- Email: [email protected]
A method for assaying the activity of the endopeptidase which excises the nonhelical carboxyterminal extensions from type I procollagen
ANALYTICAL
BIOCHEMISTRY
86,
463-469 (1978)
A Method for Assaying the Activity of the Endopeptidase Which Excises the Nonhelical Carboxyterminal Extensions from Type I Procollagen EFRAT Department
of Pathology, Received
KESSLER New
AND
BURTON
York University
Medical
August
GOLDBERG Center,
New
York,
Ne~z York 10016
1, 1977: accepted December I, 1977
A new method for measuring the rate of enzymatic excision of the carboxy-terminal, nonhelical fragment from type I procollagen is presented. Human procollagen containing [3H]tryptophan-labeled carboxy-terminal extensions was used as the substrate, and the enzyme was derived from the culture medium of mouse 3T6 fibroblasts. Incubation mixtures of substrate with enzyme were made 25% in ethanol which left the excised radiolabeled carboxy-terminal fragment in solution, whereas all other radiolabeled components were precipitated. Enzymatic activity was measured by radioactive counting of the ethanol supernatant. The assay is simple, rapid, sensitive and generates valid kinetic data.
Soluble secreted precursors (procollagens) have been demonstrated for each of the four major genetic types of collagen (1,2 for general reviews). To date, the conversion of type I procollagen to collagen has been studied in greatest detail and has been shown to require the enzymatic excision of nonhelical peptides from both ends of the precursor molecule. Available data indicate that at least some of these excisions involve endopeptidases; cleavages at the aminoterminus release peptide chains of about 11,500 and 17,500 daltons (3), whereas, the cleavages at the carboxy-terminus generate a three-chain, disulfide-linked fragment of approximately 75,000 daltons (45). Purification of the enzymes involved in the conversion of type I procollagen to collagen has been hampered by lack of practical assays for these activities. The gel electrophoresis (3,4) ion-exchange, and immunoprecipitation techniques applied to date are laborious or require complete conversion of the precursor substrate (6). In this paper we describe a simple and rapid assay for the endopeptidase(s) which releases the three-chain fragment from the carboxy-terminal end of type I procollagen. The method is based on the observation that the excised fragment is soluble in 25% ethanol, whereas the undigested substrate, digestion intermediates, and collagen are insoluble in this concentration of ethanol. 463
0003-2697/78/0862-0463$02.00/O Copyright All rights
& 1978 by Academic Press, Inc. of reproductnon in any form reserved.
KESSLER
MATERIALS
AND
GOLDBERG
AND METHODS
Cells and culture conditions. Human skin fibroblasts (CRL 1121, American Type Culture Collection) and mouse 3T6 fibroblasts (7) were grown to confluence in Dulbecco-Vogt medium supplemented with 10% fetal calf serum and sodium ascorbate (75 pug/ml). Prior to preparation of radiolabeled substrate or enzyme, the cell layers were washed and overlaid with serum-free ascorbate-supplemented medium. Radiolabeled substrate. Roller bottles, each containing approximately lo* human fibroblasts at confluence, were incubated for 24 hr with 200 PCi of L-[G-3H]tryptophan (New England Nuclear). The medium was collected, chilled, clarified by centrifugation, and made 0.5 M in acetic acid. All subsequent procedures were performed at 4°C. The acidified medium was allowed to stand for 2 to 3 hr, exhaustively dialyzed against water, and then lyophilized. The lyophilate was suspended in buffer (3 mg/ml in 0.05 M Tris-HCl, pH 7.5, 0.15 M NaCl) and stirred for 18 hr. Undissolved material was removed by centrifugation (12,OOOg, 15 min) and ammonium sulfate (Schwarz/Mann, ultrapure, 176 mg/ml) was added to the supernate. After 18 hr, the precipitate was collected by centrifugation (27,OOOg, 30 min) and dissolved in and dialyzed against the above Tris buffer. The concentration of procollagen in the final solution was determined by a radioimmunoassay as previously described (8,9). The specific activity of the procollagen was 3.3 to 4.4 x lo6 cpm/mg. Occasionally double-labeled substrate was prepared by adding 25 /.Ki of [14C]proline, 25 PCi of [14C]glycine, and 100 &i of [3H]tryptophan to the cultures. Enzyme preparation. A crude enzyme preparation was obtained from the medium of 3T6 mouse cultures by ammonium sulfate precipitation as previously described (4). Standard enzyme assay. Incubation of substrate with enzyme (1 hr at 37°C) was performed in 0.2 ml of standard assay buffer (0.05 M Tris-HCl, pH 7.5; 0.15 M NaCl; 0.005 M CaCl,) in conical polystyrene microtubes (Fisher). Reactions were initiated by adding 50 ~1 of [3H]tryptophanlabeled substrate solution (30,000 cpm in the assay buffer) to 150 ~1 of enzyme solution (20-360 pg of enzyme protein in the assay buffer). To stop the reaction, 40 ~1 of a cold fibrinogen-disodium ethylenediaminetetraacetate (EDTA) solution (bovine fibrinogen, BDH Biochemicals, 10 mg/ml; 0.1 M Na, EDTA; 0.05 M Tris-HCl, pH 7.5; 0.15 M NaCl) was added to each tube, and the tubes were immediately placed in an ice bath. Eighty microliters of cold absolute ethanol were then added to each tube, and after thorough mixing the tubes were allowed to stand for 2.5 hr at 4°C. The EDTA inactivated the enzyme, and the fibrinogen served as a carrier for the precipitate which formed after the addition of ethanol. The precipitates were removed by centrifugation (27,OOOg, 30 min, 4°C) and 200-~1 aliquots
ASSAY
FOR
PROCOLLAGEN
465
PEPTIDASE
of the supernatants were added to Aquasol (New England Nuclear) and radioactivity measured in a scintillation spectrometer. Under these conditions quenching of radioactivity was 50%. Control tubes contained substrate and heat-inactivated enzyme (5 min in a boiling water bath). Electrophoresis
in sodium dodecyl sulfate (SDS) -urea
polyacrylamide
gels. Electrophoresis in 5% gels and measurement of radioactivity slices were performed as previously described (IO). RESULTS
in gel
AND DISCUSSION
The radiolabeled procollagen accumulating in the medium of the human fibroblast cultures has nonhelical extensions at only the carboxyterminal end of the molecule (unpublished data), and [3H]tryptophan is exclusively incorporated into these nonhelical sequences (1 I). Figure 1A shows the gel pattern given by the radiolabeled substrate prepared from culture medium. Approximately 58% of the radioactivity in the gel is found in the procollagen species between 8 and 19 mm. To test the effect of 25% ethanol on the components of an enzyme-substrate reaction mixture at an intermediate stage of the reaction, the substrate was incubated with enzyme for 3 hr and the solution was divided into two equal portions. One sample was heat-denatured and subjected to polyacrylamide gel electrophoresis. A fibrinogen-EDTA solution was added to the other
0
10
20 30 m m FROM
40 50 ORIGIN
60
0
10
20 mm
30 FROM
40 50 ORIGIN
60
FIG. I. (A) SDS-urea-polyacrylamide gel electrophoresis of the undigested [“Hltryptophan-labeled procollagen substrate. The substrate (25,000 cpm in 0.1 ml of the standard assay buffer) was incubated without enzyme for 3 hr at 37”C, and then heat-denatured forge] electrophoresis (60°C. 1 hr; 1% SDS and 0.5 M urea in 0.01 M phosphate buffer, pH 7.2). (B) Gel patterns of an enzyme-substrate reaction mixture before and after ethanol treatment. [YH]Tryptophan-labeled procollagen (50,000 cpm in 0.2 ml of standard assay buffer) was incubated with 140 fig of enzyme protein for 3 hr at 37°C. and the solution was equally divided. One sample was made 1% in SDS, heat-denatured, and applied to a gel (0 0). A fibrinogen-EDTA solution was added to the other sample which was then made 25% in ethanol and centrifuged (see Materials and Methods). The supernatant was dried in ~cuo. denatured in electrophoresis buffer, and applied to a gel (0 - - - 0).
466
KESSLER
AND
GOLDBERG
sample, which was then made 25% in ethanol. The resultant precipitate and supernatant were each processed for gel electrophoresis. The solid line in Fig. 1B depicts the gel pattern given by the sample which was not treated with ethanol. The main digestion product is the 75,000 dalton carboxyterminal fragment centered at 52 mm, hereafter referred to as the propeptide. The other radioactive peaks represent undigested substrate (at 11 mm) and three intermediates (at 15,23, and 32 mm) which are generated when the enzyme successively cleaves the three chains of the substrate (see Ref 4). The dotted line in Fig. 1B is the gel pattern given by the supernatant obtained from the ethanol-treated sample. It is evident that of all the radioactive components in the original digest, only the propeptide was soluble in 25% ethanol and that about 90% of it was recovered in the supernatant. The electrophoretic pattern of the ethanol precipitate (not shown) showed quantitative recoveries of the substrate and reaction intermediates and only a trace amount of the propeptide. A similar experiment was performed with substrate labeled at the nonhelical carboxyterminus with [3H]tryptophan and in the helical portion with [14C]proline and [14C]glycine. In this instance the 14C-labeled collagen chains generated in the reaction were quantitatively recovered in the ethanol precipitate. To verify that the fragment soluble in 25% ethanol was the disulfide-linked carboxy-terminal peptide, its behavior upon reduction was studied. The radiolabeled substrate was incubated with enzyme, and a 25% ethanol supernatant of the incubation solution was prepared. Aliquots of the supernatant were denatured in the presence or absence of 2-mercaptoethanol and then subjected to gel electrophoresis (Fig. 2). As expected, the unreduced radioactive propeptide migrated as a molecule of approximately 75,000 molecular weight, whereas after reduction, the
0
50
60 70 80 m m FROM ORIGIN
90
lee
FIG. 2. Effect of reduction on the ethanol supernatant of an enzyme-substrate reaction mixture. Aliquots of a 25% ethanol supernatant. each containing 9000 cpm. were dried in vacua. The samples were heat-denatured in electrophoresis buffer in the presence (0 - - - 0) or absence (0 0) of2-mercaptoethanol anti applied to gels. The inserted numbers are the total counts per minute in each peak.
ASSAY
FOR
PROCOLLAGEN
467
PEPTIDASE
radioactivity was quantitatively recovered in a band migrating as a protein of one-third the molecular weight (5). An aliquot of the above ethanol supernatant was also incubated with rabbit antiserum specific for the nonhelical, carboxy-terminal extensions of human type I procollagen (8). The antiserum precipitated about 80% of the radioactivity in the sample, and when the immune precipitate was denatured and subjected to gel electrophoresis, all the applied radioactivity was recovered in a single peak migrating to the position of the propeptide (data not shown). All these data indicated that the ethanol treatment of the reaction mixtures left only the propeptide in solution and that radioactive counting of these supernatants would therefore be a measure of the amount of propeptide generated by the enzyme. Accordingly, a standard assay procedure was established (see Materials and Methods) which was used to study the kinetics of the reaction. Figure 3A shows the relation between degree of conversion of the substrate and enzyme protein concentration; Fig. 3B shows the percentage conversion as a function of incubation time. In both cases characteristic and unambiguous curves were obtained, showing that the radioactivity in the ethanol supernatant was a reliable measure of enzyme activity. The curve of Fig. 3A is linear up to 9% conversion whereas the progress curve (Fig. 3B) is linear up to 25% conversion. This difference might be due to the presence of inhibitory substances in the crude enzyme preparation. The rate of generation of propeptide as a function of substrate I
I
200
400
I
’
I
I
1
6
8
2o (A)
0 //g
PROTEIN
0
4
2 TIME
(HOURS)
FIG. 3. (A) Dependence of activity on enzyme protein. Radiolabeled substrate was incubated with increasing amounts of enzyme protein under the standard assay conditions (see Materials and Methods). Enzyme activity is expressed as the percentage of conversion of substrate to propeptide (15,000 cpm = 100% conversion). Each point represents the average of two experiments. (B) Dependence of activity on time of incubation. The reaction solution (2.0 ml) contained 300.000 cpm of substrate and 0.9 mg of enzyme protein. Aliquots (200 ~1) were removed after different times of incubation and processed as described in Materials and Methods. Percentage conversion was determined as in A. Each point represents the average of two experiments.
468
KESSLER
AND GOLDBERG
FIG. 4. Double-reciprocal plot of initial velocity against substrate concentration. Ninety micrograms of enzyme protein were incubated with increasing amounts of radiolabeled substrate for I hr as described in Materials and Methods. Velocity (v) is expressed in counts per minute per hour. [S] is expressed in moles per liter of substrate, assuming a molecular weight of 360,000 for the procollagen substrate. Each point represents the average of two experiments.
concentration was also studied and the results were plotted according to Lineweaver and Burk (12). An apparent Michaelis-Menten constant (K,) of 2.5 x lop6 M was derived (Fig. 4). It is interesting to note that a similar K, value (10d6 M) was reported by Kohn et al. (3) for the enzymatic excision of the amino-terminal nonhelical extensions from dermatosparactic procollagen. In the assay, duplicate samples differ by no more than 5%, and background radioactivity (substrate plus boiled enzyme) typically does not exceed 10% of the total substrate radioactivity. With our enzyme preparation the assay is specific, since essentially all the radioactivity in the supernatant originates from the propeptide. This also indicates that our enzyme preparation contains little or no nonspecific protease activity. If the assay is applied to enzymes prepared from other sources, the degree of specificity should first be determined by characterizing the radioactive contents of the ethanol supernatant by gel electrophoresis. The assay is highly sensitive, allowing measurements of just a few hundred counts per minute in propeptide. By contrast, such low amounts of radioactivity cannot be measured precisely in assays using polyacrylamide gel electrophoresis or ion-exchange chromatography. Other advantages of the assay are simplicity and rapidity, so that it lends itself to efficient analysis of multiple fractions obtained in the course of enzyme purification. Additionally, the method might possibly be adapted to measurement of the peptidase activities which cleave the amino-terminal extensions from procollagen. ACKNOWLEDGMENTS This work was supported by NIH Grants 5 ROI HLl7551 and 5 SO7 RR05399. The authors gratefully acknowledge the excellent assistance of Sheila Heitner.
ASSAY
FOR
PROCOLLAGEN
PEPTIDASE
469
REFERENCES 1. Miller. E. J. (1976) Mol. Cell. Biochem. 13, 165-192. 2. Kefalides, N. A. (1975) J. Invesr. Dermatol. 65, 85-92. 3. Kohn, L. D., Isersky, C.. Zupkin. J.. Lenaers. A., Lee, G.. and Lap&e, C. M. (1974) Proc. Nat. Acad. Sri. USA 71, 40-44. 4. Goldberg, B.. Taubman. M. B.. and Radin. A. (1975) Cell 4, 45-50. 5. Sherr. C. J., Taubman. M. B., and Goldberg, B. (1973)J. Biol. Chem. 248, 7033-7038. 6. Bornstein, P.. Davidson, J. M., and Monson, J. M. (1975) in Proteases and Biological Control, (Reich, E.. Ritkin. D. B., and Shaw, E., eds.), pp. 579-590. Cold Spring Harbor Laboratory, N. Y. 7. Todaro, G. J., and Green, H. (1963) J. Cell. Biol. 17, 299-313. 8. Taubman, M. B., Goldberg, B., and Sherr, C. J. (1974) Science 186, I1 15- 1117. 9. Taubman, M. B.. Kammerman, S.. and Goldberg, B. (1976) Proc. SM. Exp. Biol. Med. 152, 284-287. 10. Goldberg, B., Epstein, E. H. Jr., and Sherr. C. J. (1972) Proc. Nat. Acud. Sci. I/S4 69, 3655-3659. II. Uitto. J.. Lichtenstein, J. R., and Bauer. E. A. (1976) Biochemistry 15, 4935-4942. 12. Lineweaver, H., and Burk, D. (1934) J. Amer. Chem. Sot. 56, 658-666.
BIOCHEMISTRY
86,
463-469 (1978)
A Method for Assaying the Activity of the Endopeptidase Which Excises the Nonhelical Carboxyterminal Extensions from Type I Procollagen EFRAT Department
of Pathology, Received
KESSLER New
AND
BURTON
York University
Medical
August
GOLDBERG Center,
New
York,
Ne~z York 10016
1, 1977: accepted December I, 1977
A new method for measuring the rate of enzymatic excision of the carboxy-terminal, nonhelical fragment from type I procollagen is presented. Human procollagen containing [3H]tryptophan-labeled carboxy-terminal extensions was used as the substrate, and the enzyme was derived from the culture medium of mouse 3T6 fibroblasts. Incubation mixtures of substrate with enzyme were made 25% in ethanol which left the excised radiolabeled carboxy-terminal fragment in solution, whereas all other radiolabeled components were precipitated. Enzymatic activity was measured by radioactive counting of the ethanol supernatant. The assay is simple, rapid, sensitive and generates valid kinetic data.
Soluble secreted precursors (procollagens) have been demonstrated for each of the four major genetic types of collagen (1,2 for general reviews). To date, the conversion of type I procollagen to collagen has been studied in greatest detail and has been shown to require the enzymatic excision of nonhelical peptides from both ends of the precursor molecule. Available data indicate that at least some of these excisions involve endopeptidases; cleavages at the aminoterminus release peptide chains of about 11,500 and 17,500 daltons (3), whereas, the cleavages at the carboxy-terminus generate a three-chain, disulfide-linked fragment of approximately 75,000 daltons (45). Purification of the enzymes involved in the conversion of type I procollagen to collagen has been hampered by lack of practical assays for these activities. The gel electrophoresis (3,4) ion-exchange, and immunoprecipitation techniques applied to date are laborious or require complete conversion of the precursor substrate (6). In this paper we describe a simple and rapid assay for the endopeptidase(s) which releases the three-chain fragment from the carboxy-terminal end of type I procollagen. The method is based on the observation that the excised fragment is soluble in 25% ethanol, whereas the undigested substrate, digestion intermediates, and collagen are insoluble in this concentration of ethanol. 463
0003-2697/78/0862-0463$02.00/O Copyright All rights
& 1978 by Academic Press, Inc. of reproductnon in any form reserved.
KESSLER
MATERIALS
AND
GOLDBERG
AND METHODS
Cells and culture conditions. Human skin fibroblasts (CRL 1121, American Type Culture Collection) and mouse 3T6 fibroblasts (7) were grown to confluence in Dulbecco-Vogt medium supplemented with 10% fetal calf serum and sodium ascorbate (75 pug/ml). Prior to preparation of radiolabeled substrate or enzyme, the cell layers were washed and overlaid with serum-free ascorbate-supplemented medium. Radiolabeled substrate. Roller bottles, each containing approximately lo* human fibroblasts at confluence, were incubated for 24 hr with 200 PCi of L-[G-3H]tryptophan (New England Nuclear). The medium was collected, chilled, clarified by centrifugation, and made 0.5 M in acetic acid. All subsequent procedures were performed at 4°C. The acidified medium was allowed to stand for 2 to 3 hr, exhaustively dialyzed against water, and then lyophilized. The lyophilate was suspended in buffer (3 mg/ml in 0.05 M Tris-HCl, pH 7.5, 0.15 M NaCl) and stirred for 18 hr. Undissolved material was removed by centrifugation (12,OOOg, 15 min) and ammonium sulfate (Schwarz/Mann, ultrapure, 176 mg/ml) was added to the supernate. After 18 hr, the precipitate was collected by centrifugation (27,OOOg, 30 min) and dissolved in and dialyzed against the above Tris buffer. The concentration of procollagen in the final solution was determined by a radioimmunoassay as previously described (8,9). The specific activity of the procollagen was 3.3 to 4.4 x lo6 cpm/mg. Occasionally double-labeled substrate was prepared by adding 25 /.Ki of [14C]proline, 25 PCi of [14C]glycine, and 100 &i of [3H]tryptophan to the cultures. Enzyme preparation. A crude enzyme preparation was obtained from the medium of 3T6 mouse cultures by ammonium sulfate precipitation as previously described (4). Standard enzyme assay. Incubation of substrate with enzyme (1 hr at 37°C) was performed in 0.2 ml of standard assay buffer (0.05 M Tris-HCl, pH 7.5; 0.15 M NaCl; 0.005 M CaCl,) in conical polystyrene microtubes (Fisher). Reactions were initiated by adding 50 ~1 of [3H]tryptophanlabeled substrate solution (30,000 cpm in the assay buffer) to 150 ~1 of enzyme solution (20-360 pg of enzyme protein in the assay buffer). To stop the reaction, 40 ~1 of a cold fibrinogen-disodium ethylenediaminetetraacetate (EDTA) solution (bovine fibrinogen, BDH Biochemicals, 10 mg/ml; 0.1 M Na, EDTA; 0.05 M Tris-HCl, pH 7.5; 0.15 M NaCl) was added to each tube, and the tubes were immediately placed in an ice bath. Eighty microliters of cold absolute ethanol were then added to each tube, and after thorough mixing the tubes were allowed to stand for 2.5 hr at 4°C. The EDTA inactivated the enzyme, and the fibrinogen served as a carrier for the precipitate which formed after the addition of ethanol. The precipitates were removed by centrifugation (27,OOOg, 30 min, 4°C) and 200-~1 aliquots
ASSAY
FOR
PROCOLLAGEN
465
PEPTIDASE
of the supernatants were added to Aquasol (New England Nuclear) and radioactivity measured in a scintillation spectrometer. Under these conditions quenching of radioactivity was 50%. Control tubes contained substrate and heat-inactivated enzyme (5 min in a boiling water bath). Electrophoresis
in sodium dodecyl sulfate (SDS) -urea
polyacrylamide
gels. Electrophoresis in 5% gels and measurement of radioactivity slices were performed as previously described (IO). RESULTS
in gel
AND DISCUSSION
The radiolabeled procollagen accumulating in the medium of the human fibroblast cultures has nonhelical extensions at only the carboxyterminal end of the molecule (unpublished data), and [3H]tryptophan is exclusively incorporated into these nonhelical sequences (1 I). Figure 1A shows the gel pattern given by the radiolabeled substrate prepared from culture medium. Approximately 58% of the radioactivity in the gel is found in the procollagen species between 8 and 19 mm. To test the effect of 25% ethanol on the components of an enzyme-substrate reaction mixture at an intermediate stage of the reaction, the substrate was incubated with enzyme for 3 hr and the solution was divided into two equal portions. One sample was heat-denatured and subjected to polyacrylamide gel electrophoresis. A fibrinogen-EDTA solution was added to the other
0
10
20 30 m m FROM
40 50 ORIGIN
60
0
10
20 mm
30 FROM
40 50 ORIGIN
60
FIG. I. (A) SDS-urea-polyacrylamide gel electrophoresis of the undigested [“Hltryptophan-labeled procollagen substrate. The substrate (25,000 cpm in 0.1 ml of the standard assay buffer) was incubated without enzyme for 3 hr at 37”C, and then heat-denatured forge] electrophoresis (60°C. 1 hr; 1% SDS and 0.5 M urea in 0.01 M phosphate buffer, pH 7.2). (B) Gel patterns of an enzyme-substrate reaction mixture before and after ethanol treatment. [YH]Tryptophan-labeled procollagen (50,000 cpm in 0.2 ml of standard assay buffer) was incubated with 140 fig of enzyme protein for 3 hr at 37°C. and the solution was equally divided. One sample was made 1% in SDS, heat-denatured, and applied to a gel (0 0). A fibrinogen-EDTA solution was added to the other sample which was then made 25% in ethanol and centrifuged (see Materials and Methods). The supernatant was dried in ~cuo. denatured in electrophoresis buffer, and applied to a gel (0 - - - 0).
466
KESSLER
AND
GOLDBERG
sample, which was then made 25% in ethanol. The resultant precipitate and supernatant were each processed for gel electrophoresis. The solid line in Fig. 1B depicts the gel pattern given by the sample which was not treated with ethanol. The main digestion product is the 75,000 dalton carboxyterminal fragment centered at 52 mm, hereafter referred to as the propeptide. The other radioactive peaks represent undigested substrate (at 11 mm) and three intermediates (at 15,23, and 32 mm) which are generated when the enzyme successively cleaves the three chains of the substrate (see Ref 4). The dotted line in Fig. 1B is the gel pattern given by the supernatant obtained from the ethanol-treated sample. It is evident that of all the radioactive components in the original digest, only the propeptide was soluble in 25% ethanol and that about 90% of it was recovered in the supernatant. The electrophoretic pattern of the ethanol precipitate (not shown) showed quantitative recoveries of the substrate and reaction intermediates and only a trace amount of the propeptide. A similar experiment was performed with substrate labeled at the nonhelical carboxyterminus with [3H]tryptophan and in the helical portion with [14C]proline and [14C]glycine. In this instance the 14C-labeled collagen chains generated in the reaction were quantitatively recovered in the ethanol precipitate. To verify that the fragment soluble in 25% ethanol was the disulfide-linked carboxy-terminal peptide, its behavior upon reduction was studied. The radiolabeled substrate was incubated with enzyme, and a 25% ethanol supernatant of the incubation solution was prepared. Aliquots of the supernatant were denatured in the presence or absence of 2-mercaptoethanol and then subjected to gel electrophoresis (Fig. 2). As expected, the unreduced radioactive propeptide migrated as a molecule of approximately 75,000 molecular weight, whereas after reduction, the
0
50
60 70 80 m m FROM ORIGIN
90
lee
FIG. 2. Effect of reduction on the ethanol supernatant of an enzyme-substrate reaction mixture. Aliquots of a 25% ethanol supernatant. each containing 9000 cpm. were dried in vacua. The samples were heat-denatured in electrophoresis buffer in the presence (0 - - - 0) or absence (0 0) of2-mercaptoethanol anti applied to gels. The inserted numbers are the total counts per minute in each peak.
ASSAY
FOR
PROCOLLAGEN
467
PEPTIDASE
radioactivity was quantitatively recovered in a band migrating as a protein of one-third the molecular weight (5). An aliquot of the above ethanol supernatant was also incubated with rabbit antiserum specific for the nonhelical, carboxy-terminal extensions of human type I procollagen (8). The antiserum precipitated about 80% of the radioactivity in the sample, and when the immune precipitate was denatured and subjected to gel electrophoresis, all the applied radioactivity was recovered in a single peak migrating to the position of the propeptide (data not shown). All these data indicated that the ethanol treatment of the reaction mixtures left only the propeptide in solution and that radioactive counting of these supernatants would therefore be a measure of the amount of propeptide generated by the enzyme. Accordingly, a standard assay procedure was established (see Materials and Methods) which was used to study the kinetics of the reaction. Figure 3A shows the relation between degree of conversion of the substrate and enzyme protein concentration; Fig. 3B shows the percentage conversion as a function of incubation time. In both cases characteristic and unambiguous curves were obtained, showing that the radioactivity in the ethanol supernatant was a reliable measure of enzyme activity. The curve of Fig. 3A is linear up to 9% conversion whereas the progress curve (Fig. 3B) is linear up to 25% conversion. This difference might be due to the presence of inhibitory substances in the crude enzyme preparation. The rate of generation of propeptide as a function of substrate I
I
200
400
I
’
I
I
1
6
8
2o (A)
0 //g
PROTEIN
0
4
2 TIME
(HOURS)
FIG. 3. (A) Dependence of activity on enzyme protein. Radiolabeled substrate was incubated with increasing amounts of enzyme protein under the standard assay conditions (see Materials and Methods). Enzyme activity is expressed as the percentage of conversion of substrate to propeptide (15,000 cpm = 100% conversion). Each point represents the average of two experiments. (B) Dependence of activity on time of incubation. The reaction solution (2.0 ml) contained 300.000 cpm of substrate and 0.9 mg of enzyme protein. Aliquots (200 ~1) were removed after different times of incubation and processed as described in Materials and Methods. Percentage conversion was determined as in A. Each point represents the average of two experiments.
468
KESSLER
AND GOLDBERG
FIG. 4. Double-reciprocal plot of initial velocity against substrate concentration. Ninety micrograms of enzyme protein were incubated with increasing amounts of radiolabeled substrate for I hr as described in Materials and Methods. Velocity (v) is expressed in counts per minute per hour. [S] is expressed in moles per liter of substrate, assuming a molecular weight of 360,000 for the procollagen substrate. Each point represents the average of two experiments.
concentration was also studied and the results were plotted according to Lineweaver and Burk (12). An apparent Michaelis-Menten constant (K,) of 2.5 x lop6 M was derived (Fig. 4). It is interesting to note that a similar K, value (10d6 M) was reported by Kohn et al. (3) for the enzymatic excision of the amino-terminal nonhelical extensions from dermatosparactic procollagen. In the assay, duplicate samples differ by no more than 5%, and background radioactivity (substrate plus boiled enzyme) typically does not exceed 10% of the total substrate radioactivity. With our enzyme preparation the assay is specific, since essentially all the radioactivity in the supernatant originates from the propeptide. This also indicates that our enzyme preparation contains little or no nonspecific protease activity. If the assay is applied to enzymes prepared from other sources, the degree of specificity should first be determined by characterizing the radioactive contents of the ethanol supernatant by gel electrophoresis. The assay is highly sensitive, allowing measurements of just a few hundred counts per minute in propeptide. By contrast, such low amounts of radioactivity cannot be measured precisely in assays using polyacrylamide gel electrophoresis or ion-exchange chromatography. Other advantages of the assay are simplicity and rapidity, so that it lends itself to efficient analysis of multiple fractions obtained in the course of enzyme purification. Additionally, the method might possibly be adapted to measurement of the peptidase activities which cleave the amino-terminal extensions from procollagen. ACKNOWLEDGMENTS This work was supported by NIH Grants 5 ROI HLl7551 and 5 SO7 RR05399. The authors gratefully acknowledge the excellent assistance of Sheila Heitner.
ASSAY
FOR
PROCOLLAGEN
PEPTIDASE
469
REFERENCES 1. Miller. E. J. (1976) Mol. Cell. Biochem. 13, 165-192. 2. Kefalides, N. A. (1975) J. Invesr. Dermatol. 65, 85-92. 3. Kohn, L. D., Isersky, C.. Zupkin. J.. Lenaers. A., Lee, G.. and Lap&e, C. M. (1974) Proc. Nat. Acad. Sri. USA 71, 40-44. 4. Goldberg, B.. Taubman. M. B.. and Radin. A. (1975) Cell 4, 45-50. 5. Sherr. C. J., Taubman. M. B., and Goldberg, B. (1973)J. Biol. Chem. 248, 7033-7038. 6. Bornstein, P.. Davidson, J. M., and Monson, J. M. (1975) in Proteases and Biological Control, (Reich, E.. Ritkin. D. B., and Shaw, E., eds.), pp. 579-590. Cold Spring Harbor Laboratory, N. Y. 7. Todaro, G. J., and Green, H. (1963) J. Cell. Biol. 17, 299-313. 8. Taubman, M. B., Goldberg, B., and Sherr, C. J. (1974) Science 186, I1 15- 1117. 9. Taubman, M. B.. Kammerman, S.. and Goldberg, B. (1976) Proc. SM. Exp. Biol. Med. 152, 284-287. 10. Goldberg, B., Epstein, E. H. Jr., and Sherr. C. J. (1972) Proc. Nat. Acud. Sci. I/S4 69, 3655-3659. II. Uitto. J.. Lichtenstein, J. R., and Bauer. E. A. (1976) Biochemistry 15, 4935-4942. 12. Lineweaver, H., and Burk, D. (1934) J. Amer. Chem. Sot. 56, 658-666.