Purification and characterization of a gelatinolytic metalloproteinase from the skeletal muscle of red sea bream (Pagrus major).

A gelatinolytic metalloproteinase (gMP) from red sea bream ( Pagrus major ) skeletal muscle was highly purified by ammonium sulfate fractionation and column chromatographies including (diethylamino)ethyl (DEAE)-Sephacel, phenyl-Sepharose, and gelatin-Sepharose. Purified gMP revealed two bands with molecular masses of 52 and 55 kDa as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions. The 55 kDa band is quite possibly a glycosylated form of the 52 kDa band. The proteinase revealed optimal activity at 40 degrees C and pH 8.0. Metalloproteinase inhibitors including ethylenediaminetetraacetic acid (EDTA), ethylene glycol bis(2-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), and 1,10-phenanthroline specifically suppressed its activity. gMP was also significantly inhibited by cysteine and dithiothreitol. Divalent metal ion Ca(2+) is essential for its gelatinolytic activity. Thus, the proteinase is regarded as a matrix metalloproteinase-like proteinase. Furthermore, gMP hydrolyzed gelatin and type-I collagen effectively even at 4 degrees C, suggesting the possibility of its involvement in the texture tenderization of fish muscle during the post-mortem stage.