Type I procollagen N-proteinase from whole chick embryos. Cleavage of a homotrimer of pro-alpha 1(I) chains and the requirement for procollagen with a triple-helical conformation.
Procollagen N-proteinase, the enzyme which cleaves the NH2-terminal propeptides from type I procollagen, was purified over 15,000-fold from extracts of chick embryos by chromatography on columns of DEAE-cellulose, concanavalin A-agarose, heparin-agarose, pN-collagen-agarose, and a filtration gel. The purified enzyme had an apparent molecular weight of 320,000 as estimated by gel filtration and a pH optimum for activity of 7.4 to 9.0. The enzyme was inhibited by metal chelators and the thiol reagent dithiothreitol. Addition of calcium was required for maximal activity under the standard assay conditions, and the presence of calcium decreased thermal inactivation at 37 degrees C. The purified enzyme cleaved a homotrimer of pro-alpha 1(I) chains, an observation which indicated that the presence of pro-alpha 2(I) chain is not essential for the enzymic cleavage of NH2-terminal propeptides. Previous observations suggesting that the enzyme requires a substrate with a native conformation were explored further by reacting the enzyme with type I procollagen at different temperatures. Type I procollagen from chick embryo fibroblasts became resistant to cleavage at about 43 degrees C. Type I procollagen from human skin fibroblasts, which was previously shown to have a slightly lower thermal stability than chick embryo type I procollagen, became resistant to cleavage at temperatures that were about 2 degrees C lower. The results suggested that the enzyme is a sensitive probe for the three-dimensional structure of the NH2-terminal region of the procollagen molecule and that it requires the protein substrate to be triple helical.
D000818:Animals; D002118:Calcium / Q000494:pharmacology; D002642:Chick Embryo; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D011347:Procollagen / Q000378:metabolism*; D011348:Procollagen N-Endopeptidase; D011487:Protein Conformation; D012756:Sheep; D013696:Temperature
D011347:Procollagen; D010450:Endopeptidases; D011348:Procollagen N-Endopeptidase; D002118:Calcium