Partial purification and characterization of a procollagen C-proteinase from the culture medium of mouse fibroblasts.
A procollagen C-proteinase was purified about 100-fold from the medium of cultured mouse fibroblasts by a combination of ammonium sulfate precipitation, gel-filtration, 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 alpha 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 alpha 1(I), pro alpha 2(I) and pro alpha 1(III) chains showed that the cleavage occurred at the physiological site, i.e. at the specific Ala-Asp bond in the pro alpha 1(I) and pro alpha 2(I) chains, and at the specific Gly-Asp bond in the pro alpha 1(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 metal-chelators, 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.
D000595:Amino Acid Sequence; D000818:Animals; D055395:Bone Morphogenetic Protein 1; D019485:Bone Morphogenetic Proteins; D002478:Cells, Cultured; D003470:Culture Media; D010450:Endopeptidases / Q000302:isolation & purification* / Q000378:metabolism; D005347:Fibroblasts / Q000201:enzymology; D006801:Humans; D007700:Kinetics; D008666:Metalloendopeptidases; D051379:Mice; D010447:Peptide Hydrolases / Q000378:metabolism; D011347:Procollagen / Q000378:metabolism; D013379:Substrate Specificity
D019485:Bone Morphogenetic Proteins; D003470:Culture Media; D011347:Procollagen; D010450:Endopeptidases; D010447:Peptide Hydrolases; D008666:Metalloendopeptidases; C525705:BMP1 protein, human; C525707:Bmp1 protein, mouse; D055395:Bone Morphogenetic Protein 1