Calf Tendon Procollagen Peptidase: Its Purification and Endopeptidase Mode of Action (Dermatosparaxis/Antiprocollagen Peptidase/Antiprocollagen) LEONARD D
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Proc. Nat. Acad. Sci. USA Vol. 71, No. 1, pp. 40-44, January 1974 Calf Tendon Procollagen Peptidase: Its Purification and Endopeptidase Mode of Action (dermatosparaxis/antiprocollagen peptidase/antiprocollagen) LEONARD D. KOHN*, CHAVIVA ISERSKY*, JAMES ZUPNIKt, ALBERT LENAERSt, GEORGE LEE*t, AND CHARLES M. LAPIEREt *National Institute of Arthritis, Metabolism, and Digestive Diseases, National Institutes of Health, Bethesda, Maryland 20014; and tService de Dermatologie, Hopital de Baviere, University of Liege, 4000 Liege, Belgium Communicated by Leon A. Heppel, September 4, 1973 ABSTRACT The procollagen peptidase activity of calf MATERIALS AND METHODS tendon has been purified. The enzyme has a high degree of specificity for native procollagen and converts both Collagen Substrates and Enzyme Assay. Dermatosparaxic pro al and pro a2 to al and a2, respectively. The purified procollagen served as the substrate in all reactions; its prepa- enzyme is an endopeptidase which excises the amino ration has been described (5, 13). The standard enzyme assay terminal peptide extensions of the precursor chains in ml solution (0.6 mg of native block; the molecular size and amino-acid composition of included 0.1 of the substrate the excised peptides compare favorably with those pre- procollagen per ml), 0.05 ml of M glycyl glycine buffer (pH dicted in previous reports. Antisera to the enzyme and to 7.5), and appropriate aliquots of enzyme, in a final volume procollagen have been prepared and have been used to of 0.25 ml. Tubes were incubated at 26° for 6 hr unless other- characterize the enzyme, the enzymatically excised pep- wise noted. Reactions were stopped and analyzed by disc-gel tides, and the enzyme-peptide complex in reaction mix- tures. electrophoresis as described (13). All assays were run at mul- tiple protein concentrations and in duplicate. Dermatosparaxis is an inherited connective tissue disorder of Enzyme activity was quantitated by calculating the frac- cattle which is characterized by extreme fragility of the skin tion of pro al or pro a2 converted to al or a2 when compared (1, 2). The dermis of these animals contains disorganized to control incubations without enzyme. Since each incubation collagen bundles which demonstrate an impaired parallel mixture contained 60 jig of intact native procollagen, the packing of individual collagen filaments within the collagen fractional conversion was readily expressed in absolute terms, fibers (3, 4). These abnormal fibers are presumed to result i.e., one unit of enzyme activity was defined as the conversion of from the accumulation of procollagen, the triple helical 1 Mig of native procollagen per 6 hr per assay volume. Specific precursor of collagen which is composed of pro al and pro activity correlated these units to enzyme protein measured a2 chains (5-9). Pro al and pro a2 chains are ribosomal calorimetrically (15). which contain an additional peptide of 100 products (10, 11) Immunization and Evaluation of Antisera. New Zealand residues at the amino termini of their respective a to 200 rabbits were immunized with native procollagen using sched- chains (5, 8, 9, 12). described elsewhere (16). Antisera were In a previous report (13) we described an enzyme activity ules and techniques which excised the amino-terminal regularly evaluated by microimmunodiffusion (16, 17) or in normal bovine tissues immunoelectrophoresis (16,18,19). extension of the precursor chains to yield electrophoretically used in this chains. We further showed that this activity was The unadsorbed, antiserum to procollagen normal report reacted with native procollagen but gave no reaction absent or markedly reduced in dermatosparaxic tissues, i.e., and the resultant tissue with collagen or normal calf serum in immunodiffusion or by the accumulation of procollagen radioimmunoassay (16). changes were apparently caused by a defect in a specific enzyme, "procollagen peptidase." The "procollagen pep- RESULTS tidase" activity was associated with a very low level of non- Purification of the enzyme protease activity in contrast to pepsin and chymo- specific of killed calves were trypsin, two enzymes used in vitro by several laboratories Tendons obtained from the legs freshly al carefully cleaned of contaminating muscle and were washed to convert pro al to chains (6, 7, 14). M NaCl. In we describe the of calf- three times with 0.05 M Tris HCl (pH 7.4)-0.15 the present report purification 0-2' as tendon procollagen peptidase, characterize several of its The dissecting and washing procedure was done at properties, and show that the enzyme is an endopeptidase were all subsequent manipulations. Tendon preparations were the amino-terminal of in either processed immediately or stored at -70° for subsequent which excises peptide procollagen in results. block rather than in multiple fragments. extractions with no significant difference Step 1: Homogenization. Tendon pieces were homogenized in a Hobart commercial meat To cool I This work was performed in partial fulfillment of the degree by grinding grinder. requirements for the Department of Chemistry, American the tissue during this procedure, and to aid in its processing, University, Washington, D.C. 20016. frozen pieces of the wash buffer were alternated with tissue 40 Proc. Nat. Acad. Sci. USA 71 (1974) Tendon Procollagen Peptidase 41 aliquots. The total amount of frozen buffer used was two to three times the weight of the tendon homogenized. was Step 2: Centrifugation of Homogenate. The homogenate 2. centrifuged at 7500 X g for 15 min. The supernatant solution was pooled and could be stored at -20° for as long as 3 months with no significant change in enzyme activity. Step 3: Ammonium Sulfate Precipitation. Ammonium sul- oioo--~ o-o-o\ J fate, 39 g/100 ml, was slowly added to the thawed supernatant 10- St.2 ~2 3 4 5 6 1200 of Step 2. The stirred suspension was equilibrated overnight 1.0 100 and the precipitate was harvested by centrifugation at co 40,000 X g for 20 min. The precipitate was solubilized in < I.A°*,-*st 11000 0.05 M Tris HCl (pH 7.4)-i mM mercaptoethanol-0.15 M NaCl-10 mM CaC12. CC 0I8 800 Step 4: Dialysis. By dialysis for 24 hr against two separate 0.6 '_ 0- 600 6-liter volumes, the solubilized precipitate was equilibrated with the buffer used in Step 3 minus NaCl. The flocculant - precipitate which formed under these conditions was removed 0.4- B C D 20H400 by centrifugation at 40,000. X g for 10 min. 0.2 -l10 200 Step 5: DEAE-Cellulose Chromatography. The supernatant from was a X 4-cm solution Step 4 applied (1 ml/min) to 100 -~~~~- column of microgranular DEAE previously equilibrated with 0 100 200 FRACTION 0.05 M Tris HCl (pH 7.4),-1 mM mercaptoethanol. The FIG. 1. (A) DEAE-Cellulose chromatography of procollagen was a column eluted with linear gradient consisting of 0-0.3 peptidase. Fractions of 20 ml were collected at a flow rate of 1-2 M NaCl in equilibrating buffer (Fig. 1A). ml/min. Procollagen peptidase activity (@--*) was eluted as a peak between fractions 220 and 280. Arrow indicates the ini- Step 6: Ammonium Sulfate Extraction. Enzyme activity tiation of the NaCl gradient. The ordinate on the right is units eluted from the DEAE column (Fig. IA, black bar) was pooled, of enzyme activity. (B) Sephadex G-200 chromatography of concentrated by ultrafiltration, and precipitated by the addi- DEAE-cellulose purified enzyme. Fractions of 12 ml were col- tion of ammonium sulfate to a final concentration of 60%. lected at a flow rate of 0.5 ml/min. Disc-gel inserts are: (1) the The precipitate was harvested by centrifugation at 40,000 X 10% ammonium sulfate extract (Step 6) of the DEAE-cellulose g and was consecutively extracted by the following solutions: enzyme pool, 50 yg; (2-4) peaks A, B, and C, respectively, of 40, 30, 25, 20, and 10% ammonium sulfate in equilibrating the Sephadex G-200 chromatography procedure, 50Mg; (5) peak buffer (Step 5) and equilibrating buffer alone. The supernatant A of the Sephadex G-200 procedure after incubation with 1% of the extraction with the highest specific activity (usually sodium dodecylsulfate, 50 Mg; and (6) peak A of the Sephadex G-200 procedure after incubation with 1% sodium dodecylsulfate the 10% extract) was dialyzed against 0.05 M Trisp Cl, (pH plus 0.1 M mercaptoethanol, 50Oug. The first four gels use a pH 7.5)-i mM mercaptoethanol-10 mM CaCl2-0.15 M NaCl. 8.5 system (20); the last two use a sodium dodecylsulfate gel system described by Maizel (21). The anode is at the bottom Step 7: Sephadex G-200 Chromatography. The enzyme con- of the gel in all cases; the stain used was Coomassie blue (22). centrate from Step 6 was applied to a 200 X 2.5-cm column of Sephadex G-200 previously equilibrated with the dialysis A summary of the purification procedure is presented in buffer given in Step 6. Elution was at 20-30 ml/hr (Fig. 1B). Table 1. The addition of phenylmethylsulfonyl fluoride (0.1 TABLE 1. Purification of calf-tendon procollagen peptidase Total Total Specific Specific Volume protein activity* % activity activity Step (ml) (i-ng) (U) Recovered (U/mg) increase 1. Crude 3,400 1,145,000 480,000 0.4 1 2. Crude supernate 3,000 16,5)00 450,000 94 27.3 68 3. Ammonium SO4 350 5,800 440,000 92 75.9 190 4. Dialysis 380 2,195 340,000 71 154.9 387 5. DEAE 820 280 169,000 35 603.6 1,509 6. Ammonium SO4 10% extract 52 130 104,000 22 800 2,000 7. Sephadex G-200 Pk A 5 68 52,760} 775.9 1,939 Pk B 5 35 26,446 21 755.6 1,889 Pk C 5 25 20,000 800 2,000 * Independent measurements of both the pro al and the pro a2 conversion gave similar results; therefore, the average of these results is presented.