NATIVE TIMP-FREE 70-KDA PROGELATINASE (MMP-2) SECRETED AT ELEVATED LEVELS BY RSV TRANSFORMED FIBROBLASTS

Rous sarcoma virus-transformed cultures of chicken embryo fibroblasts (RSVCEF) secrete elevated levels of a 70 kDa progelatinase, an avian f orm of the 72 kDa matrix metalloproteinase-2 (MMP-2). Affinity-purifie d preparations of secreted 70 kDa progelatinase are com posed of two d istinct populations of zymogen: a 70 kDa progelatinase tightly complex ed with an avian form of TIMP-2 and a native 70 kDa progelatinase free of any detectable TIMP-2. These two forms of the progelatinase can be separated by Mono Q FPLC in the absence of denaturing agents. The hom ogeneity of the two separated forms is demonstrated by both SDS-PAGE a nd nondenaturing, native gel electrophoresis. The purified TIMP-free 7 0 kDa progelatinase is stable in aqueous conditions and does not spont aneously autoactivate. Treatment of the TIMP-free progelatinase with t he organomercurial, p-aminophenylmercuric acetate (APMA), results in r apid (5-60 minutes) autolytic conversion of the 70 kDa progelatinase t o 67 kDa, 62 kDa and lower molecular weight forms of the enzyme. APMA treatment of the TIMP-free progelatinase yields a preparation that is enzymatically active with a high specific activity towards a peptide s ubstrate. Identical treatment of TIMP-complexed progelatinase with APM A results in a significantly slower conversion process in which the 70 kDa progelatinase is only 50% converted after 6-24 hours and the spec ific enzyme activity of the preparation is 8 to 18-fold lower. Purifie d avian TIMP-2 added to the TIMP-free progelatinase forms a complex wi th the progelatinase and prevents the rapid autolytic conversion induc ed by APMA. Comparative analysis of parallel cultures of transformed R SVCEF and normal CEF demonstrates that the transformed cultures contai n threefold higher levels of the TIMP-free progelatinase than the norm al CEF cultures which produce predominantly TIMP-complexed progelatina se. The presence in transformed cultures of elevated levels of a more readily activated TIMP-free progelatinase, the suppression of its rapi d activation by TIMP-2, and the potential effect of the altered balanc e between TIMP-free and TIMP-complexed 70 kDa progelatinase on the inv asive, malignant phenotype, are discussed. (C) 1994 Wiley-Liss, inc.