A metalloproteinase from human rheumatoid synovial fibroblasts that digests connective tissue matrix components. Purification and characterization.
Human rheumatoid synovial cells in culture stimulated with the conditioned culture medium of rabbit macrophages secrete three distinct latent metalloproteinases. One of them, a proteinase that digests proteoglycan and other connective tissue matrix components, was purified as two active forms after activation with 4-aminophenylmercuric acetate. The two forms were homogeneous on sodium dodecyl sulfate-gel electrophoresis with Mr = 45,000 and Mr = 28,000, whereas the latent precursor was estimated to have Mr = 51,000 by gel permeation chromatography. Both active enzymes had optimal activity at pH 7.5-7.8 and were inhibited by EDTA and 1,10-phenanthroline but not by inhibitors for cysteine, serine, or aspartic proteinases. Removal of Ca2+ from the enzyme solution resulted in a complete loss of activity that could be fully restored by the addition of 1 mM Ca2+. The activity of the apoenzyme was restored by the addition of 0.5 mM Zn2+, 5 mM Co2+, or 5 mM Mn2+ in the presence of Ca2+ but not by each metal ion alone. The identical digestion patterns of reduced, carboxymethylated protein substrates indicated that both active forms of the enzyme have the same substrate specificity. The enzyme degraded cartilage proteoglycans, type I gelatin, type IV collagen, laminin, and fibronectin, and removed the NH2-terminal propeptides from chick type I procollagen. This enzyme may play a role in the normal turnover of the connective tissue matrix as well as in the joint destruction of chronic synovitis.
D000818:Animals; D001172:Arthritis, Rheumatoid / Q000378:metabolism*; D002118:Calcium / Q000494:pharmacology; D003035:Cobalt / Q000494:pharmacology; D003238:Connective Tissue / Q000378:metabolism*; D003470:Culture Media; D004492:Edetic Acid / Q000494:pharmacology; D010450:Endopeptidases / Q000378:metabolism*; D005109:Extracellular Matrix / Q000378:metabolism*; D005347:Fibroblasts / Q000201:enzymology; D006801:Humans; D007854:Lead / Q000494:pharmacology; D008264:Macrophages / Q000378:metabolism; D008345:Manganese / Q000494:pharmacology; D008666:Metalloendopeptidases; D008970:Molecular Weight; D010618:Phenanthrolines / Q000494:pharmacology; D010662:Phenylmercuric Acetate / Q000031:analogs & derivatives / Q000494:pharmacology; D011480:Protease Inhibitors / Q000494:pharmacology; D011509:Proteoglycans / Q000378:metabolism; D011817:Rabbits; D013583:Synovial Membrane / Q000201:enzymology*; D015032:Zinc / Q000494:pharmacology
D003470:Culture Media; D010618:Phenanthrolines; D011480:Protease Inhibitors; D011509:Proteoglycans; D007854:Lead; D003035:Cobalt; D008345:Manganese; C010024:4-aminophenylmercuriacetate; D004492:Edetic Acid; D010450:Endopeptidases; D008666:Metalloendopeptidases; D015032:Zinc; D010662:Phenylmercuric Acetate; D002118:Calcium; C025205:1,10-phenanthroline