Procollagen type III N-terminal endopeptidase in fibroblast culture

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Abstract
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-labeled p-N-collagen type III obtained by carboxymethylation of the half-cystine residues with iodo[14C]acetamide. The released labeled labeled N-terminal extension is soluble in 27% (vol/vol) ethanol, whereas the uncleaved substrated and the collagen are precipitated under these conditions. The endopeptidase nature of p-N-collagen type III peptidase is supported by the similarity in MW 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 .times. 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 2 peaks of activity, of MW 170,000 and 100,000. 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.