Procollagen type III N-terminal endopeptidase in fibroblast culture.
A peptidase activity capable of excising in a single fragment the N-terminal extension of the precursor of collagen type III (p-N-collagen type III) was observed in calf tendon fibroblast culture medium. A new procedure was developed for detecting this peptidase (p-N-collagen type III peptidase). It is based on the use of 14C-labelled p-N-collagen type III obtained by carboxymethylation of the half-cystine residues with iodo-[14C]acetamide. The released labelled N-terminal extension is soluble in 27% (v/v) ethanol, whereas the uncleaved substrate and the collagen are precipitated under these conditions. The endopeptidase nature of p-N-collagen type III peptidase is supported by the similarity in molecular weight of the product of cleavage of p-N-collagen III by the enzyme to those obtained by cleavage with bacterial collagenase. An apparent Km of 0.3 X 10(-6)M was established. The pH optimum of p-N-collagen type III peptidase is similar to that of p-N-collagen type I peptidase, i.e. about 7.5. Both peptidases are inhibited by dithiothreitol and by Cu2+ and Zn2+, but not by other bivalent ions. p-N-collagen type III peptidase does not cleave p-N-collagen I or p-N-gelatin I. Partial purification of p-N-collagen type III peptidase from fibroblast culture medium was performed by sieve chromatography on Ultrogel AcA-34 to yield two peaks of activity, of mol.wts. 170000 and 100000. Part of the activity was retained on affinity chromatography on concanavalin A--Sepharose. Studied as a function of the age of the culture, p-N-collagen type III peptidase activity produced by tendon fibroblasts parallels that of p-N-collagen type I peptidase and collagen synthesis.
D000818:Animals; D002417:Cattle; D002478:Cells, Cultured; D003094:Collagen / Q000096:biosynthesis; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000378:metabolism*; D005347:Fibroblasts / Q000201:enzymology*; D007700:Kinetics; D008722:Methods; D003012:Microbial Collagenase / Q000378:metabolism; D010446:Peptide Fragments / Q000032:analysis; D011347:Procollagen / Q000378:metabolism; D011348:Procollagen N-Endopeptidase
D010446:Peptide Fragments; D011347:Procollagen; D003094:Collagen; D010450:Endopeptidases; D011348:Procollagen N-Endopeptidase; D003012:Microbial Collagenase