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Interchain disulfide bonds at the COOH-terminal end of procollagen synthesized by matrix-free cells from chick embryonic tendon and cartilage
ARCHIVES
OF
BIOCHEMXS~‘BY
Interchain Disulfide Synthesized
EUBRN
R. OLSEN,
Department of Biochemistry,
AND
BIOPHYSICB
175,
341-350
(1976)
Bonds at the COOH-Terminal End of Procollagen by Matrix-Free Cells from Chick Embryonic Tendon and Cartilage HANS-PETER
HOFFMANN,
AND
DARWIN
J. PROCKOP
College of Medicine and Dentistry of New Jersey, Rutgers Medical School, Piscataway, New Jersey 08854 Received
February
2, 1976
Matrix-free cells from tendons and cartilage of chick embryos were incubated in suspension, and the procollagens secreted by the cells were isolated in the presence of protease inhibitors. Tendon procollagen was shown to contain both NH,- and COOHterminal extensions and interchain dieulfide bonds were located in the COOH-terminal region. A disulfide-linked fragment previously isolated after digestion of the molecule with bacterial collagenase was shown to originate from the COOH-terminal end. Cartilage procollagen was also shown to contain interchain dieulfide bonds in the COOHterminal region.
Collagen is first synthesized as a precursor protein called procollagen which is larger than collagen because the three polypeptide chains have additional peptide sequences (for recent reviews, see 14). The structure of procollagen has been examined in a number of laboratories but the efforts have been handicapped by the fact that the molecule readily undergoes partial degradation either during biosynthesis or during purification. The problem of proteolysis in large part accounts for the differences in molecular weight and amino acid composition for similar procollagens reported by different laboratories (l-61, and by observations such as those (7) which incorrectly suggested that procollagen from membranous bone did not contain interchain disulfide bonds. Two methods have been devised for overcoming the problem of proteolysis during the synthesis and isolation of procollagen. One method has been to use freshly isolated cells (8, 9) or cell cultures (6, lo12) which synthesize procollagen but which are largely free of extracellular proteases. A second method has been to use protease inhibitors during extraction of tissues actively synthesizing procollagen 341 Copyright All rights
0 1976 by Academic Pmsa, Inc. of reproduction in any form resewed.
and collagen (13-16). Efforts in our laboratory have concentrated on the use of cells freshly isolated from embryonic tissues and incubated in suspension (8, 9). Initial studies demonstrated that the procollagen synthesized and secreted by cells from embryonic tendon contained NH,-terminal extensions which could be visualized by electron microscopy of segment-long-spacing aggregates of the protein (17). Also, radioisotopic experiments demonstrated that 30 to 60% of the polypeptides in the secreted procollagen were linked by interchain disulfide bonds. On the basis of these observations the interchain disulfide bonds were tentatively assigned to the NH,-terminal region of the molecule. Subsequently, when a disulfidelinked trimer was obtained after bacterial collagenase digestion of the procollagen, the trimer was assumed to arise from the NH,-terminal region of the molecule (18). However, more recent studies with cultured fibroblasts (12) and membranous bone (15, 16) have suggested that procollagen synthesized by these systems contain extensions at both the NH,-terminal and COOH-terminal ends of the molecule. We have therefore undertaken and here report ex-
342
OLSEN,
HOFFMANN
periments to locate directly the collagenase-resistant fragment previously isolated from tendon procollagen. Also, we have determined the location of interchain disulfide bonds in the procollagen synthesized by embryonic cartilage cells. MATERIALS AND METHODS Materials. Most materials were obtained from the sources indicated previously (9, 19). For PAGESDS,’ electrophoresis grade SDS (Bio-Rad) was used. Purified tadpole collagenase was kindly provided by Dr. Yutaka Nagai, Tokyo Medical and Dental University, Tokyo, Japan. The specific activity of the enzyme was approximately 3500 units/mg, based on the definition of 1 unit as the amount of enzyme required to digest 1 pg of collagen/min (20). Isolation don cells.
of procollagen
from
the medium
of ten-
Cells were isolated by controlled digestion of tendons or sternal cartilages from lFday-old chick embryos as described previously (8, 9, 19). From 1 to 2 x lo9 cells from 60 to 200 embryos were used for most experiments. Tendon cells were incubated at a concentration of 7.5 x 10Vml with 0.38 @i/ml of [Wlproline for 4 h at 37°C in modified Krebs medium (8) without fetal calf serum. Cartilage cells were incubated under the same conditions with 20% fetal calf serum. After the incubation, the cells were removed by centrifugation at 1200g for 10 min. The medium was cooled on ice and proteolysis was inhibited by adding protease inhibitors (13, 14) to give the following final concentrations: 25 mM disodium ethylene-diaminetetracetate (Baker); 10 mM N-ethylmaleimide (Sigma); 0.1 or 1.0 mM phenylmethylsulfonylfluoride (Sigma) and 1 mM p-aminobenzamidine-HCl (Sigma). The medium procollagen was precipitated by adding 176 mg/ml of ammonium sulfate (Baker) and stirring the solution overnight. The precipitate was removed by centrifuging at 30,OOOg for 30 min and then it was dissolved in one-fiftieth of the initial medium volume of 0.4 M NaCl and 0.1 M Tris-HCl buffer, pH 7.8 at 4°C. The solution was centrifuged at 30,OOOg for 30 min to remove insoluble material which did not contain W. The [‘4C]procollagen was either dialyzed against 0.5 M acetic acid at 4°C in preparation for pepsin digestion, or it was dialyzed against 0.4 M NaCl and 0.1 M Tris-HCl buffer, pH 7.8, in preparation for digestion with either tadpole collagenase or bacterial collagenase. Enzymic digestions. For digestion with pepsin, about 2 x lo6 cpm/ml of [14C]procollagen in 0.5 M acetic acid was incubated with 100 pg/ml of pepsin I Abbreviation used: PAGE-SDS, polyacrylemide gel electrophoresis in the presence of sodium dodecyl sulfate.
AND PROCKOP at 4°C for 6 h, and then the sample was dialyzed against 0.4 M NaCl and 0.1 M Tris-HCl buffer, pH 7.8 at 4°C. For digestion with tadpole collagenase, 1 to 2 x lo6 cpm/ml of [Wlprocollagen in 0.4 M NaCl and 0.1 M Tris-HCl buffer was diluted with an equal volume of 10 mM CaCl, in distilled water. Tadpole collagenase was added to a final concentration of 1.3 units/ ml and the sample was incubated at 22 to 23°C for 3 to 7 h. For digestion with bacterial collagenase, Wlprocollagen or [Wlprocollagen fragments obtained by cleavage with tadpole collagenase were prepared in 0.4 M NaCl and 0.1 M Tris-HCl buffer as described above, and the solutions were adjusted to 5 rnre CaCl, and 2.5 mM N-ethylmaleimide. Bacterial collagenase which was purified by gel filtration (21) was added in an amount of 50 pg/ml and the sample was incubated at 37°C for 2 h. After the enzymic digestions, samples were frozen at -20°C. Ammonium sulfate fractionation of fragments obtained by tadpole collagenuse cleavage. Ammonium
sulfate, 117 mg/ml, was added to the digest containing 1.7 x 10’ cpm in 8 ml and the sample was stirred at 4°C for 3 h. The precipitate was removed by centrifugation at 30,OOOgand 4°C for 30 min. Ammonium sulfate, 59 mg/ml, was added to the supemate and the sample was again stirred for 3 h. The second precipitate was removed by repeating the centrifugation. The first precipitate was dissolved in 4 ml and the second precipitate was dissolved in 2 ml of 0.4 M NaCl and 0.1 M Tris-HCl buffer, pH 7.8 at 4”C, by stirring at 4°C for 1 h. After centrifuging at 30,OOOg for 30 min, the supemates were stored frozen at -20°C. PAGE-SDS. Polyacrylamide gel electrophoresis was carried out on slab gels according to the procedure of King and Laemmli (22) and Studier (23). Separating gels of 2 mm thickness were prepared from either 4.5,6, or 12% polyacrylamide, and stacking gels with 4.5 or 6% polyacrylamide. The electrophoresis was carried out with about 50 mA for 3 h at 25°C. Samples were prepared by adding 10% SDS to a final concentration of 2% and immediately heating at 100°C for 3 min. The samples were then dialyzed against the following “sample buffer”: 0.125 mu Tris-HCl buffer, pH 6.8 at room temperature; 2% SDS; 10% glycerol; and 0.001% bromophenol blue. The samples were stored at -20°C. Before being applied to the gels, aliquots of about 20,000 cpm were adjusted to a volume of 100 ~1 by adding sample buffer, P-mercaptoethanol was added to a final concentration of 5%, and the samples were heated at 100°C for 2 min. For fluorography, the gels were impregnated with 2,5-diphenyloxazole (PPQ Eastman) (24). The gels were dried under vacuum and they were exposed to RP Royal “X-OMAT” X-ray film at -70°C for 6 to 24 h.
INTERCHAIN
DISULFIDE
-MSH
Gel filtration with and without SDS. For gel tiltration in the presence of SDS, samples dissolved in 0.4 M NaCl and 0.1 M Tris-HCl were adjusted to 2% SDS and 5% 2-mercaptoethanol and were heated at 100°C for 3 min before being placed on the column. Gel filtration was carried out on a 1.5 x 90 cm column of 6% agarose (Bio-Gel A-5m, 200400 mesh) which was equilibrated and eluted with 2% SDS (Sigma) in 0.125 M Tris-HCl buffer. Gel filtration without SDS was carried out in 0.1 M Tris-HCl, pH 7.8 at 4”C, on a 1.5 x 90 cm column of 8% agarose (Bio-Gel A-1.5m, 200400 mesh) (18). EXPERIMENTAL
343
BONDS AT THE COOH-TERMINAL
+MSH
Pro ‘)(
RESULTS
Isolation and characterization of procollagen from the medium of tendon cells.
Tendon cells were prepared and incubated with [14Clproline, and the [14Clprocollagen secreted into the medium was isolated. The isolation procedures were similar to those employed previously (8, 19) except that protease inhibitors (13, 14) were added to the medium immediately after the incubation period and before the proteins in the medium were precipitated with ammonium sulfate. PAGE-SDS demonstrated that most of the 14C-protein in the medium migrated as a single band which was tentatively identified as pray chains of procollagen (Fig. 1). When the 14C-protein was reduced with 2-mercaptoethanol before application to the gels, most of the radioactivity was recovered in two bands identified as pro& and proa chains of procollagen (see below). Minor bands just above and just below the procu2 band were also seen and these were subsequently shown to be the result of partial degradation of procrl and procu2 (see below). Treatment of the medium procollagen with tadpole collagenase indicated that inter-chain disulfide bonds were located in the COOH-terminal region of the procollagen molecule. Tadpole collagenase specifically cleaves collagen at a site about threequarters of the distance between the NH,and COOH-terminal ends of the triple helix (25). Therefore, the fragments al*, Lz2*, alB, and CY~~are obtained when the enzyme is used to cleave collagen or procollagen in which the additional peptide sequences are removed by limited proteolysis with pepsin. When procollagen from
*Proai - Pro a2
Dye front 1
2
1. PAGE-SDS of tendon [14Clprocollagen isolated in the presence of protease inhibitors. Electrophoresis was carried out with a 4.5% stacking gel and a 4.5% separating gel. Each sample contained about 12,000 cpm and the film was exposed overnight. Gel 1: [WlProcollagen examined without reduction. Gel 2: 1WlProcollagen examined afbr reduction. Two minor bands were present just above and below the procu2 band. FIG.
the medium was treated with tadpole collagenase and examined by PAGE-SDS, three bands were seen (Gel 1 in Fig. 2). Two of the bands had slightly lower mobilities than ~rl* and (u2* peptides obtained by cleaving pepsin-treated procollagen with tadpole collagenase (Gel 3 in Fig. 2) and these two bands were subsequently identified as proal* and procw2* (see below). The migrations of proal* and procu2* were not afkted by reduction with 2-mercaptoethanol (Gel 2 in Fig. 2). The third band (Gel 1 in Fig. 21, tentatively identified as pro-rB, had a lower mobility than pro& when electrophoresed without reduction. After reduction, proyB was converted to two bands which had electrophoretie mobilities between procu2 and alB and which were identified as proculB and
344
OLSEN, -MSH
HOFFMANN +MSH
AND PROCKOP -MSH
+MSH
1 2 3 4 FIG. 2. PAGE-SDS of tendon [Wlprocollagen cleaved with tadpole collagenase. Electrophoresis was carried out with a 6% stacking gel and a 12% separating gel. Each sample contained about 25,000 cpm and the film was exposed overnight. Gel 1: [l*C]Procollagen treated with tadpole collagenase and examined without reduction. Gel 2: [WlProcollagen treated with tadpole collagenase and examined after reduction. Gel 3: Pepsin-treated [Wlprocollagen which was cleaved with tadpole collagenase and examined without reduction. Gel 4: Pepsin-treated [14C]procollagen which was cleaved with tadpole collagenase and examined after reduction.
~roa2~ (Gel 2 in Fig. 2). The conclusion that reduction converted proyB to procylB and ~roa2~ was verified by two-dimensional gel electrophoresis (Fig. 3). Separation of proalA and prodA from proal B and pro& B. The cleavage products obtained by digestion of tendon procollagen with tadpole collagenase were separated by precipitation first with 20% ammonium sulfate and then with 30% ammonium sulfate. The two pellets were reduced with 2-mercaptoethanol and examined by gel filtration in SDS. The 20% ammonium sulfate pellet largely consisted of the NH,-terminal fragments proal* and proor2* (Figs. 4 and 5). The 30% ammonium sulfate pellet largely consisted of the COOH-terminal fragments procrlB and procu2B (Figs. 4 and 5). The nature of the fragment obtained by precipitation with 20% ammonium sulfate was further established by preparing segment-long-spacing aggregates. As shown in Fig. 6, the aggregates corresponded to the NH,-terminal fragment expected when procollagen is cleaved with tadpole collagenase.
Isolation of NH2 and COOH-terminal peptides resistant to bacterial collagenase. Tendon procollagen was cleaved with tadpole collagenase, the NH,- and COOH-terminal fragments were separated by ammonium sulfate fractionation, and the two fractions were digested with purified bacterial collagenase. Gel filtration of the digest of the COOH-terminal fraction (proyB) gave a peak (peak I in Fig. 7) which had the same elution position as the disulfide-linked peptide fragment previously obtained after digestion of tendon procollagen by bacterial collagenase (18). The radioactive material in peak I was shown to react with antibody directed against the disulfide bonded fragment previously obtained after digestion of procollagen with bacterial collagenase (18). Also, examination of the material in peak I by PAGE-SDS with and without reduction demonstrated that it contained interchain disulfide bonds. The results therefore demonstrated that the disulfidelinked fragment arose from the COOHterminal end. Gel filtration of the digest from the NH,-terminal fragments (procul*
INTERCHAIN
DISULFIDE
BONDS AT THE COOH-TERMINAL
345
Pr0aiB ProalA
Pro 42’ fDye front
FIG. 3. Two-dimensional PAGE-SDS of tendon [14Clprocollagen treated with tadpole collagenase. Separation in the first dimension (top to bottom) was carried out without reduction. After the first separation, the gel strip was cut out and immersed in “sample buffer” containing 5% 2-mercaptoethanol for 10 min and then it was placed on top of a second gel to carry out electrophoresis in the second dimension (left to right). In each case a 6% stacking gel and a 6% separating gel were used. The sample contained about 40,000 cpm and the film was exposed for 6 h. The results demonstrate that the band initially identified as proyB was converted by reduction to two spots corresponding to the positions of proolB and procu2B.
and proa2*) gave a peak (peak II in Fig. 7) which eluted after the disulfide-linked fragment. Peak II was previously obtained after digestion of tendon procollagen with bacterial collagenase and it was shown to contain peptides which were antigenically different from the disulfide-linked fragment (18). Since peak II was obtained by digestion of the NH,-terminal fragments (proal* and procu2*) with bacterial collagenase, the results presented here demonstrate that peak II (Fig. 7C) contains collagenase-resistant peptides from the NH,terminal region. Tendon procollagen without iVH2-terminul extensions. As indicated in Figs. 1 and 2, two weak minor bands with slightly lower and higher mobilities than proa chains were present in procollagen from the medium of tendon cells. These bands were shown to arise from degradation of procollagen by experiments in which the medium 14C-protein was isolated in the
absence of protease inhibitors. When the medium 14C-protein isolated in this manner was reduced and examined by PAGESDS, most of the 14C was found in two bands which were just above and below the position of the proa band and which corresponded to the minor bands seen with procollagen isolated in the presence of inhibitors (Fig. 8). When the procollagen isolated in the absence of protease inhibitors was cleaved with tadpole collagenase and the products were examined by PAGESDS without reduction, three bands were seen. One band had the same mobility as intact proyB (Fig. 8). After reduction this band was converted to two bands with the same mobilities as proculB and pro.0(y2B,indicating that the COOH-terminal end of the partially degraded molecule was largely intact. The other two bands obtained after cleavage of the partially degraded procollagen with tadpole collagenase had the same mobilities as ~rl* and
346
OLSEN, I’
’
HOFFMANN
”
AND PROCKOP
The results (Fig. 9) demonstrated that the interchain disulfide bonds were located in the COOH-terminal fragment. DISCUSSION
The difficulty of obtaining intact procollagen has confused the interpretation of a considerable amount of data on the chemical structure of the molecule, the functions of the peptide extensions, and genetic defects in the conversion of procollagen to collagen. Until rigorous criteria are established for intactness of the molecule, it will be important to examine in detail the pro-
FIG. 4. Ammonium sulfate fractionation of tendon [Wlprocollagen cleaved with tadpole collagenase. The [W]procollagen was digested with tadpole collagenase for 7 h and then precipitated with 20% ammonium sulfate and 30% ammonium sulfate. Samples containing about 200,000 cpm were reduced and treated with SDS before gel filtration in the presence of SDS (see Methods). Fractions of 1.4 ml were collected and aliquots of 0.4 ml were taken for liquid scintillation counting. (A) Total digest obtained after incubation with tadpole collagenase; (B) 20% ammonium sulfate precipitate; (C) 30% ammonium sulfate precipitate.
a2* from pepsin-treated procollagen (Fig. 8). The results indicated therefore that the tendon procollagen isolated in the absence of protease inhibitors lacked most or all of the NH,-terminal peptide extensions found in the intact molecule. Interchain disulfide bonds at the COOH-terminal end of cartilage procollagen. Matrix-free cells isolated from chick embryo sternae synthesize and secrete a cartilage procollagen (9) which contains interchain disulfide bonds (26). To locate bonds, the interchain disulfide [‘4c]procollagen from the medium of cartilage cells was also cleaved with tadpole collagenase and examined by PAGE-SDS.
ProC IA Pro(Z2A ProCClB Procc 2B Dye front 123 RG. 5. PAGE-SDS of tendon Wprocollagen cleaved with tadpole collagenase and fractionated by ammonium sulfate precipitation. Electrophoresis was carried out with a 6% stacking gel and a 6% separating gel. Each sample contained about 30,000 cpm and the film was exposed for 6 h. Gel 1: Total digest obtained after incubation with tadpole collagenase. Gel 2: 30% ammonium sulfate precipitate. Gel 3: 26% ammonium sulfate precipitate. All samples were reduced.
INTERCHAIN
DISULFIDE
BONDS AT THE COOH-TERMINAL
FIG. 6. Segment-long-spacing aggregate prepared from the 20% ammonium sulfate precipitate shown in Fig. 4B. The sample was prepared by dialyzing the precipitate against 0.01 M acetic acid containing 0.2% ATP and it was examined by electron microscopy as described previously (17). Negative staining was performed with 1% sodium phosphotungstate. The arrowheads indicate the peptide extensions on the NH,-terminal end of procollagen and the arrows indicate the region of the procollagen molecule cleaved by the tadpole collagenase. The total length of the segment is about 230 nm, and its NH,-terminal origin was confirmed by comparison with segment-long-spacing aggregates of collagen and procollagen (H.-P. Hoffmann, B. R. Olsen, H.-T. Chen, and D. J. Prockop, in preparation).
collagens which can be isolated from different biological systems. The results obtained here with tendon procollagen are generally consistent with observations which were recently made with procollagen from membranous bone of chick embryos and which were reported as the present experiments were in progress (15, 16). The procollagens from both
membranous bone and tendon are similar in that they contain both NH,-terminal and COOH-terminal extensions and in that the COOH-terminal extensions are linked by inter-chain disulfide bonds. We have recently confirmed the presence of both NH,- and COOH-terminal extensions in segment-long-spacing aggregates of purified tendon procollagen (H.-P. Hoff-
348
OLSEN,
HOFFMANN -
L
FIG. 7. Isolation of NH*- and COOH-terminal peptides resistant to bacterial collagenase. Bacterial was used to digest tendon collagenase [W]procollagen, the NH,-terminal fragments, or the COOH-terminal fragments obtained by cleavage of procollagen with tadpole collagenase. The samples were then examined by gel filtration in 0.1 M Tris-HCl buffer. Fractions of 1.6 ml were collected and aliquots of 0.4 ml were taken for liquid scintillation counting. (A) [WlProcollagen, 2.8 x lo6 cpm, digested with bacterial collagenase. (B) COOH-terminal fragment pray*, 7.1 x lo5 cpm, digested with bacterial collagenase. (C) NH,-terminal fragments proal* and proa2*, 2.1 x 1Og cpm, digested with bacterial collagenase.
mann, B. R. Olsen, H.-T. Chen, and D. J. Prockop, in preparation). Of special importance was the demonstration here that the disulfide-linked fragment previously isolated after digestion of tendon procollagen with bacterial collagenase (18) originates from the COOH-terminal end of the molecule. The new assignment of the disulfidelinked fragment helps to clarify a number
AND PROCKOP
of previous observations on the nature of the fragment and the use of specific antibodies against the fragment (10, 18, 27). For example, the new assignment of the fragment explains why specific antibodies could be used to locate procollagen in cells by electron microscopy (27) even though the antibodies did not react with partially degraded pro al chains (28). The fragments proal* and proa2* isolated from tendon procollagen were not linked by interchain disulfide bonds, suggesting that no such links are found at the NH,-terminal end of the molecule. This conclusion, however, is tentative since the data do not rigorously exclude the possibility that short peptide sequences containing inter-chain bonds were removed by intracellular proteolysis, by presence of proteases in the incubation medium, or by the tadpole collagenase used in the experiments. Similar observations have recently been made with procollagen from membranous bone, and a similar conclusion was suggested (15). The medium of freshly isolated tendon cells contained small amounts of procollagen in which the COOH-terminal end was intact but the NH,-terminal extensions were either absent or very much smaller in size. This form of partially degraded procollagen may be similar to the “altered procollagen” which was detected in membranous bone and which was suggested to be an intermediate in the conversion of procollagen to collagen (16). The partially degraded procollagen encountered here may arise from nonspecific proteolysis during incubation of the cell system, but it may also represent the first intermediate which is formed during the conversion of procollagen to collagen in uiuo. Preliminary results with pulse chase experiments suggest this is in fact the normal sequence in intact tendons (S. Aase and B. R. Olsen, in preparation). Cartilage contains a collagen which is genetically distinct from the collagen found in either tendon or bone (29). The results presented here demonstrate that interchain disulfide bonds previously identified in cartilage procollagen (26) are located in the COOH-terminal region of the molecule. Electron microscopy of segment-
INTERCHAIN
DISULFIDE
-MSH
+MSH
Pro pro
349
BONDS AT THE COOH-TERMINAL
ai A+ a2A+
+MSH
,aiA .a2A
2a 2b la lb 3a 3b 3c 8. PAGE-SDS of partially degraded tendon procollagen isolated in the absence of protease inhibitors. The stacking and separating gels were 6% polyacrylamide. The samples contained from 17,000 to 28,006 cpm and the film was exposed for 20 h. Gel la: [14C]Procollagen isolated in the presence of inhibitors; reduced. Gel lb: Partially degraded [“Clprocollagen; reduced. Gel 2a: [14ClProcollagen isolated in the presence of protease inhibitors and cleaved with tadpole collagenase; not reduced. Gel 2b: Partially degraded [“C]procollagen cleaved with tadpole collagenase; not reduced. Gel 3a: [‘*C]Procollagen isolated in the presence of protease inhibitors and cleaved with tadpole collagenase; reduced. Gel 3b: Partially degraded [“Clprocollagen cleaved with tadpole collagenase; reduced. Gel 3c: Control sample of pepsintreated procollagen cleaved with tadpole collagenase; reduced. The fragments cylBand CX~~are in the dye front. FIG.
long-spacing aggregates of cartilage procollagen demonstrated that both ends of the molecule contain extensions not found in cartilage collagen (J. Uitto, H.-P. Hoffmann, B. R. Olsen, and D. J. Prockop, in preparation). It may be that all procollagens are similar in having both NH,- and COOH-terminal extensions and that the two types of extensions have distinct functions in triple-helix formation, in secretion, and in fiber formation. 5
IO
MIGRATION DISTANCE ICMI
FIG. 9. PAGE-SDS of cartilage procollagen cleaved with tadpole collagenase. Stacking and separating gels were 6%, each sample contained 15,000 to 40,000 cpm, and the film was exposed for 12 h. The fluorograms were scanned on Quick Scan (Helena Laboratories). (A) Tadpole collagenase digest; not reduced. (B) Tadpole collagenase digest; reduced. Because the cleavage with tadpole collagenase was incomplete, uncleaved pray chains were seen in the unreduced sample (A) and uncleaved proa chains were seen in the reduced sample (B).
ACKNOWLEDGMENTS The work was supported in part by Research Grant AM-16516 from the National Institutes of Health of the U.S. Public Health Service. The authors gratefully acknowledge the expert assistance of Ms. Chris Condit and Ms. Nancy Kedersha. REFERENCES 1. PROCKOP, D. J., BERG, R. A., KIVIRIKKO, K. I., AND Urrro, J. (1976) in Biochemistry of Collagen (Ramachandran, G. N. & Reddi, A. H., eds.), Plenum, New York (in press).
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OLSEN,
HOFFMANN
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OF
BIOCHEMXS~‘BY
Interchain Disulfide Synthesized
EUBRN
R. OLSEN,
Department of Biochemistry,
AND
BIOPHYSICB
175,
341-350
(1976)
Bonds at the COOH-Terminal End of Procollagen by Matrix-Free Cells from Chick Embryonic Tendon and Cartilage HANS-PETER
HOFFMANN,
AND
DARWIN
J. PROCKOP
College of Medicine and Dentistry of New Jersey, Rutgers Medical School, Piscataway, New Jersey 08854 Received
February
2, 1976
Matrix-free cells from tendons and cartilage of chick embryos were incubated in suspension, and the procollagens secreted by the cells were isolated in the presence of protease inhibitors. Tendon procollagen was shown to contain both NH,- and COOHterminal extensions and interchain dieulfide bonds were located in the COOH-terminal region. A disulfide-linked fragment previously isolated after digestion of the molecule with bacterial collagenase was shown to originate from the COOH-terminal end. Cartilage procollagen was also shown to contain interchain dieulfide bonds in the COOHterminal region.
Collagen is first synthesized as a precursor protein called procollagen which is larger than collagen because the three polypeptide chains have additional peptide sequences (for recent reviews, see 14). The structure of procollagen has been examined in a number of laboratories but the efforts have been handicapped by the fact that the molecule readily undergoes partial degradation either during biosynthesis or during purification. The problem of proteolysis in large part accounts for the differences in molecular weight and amino acid composition for similar procollagens reported by different laboratories (l-61, and by observations such as those (7) which incorrectly suggested that procollagen from membranous bone did not contain interchain disulfide bonds. Two methods have been devised for overcoming the problem of proteolysis during the synthesis and isolation of procollagen. One method has been to use freshly isolated cells (8, 9) or cell cultures (6, lo12) which synthesize procollagen but which are largely free of extracellular proteases. A second method has been to use protease inhibitors during extraction of tissues actively synthesizing procollagen 341 Copyright All rights
0 1976 by Academic Pmsa, Inc. of reproduction in any form resewed.
and collagen (13-16). Efforts in our laboratory have concentrated on the use of cells freshly isolated from embryonic tissues and incubated in suspension (8, 9). Initial studies demonstrated that the procollagen synthesized and secreted by cells from embryonic tendon contained NH,-terminal extensions which could be visualized by electron microscopy of segment-long-spacing aggregates of the protein (17). Also, radioisotopic experiments demonstrated that 30 to 60% of the polypeptides in the secreted procollagen were linked by interchain disulfide bonds. On the basis of these observations the interchain disulfide bonds were tentatively assigned to the NH,-terminal region of the molecule. Subsequently, when a disulfidelinked trimer was obtained after bacterial collagenase digestion of the procollagen, the trimer was assumed to arise from the NH,-terminal region of the molecule (18). However, more recent studies with cultured fibroblasts (12) and membranous bone (15, 16) have suggested that procollagen synthesized by these systems contain extensions at both the NH,-terminal and COOH-terminal ends of the molecule. We have therefore undertaken and here report ex-
342
OLSEN,
HOFFMANN
periments to locate directly the collagenase-resistant fragment previously isolated from tendon procollagen. Also, we have determined the location of interchain disulfide bonds in the procollagen synthesized by embryonic cartilage cells. MATERIALS AND METHODS Materials. Most materials were obtained from the sources indicated previously (9, 19). For PAGESDS,’ electrophoresis grade SDS (Bio-Rad) was used. Purified tadpole collagenase was kindly provided by Dr. Yutaka Nagai, Tokyo Medical and Dental University, Tokyo, Japan. The specific activity of the enzyme was approximately 3500 units/mg, based on the definition of 1 unit as the amount of enzyme required to digest 1 pg of collagen/min (20). Isolation don cells.
of procollagen
from
the medium
of ten-
Cells were isolated by controlled digestion of tendons or sternal cartilages from lFday-old chick embryos as described previously (8, 9, 19). From 1 to 2 x lo9 cells from 60 to 200 embryos were used for most experiments. Tendon cells were incubated at a concentration of 7.5 x 10Vml with 0.38 @i/ml of [Wlproline for 4 h at 37°C in modified Krebs medium (8) without fetal calf serum. Cartilage cells were incubated under the same conditions with 20% fetal calf serum. After the incubation, the cells were removed by centrifugation at 1200g for 10 min. The medium was cooled on ice and proteolysis was inhibited by adding protease inhibitors (13, 14) to give the following final concentrations: 25 mM disodium ethylene-diaminetetracetate (Baker); 10 mM N-ethylmaleimide (Sigma); 0.1 or 1.0 mM phenylmethylsulfonylfluoride (Sigma) and 1 mM p-aminobenzamidine-HCl (Sigma). The medium procollagen was precipitated by adding 176 mg/ml of ammonium sulfate (Baker) and stirring the solution overnight. The precipitate was removed by centrifuging at 30,OOOg for 30 min and then it was dissolved in one-fiftieth of the initial medium volume of 0.4 M NaCl and 0.1 M Tris-HCl buffer, pH 7.8 at 4°C. The solution was centrifuged at 30,OOOg for 30 min to remove insoluble material which did not contain W. The [‘4C]procollagen was either dialyzed against 0.5 M acetic acid at 4°C in preparation for pepsin digestion, or it was dialyzed against 0.4 M NaCl and 0.1 M Tris-HCl buffer, pH 7.8, in preparation for digestion with either tadpole collagenase or bacterial collagenase. Enzymic digestions. For digestion with pepsin, about 2 x lo6 cpm/ml of [14C]procollagen in 0.5 M acetic acid was incubated with 100 pg/ml of pepsin I Abbreviation used: PAGE-SDS, polyacrylemide gel electrophoresis in the presence of sodium dodecyl sulfate.
AND PROCKOP at 4°C for 6 h, and then the sample was dialyzed against 0.4 M NaCl and 0.1 M Tris-HCl buffer, pH 7.8 at 4°C. For digestion with tadpole collagenase, 1 to 2 x lo6 cpm/ml of [Wlprocollagen in 0.4 M NaCl and 0.1 M Tris-HCl buffer was diluted with an equal volume of 10 mM CaCl, in distilled water. Tadpole collagenase was added to a final concentration of 1.3 units/ ml and the sample was incubated at 22 to 23°C for 3 to 7 h. For digestion with bacterial collagenase, Wlprocollagen or [Wlprocollagen fragments obtained by cleavage with tadpole collagenase were prepared in 0.4 M NaCl and 0.1 M Tris-HCl buffer as described above, and the solutions were adjusted to 5 rnre CaCl, and 2.5 mM N-ethylmaleimide. Bacterial collagenase which was purified by gel filtration (21) was added in an amount of 50 pg/ml and the sample was incubated at 37°C for 2 h. After the enzymic digestions, samples were frozen at -20°C. Ammonium sulfate fractionation of fragments obtained by tadpole collagenuse cleavage. Ammonium
sulfate, 117 mg/ml, was added to the digest containing 1.7 x 10’ cpm in 8 ml and the sample was stirred at 4°C for 3 h. The precipitate was removed by centrifugation at 30,OOOgand 4°C for 30 min. Ammonium sulfate, 59 mg/ml, was added to the supemate and the sample was again stirred for 3 h. The second precipitate was removed by repeating the centrifugation. The first precipitate was dissolved in 4 ml and the second precipitate was dissolved in 2 ml of 0.4 M NaCl and 0.1 M Tris-HCl buffer, pH 7.8 at 4”C, by stirring at 4°C for 1 h. After centrifuging at 30,OOOg for 30 min, the supemates were stored frozen at -20°C. PAGE-SDS. Polyacrylamide gel electrophoresis was carried out on slab gels according to the procedure of King and Laemmli (22) and Studier (23). Separating gels of 2 mm thickness were prepared from either 4.5,6, or 12% polyacrylamide, and stacking gels with 4.5 or 6% polyacrylamide. The electrophoresis was carried out with about 50 mA for 3 h at 25°C. Samples were prepared by adding 10% SDS to a final concentration of 2% and immediately heating at 100°C for 3 min. The samples were then dialyzed against the following “sample buffer”: 0.125 mu Tris-HCl buffer, pH 6.8 at room temperature; 2% SDS; 10% glycerol; and 0.001% bromophenol blue. The samples were stored at -20°C. Before being applied to the gels, aliquots of about 20,000 cpm were adjusted to a volume of 100 ~1 by adding sample buffer, P-mercaptoethanol was added to a final concentration of 5%, and the samples were heated at 100°C for 2 min. For fluorography, the gels were impregnated with 2,5-diphenyloxazole (PPQ Eastman) (24). The gels were dried under vacuum and they were exposed to RP Royal “X-OMAT” X-ray film at -70°C for 6 to 24 h.
INTERCHAIN
DISULFIDE
-MSH
Gel filtration with and without SDS. For gel tiltration in the presence of SDS, samples dissolved in 0.4 M NaCl and 0.1 M Tris-HCl were adjusted to 2% SDS and 5% 2-mercaptoethanol and were heated at 100°C for 3 min before being placed on the column. Gel filtration was carried out on a 1.5 x 90 cm column of 6% agarose (Bio-Gel A-5m, 200400 mesh) which was equilibrated and eluted with 2% SDS (Sigma) in 0.125 M Tris-HCl buffer. Gel filtration without SDS was carried out in 0.1 M Tris-HCl, pH 7.8 at 4”C, on a 1.5 x 90 cm column of 8% agarose (Bio-Gel A-1.5m, 200400 mesh) (18). EXPERIMENTAL
343
BONDS AT THE COOH-TERMINAL
+MSH
Pro ‘)(
RESULTS
Isolation and characterization of procollagen from the medium of tendon cells.
Tendon cells were prepared and incubated with [14Clproline, and the [14Clprocollagen secreted into the medium was isolated. The isolation procedures were similar to those employed previously (8, 19) except that protease inhibitors (13, 14) were added to the medium immediately after the incubation period and before the proteins in the medium were precipitated with ammonium sulfate. PAGE-SDS demonstrated that most of the 14C-protein in the medium migrated as a single band which was tentatively identified as pray chains of procollagen (Fig. 1). When the 14C-protein was reduced with 2-mercaptoethanol before application to the gels, most of the radioactivity was recovered in two bands identified as pro& and proa chains of procollagen (see below). Minor bands just above and just below the procu2 band were also seen and these were subsequently shown to be the result of partial degradation of procrl and procu2 (see below). Treatment of the medium procollagen with tadpole collagenase indicated that inter-chain disulfide bonds were located in the COOH-terminal region of the procollagen molecule. Tadpole collagenase specifically cleaves collagen at a site about threequarters of the distance between the NH,and COOH-terminal ends of the triple helix (25). Therefore, the fragments al*, Lz2*, alB, and CY~~are obtained when the enzyme is used to cleave collagen or procollagen in which the additional peptide sequences are removed by limited proteolysis with pepsin. When procollagen from
*Proai - Pro a2
Dye front 1
2
1. PAGE-SDS of tendon [14Clprocollagen isolated in the presence of protease inhibitors. Electrophoresis was carried out with a 4.5% stacking gel and a 4.5% separating gel. Each sample contained about 12,000 cpm and the film was exposed overnight. Gel 1: [WlProcollagen examined without reduction. Gel 2: 1WlProcollagen examined afbr reduction. Two minor bands were present just above and below the procu2 band. FIG.
the medium was treated with tadpole collagenase and examined by PAGE-SDS, three bands were seen (Gel 1 in Fig. 2). Two of the bands had slightly lower mobilities than ~rl* and (u2* peptides obtained by cleaving pepsin-treated procollagen with tadpole collagenase (Gel 3 in Fig. 2) and these two bands were subsequently identified as proal* and procw2* (see below). The migrations of proal* and procu2* were not afkted by reduction with 2-mercaptoethanol (Gel 2 in Fig. 2). The third band (Gel 1 in Fig. 21, tentatively identified as pro-rB, had a lower mobility than pro& when electrophoresed without reduction. After reduction, proyB was converted to two bands which had electrophoretie mobilities between procu2 and alB and which were identified as proculB and
344
OLSEN, -MSH
HOFFMANN +MSH
AND PROCKOP -MSH
+MSH
1 2 3 4 FIG. 2. PAGE-SDS of tendon [Wlprocollagen cleaved with tadpole collagenase. Electrophoresis was carried out with a 6% stacking gel and a 12% separating gel. Each sample contained about 25,000 cpm and the film was exposed overnight. Gel 1: [l*C]Procollagen treated with tadpole collagenase and examined without reduction. Gel 2: [WlProcollagen treated with tadpole collagenase and examined after reduction. Gel 3: Pepsin-treated [Wlprocollagen which was cleaved with tadpole collagenase and examined without reduction. Gel 4: Pepsin-treated [14C]procollagen which was cleaved with tadpole collagenase and examined after reduction.
~roa2~ (Gel 2 in Fig. 2). The conclusion that reduction converted proyB to procylB and ~roa2~ was verified by two-dimensional gel electrophoresis (Fig. 3). Separation of proalA and prodA from proal B and pro& B. The cleavage products obtained by digestion of tendon procollagen with tadpole collagenase were separated by precipitation first with 20% ammonium sulfate and then with 30% ammonium sulfate. The two pellets were reduced with 2-mercaptoethanol and examined by gel filtration in SDS. The 20% ammonium sulfate pellet largely consisted of the NH,-terminal fragments proal* and proor2* (Figs. 4 and 5). The 30% ammonium sulfate pellet largely consisted of the COOH-terminal fragments procrlB and procu2B (Figs. 4 and 5). The nature of the fragment obtained by precipitation with 20% ammonium sulfate was further established by preparing segment-long-spacing aggregates. As shown in Fig. 6, the aggregates corresponded to the NH,-terminal fragment expected when procollagen is cleaved with tadpole collagenase.
Isolation of NH2 and COOH-terminal peptides resistant to bacterial collagenase. Tendon procollagen was cleaved with tadpole collagenase, the NH,- and COOH-terminal fragments were separated by ammonium sulfate fractionation, and the two fractions were digested with purified bacterial collagenase. Gel filtration of the digest of the COOH-terminal fraction (proyB) gave a peak (peak I in Fig. 7) which had the same elution position as the disulfide-linked peptide fragment previously obtained after digestion of tendon procollagen by bacterial collagenase (18). The radioactive material in peak I was shown to react with antibody directed against the disulfide bonded fragment previously obtained after digestion of procollagen with bacterial collagenase (18). Also, examination of the material in peak I by PAGE-SDS with and without reduction demonstrated that it contained interchain disulfide bonds. The results therefore demonstrated that the disulfidelinked fragment arose from the COOHterminal end. Gel filtration of the digest from the NH,-terminal fragments (procul*
INTERCHAIN
DISULFIDE
BONDS AT THE COOH-TERMINAL
345
Pr0aiB ProalA
Pro 42’ fDye front
FIG. 3. Two-dimensional PAGE-SDS of tendon [14Clprocollagen treated with tadpole collagenase. Separation in the first dimension (top to bottom) was carried out without reduction. After the first separation, the gel strip was cut out and immersed in “sample buffer” containing 5% 2-mercaptoethanol for 10 min and then it was placed on top of a second gel to carry out electrophoresis in the second dimension (left to right). In each case a 6% stacking gel and a 6% separating gel were used. The sample contained about 40,000 cpm and the film was exposed for 6 h. The results demonstrate that the band initially identified as proyB was converted by reduction to two spots corresponding to the positions of proolB and procu2B.
and proa2*) gave a peak (peak II in Fig. 7) which eluted after the disulfide-linked fragment. Peak II was previously obtained after digestion of tendon procollagen with bacterial collagenase and it was shown to contain peptides which were antigenically different from the disulfide-linked fragment (18). Since peak II was obtained by digestion of the NH,-terminal fragments (proal* and procu2*) with bacterial collagenase, the results presented here demonstrate that peak II (Fig. 7C) contains collagenase-resistant peptides from the NH,terminal region. Tendon procollagen without iVH2-terminul extensions. As indicated in Figs. 1 and 2, two weak minor bands with slightly lower and higher mobilities than proa chains were present in procollagen from the medium of tendon cells. These bands were shown to arise from degradation of procollagen by experiments in which the medium 14C-protein was isolated in the
absence of protease inhibitors. When the medium 14C-protein isolated in this manner was reduced and examined by PAGESDS, most of the 14C was found in two bands which were just above and below the position of the proa band and which corresponded to the minor bands seen with procollagen isolated in the presence of inhibitors (Fig. 8). When the procollagen isolated in the absence of protease inhibitors was cleaved with tadpole collagenase and the products were examined by PAGESDS without reduction, three bands were seen. One band had the same mobility as intact proyB (Fig. 8). After reduction this band was converted to two bands with the same mobilities as proculB and pro.0(y2B,indicating that the COOH-terminal end of the partially degraded molecule was largely intact. The other two bands obtained after cleavage of the partially degraded procollagen with tadpole collagenase had the same mobilities as ~rl* and
346
OLSEN, I’
’
HOFFMANN
”
AND PROCKOP
The results (Fig. 9) demonstrated that the interchain disulfide bonds were located in the COOH-terminal fragment. DISCUSSION
The difficulty of obtaining intact procollagen has confused the interpretation of a considerable amount of data on the chemical structure of the molecule, the functions of the peptide extensions, and genetic defects in the conversion of procollagen to collagen. Until rigorous criteria are established for intactness of the molecule, it will be important to examine in detail the pro-
FIG. 4. Ammonium sulfate fractionation of tendon [Wlprocollagen cleaved with tadpole collagenase. The [W]procollagen was digested with tadpole collagenase for 7 h and then precipitated with 20% ammonium sulfate and 30% ammonium sulfate. Samples containing about 200,000 cpm were reduced and treated with SDS before gel filtration in the presence of SDS (see Methods). Fractions of 1.4 ml were collected and aliquots of 0.4 ml were taken for liquid scintillation counting. (A) Total digest obtained after incubation with tadpole collagenase; (B) 20% ammonium sulfate precipitate; (C) 30% ammonium sulfate precipitate.
a2* from pepsin-treated procollagen (Fig. 8). The results indicated therefore that the tendon procollagen isolated in the absence of protease inhibitors lacked most or all of the NH,-terminal peptide extensions found in the intact molecule. Interchain disulfide bonds at the COOH-terminal end of cartilage procollagen. Matrix-free cells isolated from chick embryo sternae synthesize and secrete a cartilage procollagen (9) which contains interchain disulfide bonds (26). To locate bonds, the interchain disulfide [‘4c]procollagen from the medium of cartilage cells was also cleaved with tadpole collagenase and examined by PAGE-SDS.
ProC IA Pro(Z2A ProCClB Procc 2B Dye front 123 RG. 5. PAGE-SDS of tendon Wprocollagen cleaved with tadpole collagenase and fractionated by ammonium sulfate precipitation. Electrophoresis was carried out with a 6% stacking gel and a 6% separating gel. Each sample contained about 30,000 cpm and the film was exposed for 6 h. Gel 1: Total digest obtained after incubation with tadpole collagenase. Gel 2: 30% ammonium sulfate precipitate. Gel 3: 26% ammonium sulfate precipitate. All samples were reduced.
INTERCHAIN
DISULFIDE
BONDS AT THE COOH-TERMINAL
FIG. 6. Segment-long-spacing aggregate prepared from the 20% ammonium sulfate precipitate shown in Fig. 4B. The sample was prepared by dialyzing the precipitate against 0.01 M acetic acid containing 0.2% ATP and it was examined by electron microscopy as described previously (17). Negative staining was performed with 1% sodium phosphotungstate. The arrowheads indicate the peptide extensions on the NH,-terminal end of procollagen and the arrows indicate the region of the procollagen molecule cleaved by the tadpole collagenase. The total length of the segment is about 230 nm, and its NH,-terminal origin was confirmed by comparison with segment-long-spacing aggregates of collagen and procollagen (H.-P. Hoffmann, B. R. Olsen, H.-T. Chen, and D. J. Prockop, in preparation).
collagens which can be isolated from different biological systems. The results obtained here with tendon procollagen are generally consistent with observations which were recently made with procollagen from membranous bone of chick embryos and which were reported as the present experiments were in progress (15, 16). The procollagens from both
membranous bone and tendon are similar in that they contain both NH,-terminal and COOH-terminal extensions and in that the COOH-terminal extensions are linked by inter-chain disulfide bonds. We have recently confirmed the presence of both NH,- and COOH-terminal extensions in segment-long-spacing aggregates of purified tendon procollagen (H.-P. Hoff-
348
OLSEN,
HOFFMANN -
L
FIG. 7. Isolation of NH*- and COOH-terminal peptides resistant to bacterial collagenase. Bacterial was used to digest tendon collagenase [W]procollagen, the NH,-terminal fragments, or the COOH-terminal fragments obtained by cleavage of procollagen with tadpole collagenase. The samples were then examined by gel filtration in 0.1 M Tris-HCl buffer. Fractions of 1.6 ml were collected and aliquots of 0.4 ml were taken for liquid scintillation counting. (A) [WlProcollagen, 2.8 x lo6 cpm, digested with bacterial collagenase. (B) COOH-terminal fragment pray*, 7.1 x lo5 cpm, digested with bacterial collagenase. (C) NH,-terminal fragments proal* and proa2*, 2.1 x 1Og cpm, digested with bacterial collagenase.
mann, B. R. Olsen, H.-T. Chen, and D. J. Prockop, in preparation). Of special importance was the demonstration here that the disulfide-linked fragment previously isolated after digestion of tendon procollagen with bacterial collagenase (18) originates from the COOH-terminal end of the molecule. The new assignment of the disulfidelinked fragment helps to clarify a number
AND PROCKOP
of previous observations on the nature of the fragment and the use of specific antibodies against the fragment (10, 18, 27). For example, the new assignment of the fragment explains why specific antibodies could be used to locate procollagen in cells by electron microscopy (27) even though the antibodies did not react with partially degraded pro al chains (28). The fragments proal* and proa2* isolated from tendon procollagen were not linked by interchain disulfide bonds, suggesting that no such links are found at the NH,-terminal end of the molecule. This conclusion, however, is tentative since the data do not rigorously exclude the possibility that short peptide sequences containing inter-chain bonds were removed by intracellular proteolysis, by presence of proteases in the incubation medium, or by the tadpole collagenase used in the experiments. Similar observations have recently been made with procollagen from membranous bone, and a similar conclusion was suggested (15). The medium of freshly isolated tendon cells contained small amounts of procollagen in which the COOH-terminal end was intact but the NH,-terminal extensions were either absent or very much smaller in size. This form of partially degraded procollagen may be similar to the “altered procollagen” which was detected in membranous bone and which was suggested to be an intermediate in the conversion of procollagen to collagen (16). The partially degraded procollagen encountered here may arise from nonspecific proteolysis during incubation of the cell system, but it may also represent the first intermediate which is formed during the conversion of procollagen to collagen in uiuo. Preliminary results with pulse chase experiments suggest this is in fact the normal sequence in intact tendons (S. Aase and B. R. Olsen, in preparation). Cartilage contains a collagen which is genetically distinct from the collagen found in either tendon or bone (29). The results presented here demonstrate that interchain disulfide bonds previously identified in cartilage procollagen (26) are located in the COOH-terminal region of the molecule. Electron microscopy of segment-
INTERCHAIN
DISULFIDE
-MSH
+MSH
Pro pro
349
BONDS AT THE COOH-TERMINAL
ai A+ a2A+
+MSH
,aiA .a2A
2a 2b la lb 3a 3b 3c 8. PAGE-SDS of partially degraded tendon procollagen isolated in the absence of protease inhibitors. The stacking and separating gels were 6% polyacrylamide. The samples contained from 17,000 to 28,006 cpm and the film was exposed for 20 h. Gel la: [14C]Procollagen isolated in the presence of inhibitors; reduced. Gel lb: Partially degraded [“Clprocollagen; reduced. Gel 2a: [14ClProcollagen isolated in the presence of protease inhibitors and cleaved with tadpole collagenase; not reduced. Gel 2b: Partially degraded [“C]procollagen cleaved with tadpole collagenase; not reduced. Gel 3a: [‘*C]Procollagen isolated in the presence of protease inhibitors and cleaved with tadpole collagenase; reduced. Gel 3b: Partially degraded [“Clprocollagen cleaved with tadpole collagenase; reduced. Gel 3c: Control sample of pepsintreated procollagen cleaved with tadpole collagenase; reduced. The fragments cylBand CX~~are in the dye front. FIG.
long-spacing aggregates of cartilage procollagen demonstrated that both ends of the molecule contain extensions not found in cartilage collagen (J. Uitto, H.-P. Hoffmann, B. R. Olsen, and D. J. Prockop, in preparation). It may be that all procollagens are similar in having both NH,- and COOH-terminal extensions and that the two types of extensions have distinct functions in triple-helix formation, in secretion, and in fiber formation. 5
IO
MIGRATION DISTANCE ICMI
FIG. 9. PAGE-SDS of cartilage procollagen cleaved with tadpole collagenase. Stacking and separating gels were 6%, each sample contained 15,000 to 40,000 cpm, and the film was exposed for 12 h. The fluorograms were scanned on Quick Scan (Helena Laboratories). (A) Tadpole collagenase digest; not reduced. (B) Tadpole collagenase digest; reduced. Because the cleavage with tadpole collagenase was incomplete, uncleaved pray chains were seen in the unreduced sample (A) and uncleaved proa chains were seen in the reduced sample (B).
ACKNOWLEDGMENTS The work was supported in part by Research Grant AM-16516 from the National Institutes of Health of the U.S. Public Health Service. The authors gratefully acknowledge the expert assistance of Ms. Chris Condit and Ms. Nancy Kedersha. REFERENCES 1. PROCKOP, D. J., BERG, R. A., KIVIRIKKO, K. I., AND Urrro, J. (1976) in Biochemistry of Collagen (Ramachandran, G. N. & Reddi, A. H., eds.), Plenum, New York (in press).
350
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HOFFMANN
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