CHARACTERIZATION AND NOVEL ACTIVATION OF 72-KDA METALLOPROTEINASE IN RETINAL INTERPHOTORECEPTOR MATRIX AND Y-79 CELL-CULTURE MEDIUM

Analysis of bovine interphotoreceptor matrix and conditioned medium fr om human Y-79 retinoblastoma cells by gelatin SDS-PAGE zymography reve als abundant activity of a 72-kDa M(r) gelatinase. The 72-kDa gelatina se from either source is inhibited by EDTA but not aprotinin or NEM, i ndicating that it is a metalloproteinase (MMP). The 72-kDa MMP is conv erted to a 62-kDa species with APMA treatment after gelatin sepharose affinity purification, typical of previously described gelatinase MMP- 2. The latent 72-kDa gelatinase from either bovine IPM or Y-79 media a utoactivates without APMA in the presence of calcium and zinc after 72 hr at 37 degrees C, producing a fully active mixture of proteinase sp ecies, 50 (48 in Y-79 medium), 38 and 35 kDa in size. The presence of inhibitory activity was examined in both whole bovine IPM and IPM frac tions separated by SDS-PAGE. Whole IPM inhibited gelatinolytic activit y of autoactivated Y-79-derived MMP in a dose-dependent manner. Inhibi tory activities are observed in two protein fractions of 27-42 and 20- 25 kDa. Western blots using antibodies to human tissue inhibitor of me talloproteinase 1 and 2 (TIMP-1 and -2) reveal the presence of two TIM P-1-like proteins at 21 and 29 kDa in inhibitory fractions of the bovi ne IPM. TIMP-2 was not detected in the inhibitory IPM fractions, consi stent with the observed autoactivation of bovine IPM 72-kDa gelatinase . Potential roles for this IPM MMP-TIMP system include physiologic rem odelling of the neural retina-RPE cell interface and digestion of shed rod outer segment as well as pathological processes such as retinal d etachment, PE cell migration, neovascularization and tumor progression , Cultured Y-79 cells appear to be a good model for studying the produ ction and regulation of this proteinase system.