Partial Purification and Characterization of a Procollagen C-Proteinase from the Culture Medium of Mouse Fibroblasts

Abstract A procollagen C-proteinase was purified about 100-fold from the medium of cultured mouse fibroblasts by a combination of ammonium sulfate precipitation, gelfiltration, and affinity chromatography on a column of Sepharose coupled to the carboxyl propeptide of type I procollagen. The purified enzyme did not exhibit other proteolytic activities, and it cleaved type I, II and III procollagens to produce the corresponding pN a chains and carboxyl propeptides as the only products. Amino acid sequencing of the first 14-18 residues at the N-terminus of the carboxyl propeptides generated by the enzyme from human pro al(l), pro a 2(I) and pro a 1(III) chains showed that the cleavage occurred at the physiological site, i. e. at the specific Ala-Asp bond in the pro a1 (I) and pro a2(I) chains, and at the specific Gly-Asp bond in the pro a1(III) chain. The pH optimum of the enzyme is 8.5 and its molecular weight as estimated by gel-filtration is about 125,000 daltons. The enzyme is inhibited by metalchelators, various amines, dithiothreitol, N-ethylmaleimide and serum, but it is insensitive to pepstatin, leupeptin and serine proteases inhibitors. The enzyme differs from the C-proteinase described by Njieha et al. (Biochemistry 21: 757–764, 1982), and the catheptic activities reported by Davidson et al. (Eur. J. Biochem 100: 551–558, 1979) and Helseth and Veis (Proc. Natl. Acad. Sci. USA 81: 3302–3306, 1984). The specificity of the enzyme is offered as evidence for a unique, C-proteinase, and its recovery from culture medium supports an extracellular location for procollagen processing.