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 thr ee sources: articular cartilage and conditioned media from synovial fi broblasts and Chinese hamster ovary cells expressing recombinant pro-M MP-3. All three preparations reacted with two monoclonal antibodies sp ecific 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-c hain at Ala14-Leu15 and Tyr16-Leu17 in a ratio of 3:1; and heat stabil ity at neutral pH. Further characterization of MMP-3 establishes that the acid pH optimum for cartilage aggrecan is not due to substrate den aturation since the same optimum is found by viscosity assay, by SDS-p olyacrylamide gel electrophoresis assay of G1 domain, and by digestion of aggrecan in fresh cartilage fragments in vitro. Fibronectin was al so digested optimally at pH 5.5 and NH2-terminal sequence analysis rev ealed no pH change in a major proteolytic site of cleavage at the Pro6 89-Leu690 bond. The specificity constant k(cat)/K(m) is maximal at pH 5.5 as determined in a quenched fluorescence peptide assay. This is du e to an increase in k(cat) at pH 5.5 without any substantial effect on K(m). The affinity of MMP-3 for calcium is decreased about 10-fold at pH 5.3 compared to neutral pH. Finally, the neutral cartilage metallo proteinase is identified as 72-kDa pro-MMP-2 based on M(r), specificit y of insulin B-chain cleavage, and reactivity with a specific polycona l antibody to human MMP-2.