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.
Wu
Guo-Ping
GP
Fisheries College, The Key Laboratory of Science and Technology for Aquaculture and Food Safety, Jimei University, Xiamen, China.
Cao
Min-Jie
MJ
Chen
Su-Hua
SH
Weng
Wu-Yin
WY
Cai
Qiu-Feng
QF
Su
Wen-Jin
WJ
D000818:Animals; D004492:Edetic Acid / Q000494:pharmacology; D004533:Egtazic Acid / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D005780:Gelatin / Q000737:chemistry*; D006868:Hydrolysis; D045726:Metalloproteases / Q000737:chemistry / Q000302:isolation & purification*; D018482:Muscle, Skeletal / Q000201:enzymology*; D010618:Phenanthrolines / Q000494:pharmacology; D011480:Protease Inhibitors / Q000494:pharmacology; D021541:Sea Bream
D010618:Phenanthrolines; D011480:Protease Inhibitors; D004533:Egtazic Acid; D005780:Gelatin; D004492:Edetic Acid; D045726:Metalloproteases; C025205:1,10-phenanthroline