Purification and characterization of bovine dental pulp collagenase inhibitor.
Root pulps from bovine unerupted wisdom teeth produce a potent collagenase inhibitor together with latent collagenase when cultured in Eagle's minimal essential medium (Biochem. Int. 5, 763, 1982). The inhibitor was purified more than 700-fold from the explant medium using Con A-Sepharose, Ultrogel AcA 44 and DE-52 cellulose columns. It showed a single band (MW = 36,000) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, but showed multiple bands on basic (pH 8.3) polyacrylamide gel electrophoresis and electrofocusing. The inhibitor is a sialo-glycoprotein containing approx. 20% carbohydrate by weight and its composition suggests that it contains complex-type oligosaccharides. The electrophoretic heterogeneity of the inhibitor was proved to be due to the attachment of different numbers of sialic acid residues. All the SH groups were demonstrated to exist as six disulfide linkages which might be involved in the inhibitory activity. The bovine pulp inhibitor does not combine with collagen. The addition of the inhibitor to activated collagenase resulted in dose-dependent inhibition of the enzyme activity, but the interaction between the inhibitor and activated collagenase is not tight enough for the complex to remain intact during gel filtration column chromatography. A rabbit antiserum was prepared against the inhibitor, and immunoglobulin purified from the antiserum can completely abolish the inhibitory activity of the inhibitor.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002241:Carbohydrates / Q000032:analysis; D002417:Cattle; D003782:Dental Pulp / Q000032:analysis*; D004791:Enzyme Inhibitors / Q000302:isolation & purification* / Q000494:pharmacology; D006801:Humans; D005779:Immunodiffusion; D007122:Immunoelectrophoresis; D003012:Microbial Collagenase / Q000037:antagonists & inhibitors*; D008964:Molar, Third / Q000032:analysis; D008970:Molecular Weight; D009928:Organ Specificity; D013045:Species Specificity; D019714:Tissue Inhibitor of Metalloproteinases; D014097:Tooth, Unerupted / Q000032:analysis
D000596:Amino Acids; D002241:Carbohydrates; D004791:Enzyme Inhibitors; D019714:Tissue Inhibitor of Metalloproteinases; D003012:Microbial Collagenase