Human skin fibroblast collagenase inhibitor. Purification and biochemical characterization.
Human skin fibroblasts maintained in cell culture secrete a collagenase inhibitor which has been purified to homogeneity from serum-containing culture medium using a combination of salt precipitation, and ion-exchange, phenylboronate-agarose, gel filtration, and high pressure liquid chromatography. The pure inhibitor retained full activity and exhibited a 1:1 molar inhibition of collagenase. A single band of Mr = 28,500 was seen on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The amino acid composition was remarkable for the presence of 24 half-cystine residues. No free sulfhydryls were present and the resultant 12 disulfide bridges may account for the remarkable stability of this protein to extremes of pH, temperature, pressure, as well as to denaturing agents. A total of 13 hexose residues/molecule were found. NH2-terminal sequence analysis revealed the presence of a single polypeptide chain and the first 23 residues were identified. A monospecific antibody was produced which abolished the functional activity of the inhibitor. Fibroblast inhibitor was found to migrate with the beta-globulins by immunoelectrophoresis. A chromatographically and immunologically identical collagenase inhibitor was partially purified from human serum, suggesting the possibility that the fibroblast-derived inhibitor and the previously reported serum beta 1-anticollagenase (Woolley, D. E., Roberts, D. R., and Evanson, J. M., (1975) Biochem. Biophys. Res. Commun. 66, 747-754) are similar, if not identical, proteins. The fibroblast collagenase inhibitor was found to be clearly distinct from other collagenase inhibitors of leukocyte and serum origin.
D000596:Amino Acids / Q000032:analysis; D002460:Cell Line; D004220:Disulfides / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000302:isolation & purification* / Q000494:pharmacology; D005347:Fibroblasts / Q000201:enzymology; D006801:Humans; D005779:Immunodiffusion; D007122:Immunoelectrophoresis; D003012:Microbial Collagenase / Q000037:antagonists & inhibitors*; D010944:Plants / Q000201:enzymology; D012867:Skin / Q000201:enzymology*; D019714:Tissue Inhibitor of Metalloproteinases; D003313:Zea mays / Q000201:enzymology
D000596:Amino Acids; D004220:Disulfides; D004791:Enzyme Inhibitors; D019714:Tissue Inhibitor of Metalloproteinases; D003012:Microbial Collagenase