The carboxyl fragment released by bacterial collagenase from human type I procollagen: antibodies to the propeptide determinants.
A protocol is offered for the isolation of the carboxyterminal propeptides of human type I procollagen and the development of an antibody specific for these propeptides. Type I procollagen was harvested from the media of cultured human fibroblasts. Digestion with bacterial collagenase released a carboxyterminal fragment that was isolated by ion-exchange chromatography. The fragment contained telopeptides joined to propeptides and could be cleaved by a carboxyl procollagen peptidase. Rabbit antibodies raised to the collagenase-generated fragment were sequentially adsorbed on affinity columns of the reference antigen and human type I collagen. The antibody obtained was shown by sensitive radioimmunoassays to recognize conformational carboxyl propeptide determinants and not to react with triple helical and telopeptide determinants of human type I collagen. Indirect immunofluorescence and indirect immunoperoxidase staining of cultured fibroblasts localized the antigen in the cytoplasm, at the cell surface, and in the extracellular matrix. A radioimmunoassay with the same antibody has reported altered concentrations of the antigen in the sera of patients with diseases affecting collagen metabolism (Taubman, et al., 1976; Savolainen et al., 1984; Carey et al., 1985).
D000818:Animals; D000906:Antibodies / Q000302:isolation & purification; D001419:Bacteria / Q000201:enzymology; D002460:Cell Line; D002478:Cells, Cultured; D002645:Chickens; D002848:Chromatography, DEAE-Cellulose; D003429:Cross Reactions; D004591:Electrophoresis, Polyacrylamide Gel; D005347:Fibroblasts / Q000166:cytology; D005455:Fluorescent Antibody Technique; D006801:Humans; D007124:Immunoenzyme Techniques; D003012:Microbial Collagenase / Q000378:metabolism*; D010446:Peptide Fragments / Q000032:analysis*; D011347:Procollagen / Q000378:metabolism*; D051381:Rats; D012867:Skin / Q000166:cytology
D000906:Antibodies; D010446:Peptide Fragments; D011347:Procollagen; D003012:Microbial Collagenase