Matrix metalloproteinase-3 (stromelysin-1). Identification as the cartilage acid metalloprotease and effect of pH on catalytic properties and calcium affinity.
Human pro-MMP-3 (pro-matrix metalloproteinase-3) was purified from three sources: articular cartilage and conditioned media from synovial fibroblasts and Chinese hamster ovary cells expressing recombinant pro-MMP-3. All three preparations reacted with two monoclonal antibodies specific for human fibroblast pro-MMP-3. Each preparation of active MMP-3 possessed properties identical to those previously reported for the cartilage acid metalloproteinase (MMP-6; Azzo and Woessner, J. F., Jr. (1986) J. Biol. Chem. 261, 5434-5441): an acid pH optimum of 5.3-5.5 for digestion of cartilage aggrecan; digestion of oxidized insulin B-chain at Ala14-Leu15 and Tyr16-Leu17 in a ratio of 3:1; and heat stability at neutral pH. Further characterization of MMP-3 establishes that the acid pH optimum for cartilage aggrecan is not due to substrate denaturation since the same optimum is found by viscosity assay, by SDS-polyacrylamide gel electrophoresis assay of G1 domain, and by digestion of aggrecan in fresh cartilage fragments in vitro. Fibronectin was also digested optimally at pH 5.5 and NH2-terminal sequence analysis revealed no pH change in a major proteolytic site of cleavage at the Pro689-Leu690 bond. The specificity constant kcat/Km is maximal at pH 5.5 as determined in a quenched fluorescence peptide assay. This is due to an increase in kcat at pH 5.5 without any substantial effect on Km. The affinity of MMP-3 for calcium is decreased about 10-fold at pH 5.3 compared to neutral pH. Finally, the neutral cartilage metalloproteinase is identified as 72-kDa pro-MMP-2 based on M(r), specificity of insulin B-chain cleavage, and reactivity with a specific polyclonal antibody to human MMP-2.
Wilhelm
S M
SM
Institute for Bone and Joint Disorders, Miles Research Center, Miles, Inc., West Haven, Connecticut 06516.
Shao
Z H
ZH
Housley
T J
TJ
Seperack
P K
PK
Baumann
A P
AP
Gunja-Smith
Z
Z
Woessner
J F
JF
D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D002118:Calcium / Q000378:metabolism*; D002356:Cartilage / Q000201:enzymology*; D002358:Cartilage, Articular / Q000201:enzymology; D002384:Catalysis; D006224:Cricetinae; D004591:Electrophoresis, Polyacrylamide Gel; D005347:Fibroblasts / Q000201:enzymology; D005353:Fibronectins / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007328:Insulin / Q000378:metabolism; D007700:Kinetics; D019278:Matrix Metalloproteinase 3; D008666:Metalloendopeptidases / Q000378:metabolism*; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013583:Synovial Membrane / Q000201:enzymology
D005353:Fibronectins; D007328:Insulin; D011994:Recombinant Proteins; D008666:Metalloendopeptidases; D019278:Matrix Metalloproteinase 3; D002118:Calcium