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 from human Y-79 retinoblastoma cells by gelatin SDS-PAGE zymography reveals abundant activity of a 72-kDa M(r) gelatinase. The 72-kDa gelatinase from either source is inhibited by EDTA but not aprotinin or NEM, indicating that it is a metalloproteinase (MMP). The 72-kDa MMP is converted 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 autoactivates without APMA in the presence of calcium and zinc after 72 hr at 37 degrees C, producing a fully active mixture of proteinase species, 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 fractions separated by SDS-PAGE. Whole IPM inhibited gelatinolytic activity of autoactivated Y-79-derived MMP in a dose-dependent manner. Inhibitory activities are observed in two protein fractions of 27-42 and 20-25 kDa. Western blots using antibodies to human tissue inhibitor of metalloproteinase 1 and 2 (TIMP-1 and -2) reveal the presence of two TIMP-1-like proteins at 21 and 29 kDa in inhibitory fractions of the bovine IPM. TIMP-2 was not detected in the inhibitory IPM fractions, consistent with the observed autoactivation of bovine IPM 72-kDa gelatinase. Potential roles for this IPM MMP-TIMP system include physiologic remodelling of the neural retina-RPE cell interface and digestion of shed rod outer segment as well as pathological processes such as retinal detachment, PE cell migration, neovascularization and tumor progression. Cultured Y-79 cells appear to be a good model for studying the production and regulation of this proteinase system.
Jones
B E
BE
Laboratory of Retinal Cell and Molecular Biology, National Eye Institute, National Institutes of Health, Bethesda, MD 20892.
Moshyedi
P
P
Gallo
S
S
Tombran-Tink
J
J
Arand
G
G
Reid
D A
DA
Thompson
E W
EW
Chader
G J
GJ
Waldbillig
R J
RJ
D000818:Animals; D002417:Cattle; D003470:Culture Media; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D005109:Extracellular Matrix / Q000201:enzymology; D018093:Gelatinases / Q000032:analysis; D006023:Glycoproteins / Q000032:analysis; D006801:Humans; D008666:Metalloendopeptidases / Q000032:analysis* / Q000037:antagonists & inhibitors; D008970:Molecular Weight; D011506:Proteins / Q000032:analysis; D012160:Retina / Q000201:enzymology*; D012175:Retinoblastoma / Q000201:enzymology; D019716:Tissue Inhibitor of Metalloproteinase-2; D019714:Tissue Inhibitor of Metalloproteinases; D014407:Tumor Cells, Cultured / Q000201:enzymology
D003470:Culture Media; D006023:Glycoproteins; D011506:Proteins; D019714:Tissue Inhibitor of Metalloproteinases; D019716:Tissue Inhibitor of Metalloproteinase-2; D018093:Gelatinases; D008666:Metalloendopeptidases