Purification of human procollagen type III N-proteinase from placenta and preparation of antiserum.
Procollagen type III N-proteinase, of Mr about 70,000, was detected in human placental tissue and purified from this source more than 5800-fold. It was found to be a glycoprotein, which was bound to both concanavalin A-Ultrogel and heparin-Sepharose affinity columns. Binding to a type III pN-collagen-Sepharose affinity column was used as the final step in purification. The purified enzyme accepted only native type III procollagen or [14C]carboxymethylated type III pN-collagen as its substrate; type I, type II and type IV procollagen and heat-denatured type III pN-collagen were not cleaved by the enzyme. Antibodies against this purified enzyme protein raised in rabbits demonstrated a high inhibitory effect on the enzyme activity. Immunoblotting of the denatured protein and immunoelectrophoresis of the native enzyme showed only one major antigenic component, again with an Mr of about 70,000. The antibodies cross-reacted with the enzyme preparation from foetal-calf aorta smooth-muscle cells.
D002122:Calcium Chloride / Q000494:pharmacology; D002712:Chlorides / Q000494:pharmacology; D004229:Dithiothreitol / Q000494:pharmacology; D010450:Endopeptidases / Q000276:immunology / Q000302:isolation & purification*; D005260:Female; D006801:Humans; D007106:Immune Sera / Q000276:immunology; D007122:Immunoelectrophoresis; D007074:Immunoglobulin G / Q000276:immunology; D010920:Placenta / Q000201:enzymology*; D011247:Pregnancy; D011348:Procollagen N-Endopeptidase; D011480:Protease Inhibitors; D015032:Zinc / Q000494:pharmacology; D017967:Zinc Compounds
D002712:Chlorides; D007106:Immune Sera; D007074:Immunoglobulin G; D011480:Protease Inhibitors; D017967:Zinc Compounds; C016837:zinc chloride; D010450:Endopeptidases; D011348:Procollagen N-Endopeptidase; D015032:Zinc; D002122:Calcium Chloride; D004229:Dithiothreitol