Mode of Action ... Tendons obtained from the legs of freshly killed calves were carefully cleaned ..... formed by the action of procollagen peptidase (0) or heat de-.
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 MATERIALS AND METHODS
The procollagen peptidase activity of calf ABSTRACT tendon has been purified. The enzyme has a high degree of specificity for native procollagen and converts both pro al and pro a2 to al and a2, respectively. The purified enzyme is an endopeptidase which excises the amino terminal peptide extensions of the precursor chains in block; the molecular size and amino-acid composition of the excised peptides compare favorably with those predicted in previous reports. Antisera to the enzyme and to procollagen have been prepared and have been used to characterize the enzyme, the enzymatically excised peptides, and the enzyme-peptide complex in reaction mixtures.
Collagen Substrates and Enzyme Assay. Dermatosparaxic procollagen served as the substrate in all reactions; its preparation has been described (5, 13). The standard enzyme assay included 0.1 ml of the substrate solution (0.6 mg of native procollagen per ml), 0.05 ml of M glycyl glycine buffer (pH 7.5), and appropriate aliquots of enzyme, in a final volume of 0.25 ml. Tubes were incubated at 26° for 6 hr unless otherwise noted. Reactions were stopped and analyzed by disc-gel electrophoresis as described (13). All assays were run at multiple protein concentrations and in duplicate. Enzyme activity was quantitated by calculating the fraction of pro al or pro a2 converted to al or a2 when compared to control incubations without enzyme. Since each incubation mixture contained 60 jig of intact native procollagen, the fractional conversion was readily expressed in absolute terms, i.e., one unit of enzyme activity was defined as the conversion of 1 Mig of native procollagen per 6 hr per assay volume. Specific activity correlated these units to enzyme protein measured calorimetrically (15). Immunization and Evaluation of Antisera. New Zealand rabbits were immunized with native procollagen using schedules and techniques described elsewhere (16). Antisera were regularly evaluated by microimmunodiffusion (16, 17) or immunoelectrophoresis (16,18,19). The unadsorbed, antiserum to procollagen used in this report reacted with native procollagen but gave no reaction with collagen or normal calf serum in immunodiffusion or by radioimmunoassay (16). RESULTS
Dermatosparaxis is an inherited connective tissue disorder of cattle which is characterized by extreme fragility of the skin (1, 2). The dermis of these animals contains disorganized collagen bundles which demonstrate an impaired parallel packing of individual collagen filaments within the collagen fibers (3, 4). These abnormal fibers are presumed to result from the accumulation of procollagen, the triple helical precursor of collagen which is composed of pro al and pro a2 chains (5-9). Pro al and pro a2 chains are ribosomal products (10, 11) which contain an additional peptide of 100 to 200 residues at the amino termini of their respective a chains (5, 8, 9, 12). In a previous report (13) we described an enzyme activity in normal bovine tissues which excised the amino-terminal extension of the precursor chains to yield electrophoretically normal chains. We further showed that this activity was absent or markedly reduced in dermatosparaxic tissues, i.e., the accumulation of procollagen and the resultant tissue changes were apparently caused by a defect in a specific enzyme, "procollagen peptidase." The "procollagen peptidase" activity was associated with a very low level of nonspecific protease activity in contrast to pepsin and chymotrypsin, two enzymes used in vitro by several laboratories to convert pro al to al chains (6, 7, 14). In the present report we describe the purification of calftendon procollagen peptidase, characterize several of its properties, and show that the enzyme is an endopeptidase which excises the amino-terminal peptide of procollagen in block rather than in multiple fragments.
Purification of the enzyme Tendons obtained from the legs of freshly killed calves were carefully cleaned of contaminating muscle and were washed three times with 0.05 M Tris HCl (pH 7.4)-0.15 M NaCl. The dissecting and washing procedure was done at 0-2' as were all subsequent manipulations. Tendon preparations were either processed immediately or stored at -70° for subsequent extractions with no significant difference in results.
Step 1: Homogenization. Tendon pieces were homogenized by grinding in a Hobart commercial meat grinder. To cool the tissue during this procedure, and to aid in its processing, frozen pieces of the wash buffer were alternated with tissue
I This work was performed in partial fulfillment of the degree requirements for the Department of Chemistry, American University, Washington, D.C. 20016. 40
Proc. Nat. Acad. Sci. USA 71
Tendon Procollagen Peptidase
(1974)
aliquots. The total amount of frozen buffer used three times the weight of the tendon homogenized.
was
41
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Step 2: Centrifugation of Homogenate. The homogenate was 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 sulfate, 39 g/100 ml, was slowly added to the thawed supernatant of Step 2. The stirred suspension was equilibrated overnight and the precipitate was harvested by centrifugation at 40,000 X g for 20 min. The precipitate was solubilized in 0.05 M Tris HCl (pH 7.4)-i mM mercaptoethanol-0.15 M NaCl-10 mM CaC12. Step 4: Dialysis. By dialysis for 24 hr against two separate 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 by centrifugation at 40,000. X g for 10 min. Step 5: DEAE-Cellulose Chromatography. The supernatant solution from Step 4 was applied (1 ml/min) to a 100 X 4-cm column of microgranular DEAE previously equilibrated with 0.05 M Tris HCl (pH 7.4),-1 mM mercaptoethanol. The column was eluted with a linear gradient consisting of 0-0.3 M NaCl in equilibrating buffer (Fig. 1A). Step 6: Ammonium Sulfate Extraction. Enzyme activity eluted from the DEAE column (Fig. IA, black bar) was pooled, concentrated by ultrafiltration, and precipitated by the addition of ammonium sulfate to a final concentration of 60%. The precipitate was harvested by centrifugation at 40,000 X g and was consecutively extracted by the following solutions: 40, 30, 25, 20, and 10% ammonium sulfate in equilibrating buffer (Step 5) and equilibrating buffer alone. The supernatant of the extraction with the highest specific activity (usually the 10% extract) was dialyzed against 0.05 M Trisp Cl, (pH 7.5)-i mM mercaptoethanol-10 mM CaCl2-0.15 M NaCl. Step 7: Sephadex G-200 Chromatography. The enzyme concentrate from Step 6 was applied to a 200 X 2.5-cm column of Sephadex G-200 previously equilibrated with the dialysis buffer given in Step 6. Elution was at 20-30 ml/hr (Fig. 1B).
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