c y l s u l f a t e - t r e a t e d pellet. The pe l l e t o b t a i n e d a f t e r a ~8o m i n c e n t r i f u g a t i o n of t h e h o m o g c n a t c from t w o e m b r y o s w a s r e s u s p e n d e d b y g e n t l e s t i r r i n g for 6o m i n a t 25 ¢ in a s o l u t i o n c o n t a i n i n g o.I M NaC1, o.oo 5 M T r i s - H C l (pH 7.o), a n d 0. 5 ° o s o d i u m d o d e c y l s u l f a t e . The s o l u t i o n was t h e n d i a l y z e d a g a i n s t w a t e r for 2 h while it cooled fronl zo ~' to 2 °. The n o n - d i a l y z a b l e m a t e r i a l was t h e n p l a c e d on a 5 2o % sucrose g r a d i e n t a nd cent r i f u g e d for 22 h. T h e p e a k l a b e l e d 4 S r e p r e s e n t s t h e soluble R N A , w h i l e t h e a b s o r b a n c c a t t h e b o t t o m of t h e g r a d i e n t , a t t h e left, r e p r e s e n t s t h e c o m p o n e n t s of ril)osomal R N A .

4 S characteristic of soluble RNA. Almost all the radioactivity contained in the original pellet is found associated with this fraction, suggesting that the labeled proline and hydroxyproline are actually bound covalently to sRNA and therefore constitute nascent protein.

Effect o~ puromyein treatment Puromycin is known to act oi1 protein-synthesizing systems by detaching the incomplete nascent polypeptide chains from the polysomes. This is accompanied by a disaggregation of polysomes probably as a result of the ribosomes becoming detached from the messenger RNA12, ls-2°. An experiment was carried out to determine whether the radioactive proline and hydroxyproline bound to polysomes would behave in a similar fashion. Chick embryos were incubated with [14C]proline for 20 min after which they were injected with i mg puromycin dissolved in o.i ml saline. The incubation was continued for I5 min longer after which the embryos were homogenized and subjected to gradient centrifugation. The results are shown in Fig. 13. It is evident that this treatment inhibited protein assembly and not only disaggregated Biockim. Bioph),s. dcta, 134 (i966) 411-429

425

POLYRIBOSOMAL SYNTHESIS OF COLLAGEN ~.

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Fig. 13. Sucrose d e n s i t y - g r a d i e n t analysis after p u r o m y c i n t r e a t m e n t of embryo. A 9-day-old chick e m b r y o of b o d y weight i g was injected w i t h 5 #C of L-[14C]proline and allowed to i n c u b a t e 20 rain before the injection of o.i ml saline solution containing i.o mg p u r o m y c i n after which i n c u b a t i o n was continued for an additional 15 rain. The e m b r y o was homogenized and centrifuged in the usual way. B o t h the proline and h y d r o x y p r o l i n e radioactivity h a v e been r e m o v e d from the single r i b o s o m e region, as well as from the pellet.

the polysomes ahnost completely but also detached the radioactive material from the single ribosomes as well as from the material in the pellet. Normally after 2o min incubation, a substantial amount of radioactive proline and hydroxyproline is found in the pellet; onlv a small amount remains after puromycin. Addition of puromycin in vitro, to homogenates of chick embryos previously incubated with [14C]proline had no effect on the distribution of either ultraviolet-absorbing material or radioactivity. These results are thus consistent with the interpretation that the radioactivity associated with the ribosomes in the gradient and in the pellet are indeed associated with nascent chains.

DISCUSSION

The experiments which we have described were designed with three goals in mind: (I) To establish the existence of a polyribosomal mechanism of collagen synthesis. Although it has been well established that various globular proteins are synthesized on polysomes, this mechanism has not been established for fibrous, structural proteins. (2) To determine the size of the postulated collagen polysome. This information might allow one to infer something about the size of the collagen messenger RNA('s). (3) To gain some information about the initial steps of the assembly of the triple stranded tropocollagen molecule. Collagen is an ubiquitous protein in the chick embryo. Not only is it the principal protein component of the dermis, but is found in the connective tissue which is scattered through virtually all of the organs. During the 2o min incubation period after the injection of radioactive proline which was normally used in these experiments, it is likely that a number of events occur including not only the initiation and continuation of the assembly of collagen polypeptide chains, but also the Biochim. Biophys. ~4cta, 134 (1967) 411 429

426

G. MANNER et al.

completion of some chains and their transfer from ribosomal material to other cellular and tissue components. At the end of the 2o rain incubation period in these experiments, the chick embryo is disintegrated into small fragments by Dounce homogenization. This disrupts most of the cells of the chick embryo and permits the recovery, by carrying out a low-speed centrifugation at IO ooo ×g for I5 rain, of the cytoplasmic extract free of relatively larger particles, it is not unlikely that this homogenization breaks up the bulk of the fibrous collagen-containing material into small fragments, most of which presumably is brought down in the IOO O 0 0 X ~r centrifugation. However, it is possible that a small amount of completed collagen molecules already bound into various types of connective tissue remains in the supernatant after the original low-speed centrifugation. The fragments m a y have sedimentation constants in excess of IOOO S and as such m a y be found in the pellet even though they are no longer attached to ribosomes. They m a y no longer be considered nascent protein. Some of the material accumulating in the pellet after the sucrose density centrifugation could arise from this source and accordingly, it might be considered as a possible source of some of the radioactive hydroxyproline which is retained by the pellet even after tile ribonuclease treatment. However, puromyein treatment releases most of the radioactivity from the pellet. This suggests that the amount of completed t)rotein in the pellet is small. The same conclusion can be drawn from tile fact that sodium dodecylsulfate treatment releases all the hydroxyproline radioactivity attached to a 4 S soluble RNA component. The principal findings presented by these experiments are related to the identification of nascent or growing hydroxyproline-containing collagen polypeptide chains which are attached to polyribosomes. This identification has been shown in several ways. Almost half of the hydroxyproline radioactivity in the rapidly sedimenting, pelletable material is released by ribonuclease treatment and is then found attached to single ribosomes. This pellet, on extraction with sodium dodecylsulfate, yields hydroxyproline-containing material which sediments with the 4 S soluble RNA fraction. In addition, puromycin, which attacks only nascent ribosomebound polypeptide chains, releases most of the radioactivity. The hydroxyprolinecontaining polypeptide chains can thus be identified with considerable certainty as being associated with the ribosome-bound material. Tile identification of nascent polypeptide chains in tern> of hydroxyproline shows clearly that in the intact chick embryo, the oxidation of proline occurs at some time before the completed polypeptide chain is released from tile ribosome. The experiments inwflving tyrosine incorporation make it quite clear that other proteins besides collagen are formed and present in tile rapidly sedimenting pelletable material. A portion of the radioactivity in the pelletable material is clearly polysome-bound as shown by the ribonuclease treatment. In general, ribonuclease reduces the hydroxyproline radioactivity of the pelletable material by approximately one-half. In contrast to this, the total proline incorporation is reduced to about one-third. This suggests that the ribosomal aggregates associated with the noncollagenous proteins found on very large polyribosomes m a y be more readily broken up by ribonuclease than is the case for the collagen-containing polyribosomes. it is clear from Fig. 9 that the bulk of the rapidly sedimenting pelletable material has a spectrum similar to that of polysomes. Following ribonuclease treatment perhaps 8o 85 o~, of the ribosomes are liberated. In summary, ribonuclease treatment 13iochim. Biophys. dcta, t34 ( t 9 6 7 ) 4 l~ 429

POLYRIBOSOMAL SYNTHESIS OF COLLAGEN

427

seems to release over 8o °/o of the ribosomes, about 7 ° % of the proline radioactivity which represents proteins in addition to collagen, but only 5o °//o of the hydroxyproline which represents collagen. This suggests that the small number of ribosomes which still remain in the pelletable fraction following ribonuclease treatment m a y have very long collagen molecules attached to them. We surmise that the ribosomes m a y be held in the pelletable material by these chains themselves in addition to the messenger RNA which normally maintains the integrity of the polyribosomes. It is instructive to compare /%galactosidase with collagen. The former molecule is composed of four subunits, each with a molecular weight of about 135 ooo (ref. 5). Ribonuclease treatment of the/3-galactosidase forming polysome completely destroys the polysomal structure and converts it entirely into single ribosomes which still are attached to the nascent fl-galactosidase molecules 5. The ~.-strands of collagen are of a slightly smaller molecular weight than the /5-galactosidase subunits. In contrast, ribonuclease treatment liberates only part of the radioactivity associated with nascent collagen. This m a y be related to the fact that collagen is a fibrous molecule which not only forms a highly elongated triple-stranded structure but, in addition, has an inherent tendency for lateral association. Shorter length of collagen peptides can form triple-stranded molecules, even down to molecular weights of IO ooo. It is the property of lateral association of triple-stranded molecules which eventually gives rise to collagen fibrils and the mature fibers which make up the bulk of connective tissue. It remains an attractive speculation at the present time to believe that this type of association is occurring with some of the longer collagen polypeptide chains which have not yet been completed on the polyribosomal structure. The sucrose gradient experiments carried out during short centrifugation periods shown in Figs. 7, and 8 demonstrate clearly the presence of hydroxyprolinecontaining polysomes which sediment very rapidly. Even after onlv 2o min of centrifugation, as seen in Fig. 7 there is already some accumulation of hydroxyproline radioactivity in the pellet fraction. At the same time, there appears to be a somewhat diffuse peak associated with hydroxyproline polysomes which is moving somewhat more rapidly than the absorbance contour. It m a y be that this very rapid accumulation of hydroxyproline radioactivity in the pellet is associated with collagen molecules which have been completed and detached from the ribosomes during the 2o min incubation period but are still bound to other nascent chains by lateral association. It is clear from Fig. 8b for example, that ribonuclease treatment reduces the pellet hydroxyproline radioactivity to a level which is comparable to that seen in Fig. 7b after the 2o min centrifugation. It is possible to estimate the size of the rapidly sedimenting collagen polysome peak seen in Figs. 7 and 8. An approximate sedimentation constant of 4oo S is assigned by reference to the T 2 bacteriophage virus marker which has a sedimentation constant of 7oo S. Electron-microscope experiments also enable us to make a rough estimate of the number of ribosomes in the polyribosome as a function of distance along the sucrose gradient la. This leads to the conclusion that the collagen polysome structure m a y contain in the order of IOO ribosomes. MALT AND SPEAKMAN21 have also presented evidence which suggests that collagen polysomes are very large. However, it should be pointed out that there is considerable polydispersity seen in Figs. 7 and 8. This m a y be an intrinsic property of the preparation. In partic]~iochim. Biophys. dcta, 134 (I967) 411-429

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6. MANNER el al.

ular, it m a y be that the longer collagen chains on the polysomal structure have already started to associate laterally with other completed collagen molecules and this could be the origin of the observed polydispersity which can be observed by the hydroxyproline radioactivity which appears in the pellet after only 20 rain of centrifugation. There is generally assumed to be a relation between the size of messenger RNA and the size of the polyribosomal structure. In hemoglobin synthesis, a messenger RNA large enough to code for a polypeptide chain having a molecular weight of 17 ooo is found in a polysome containing five ribosomes. If this same relationship exists between the size of the polysome and the size of the polypeptide chain which is being manufactured, it m a y be estimated that a polysome consisting of approx. 3 ° ribosomes would be adequate to synthesize one collagen ~-chain while approx. 9O-lOO ribosomes would contain enough messenger RNA information to synthesize the three collagen ~-chains. These three chains m a y in fact all have different amino acid sequences ~'~. This contrasts with the situation seen in the/#galactosidase polysome where there is one type of asymmetric protein unit with a molecular weight of about 13o ooo and the polysomes synthesizing the molecule contain approx. 4o-5 ° ribosomesS, 6. The present experiments present evidence showing that the collagen polysome is very large and polydisperse, suggesting that all three of the collagen ~-chains m a y be synthesized in the same polysomal structure. It is not possible to say from the present experiments whether this is mediated b y polycistronic messenger-RNA strands which are held together by the nascent polypeptide chains. Further experiments will have to be carried out to characterize the collagen polysome more completely. These experiments present no evidence that throws light on the question of a subunit assembly for the collagen moleculea,G, 2a. On the other hand, a preliminary report by FERNANDEZ-MADF,II) AND ~IASEI?.24 of a study of chick embryo dermis indicates that collagen is formed on very large polydisperse polysomes comparable to those described in the present study but that collagenase treatment breaks these larger structures into smaller polysomal units. They suggest that the larger polysomes m a y be aggregates of pentamer or hexamer units. ( ~ 0 L D B E R G AND G R E E N e5 have also made a preliminary report of density gradient studies of homogenates of collagen synthesizing mouse fibroblast cultures in which they find that the hydroxylation of proline residues in nascent collagen polypeptides appears to be associated with the polysomes sedimenting from 275 to 32o S. The relationship between these phenomena and collagen subunit assembly still remains to be analyzed. ACKNOWLEDGEMENTS

This research was supported by grants from the National Institutes of Health, U.S. Public Health Service and from the National Science Foundation. t~EFEREN('ES

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