Purification and Characterization of a Gelatinolytic Metalloproteinase from the Skeletal Muscle of Red Sea Bream (Pagrus major)

Abstract

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.

PARTIAL CHARACTERIZATION OF GELATINOLYTIC PROTEINASES FROM THE SKELETAL MUSCLE OF GRASS CARP (CTENOPHARYNGODON IDELLUS)

“…2008), red stingray muscle (Bae et al . 2010) and red sea bream muscle (Wu et al . 2010a,b), while it is higher than that of barnacle larvae gelatinase (Mannello et al .…”

“…2000). Gelatinolytic proteinases were detected in red sea bream muscle, gelatinolytic proteinase at 85 kDa was serine proteinase, but two gelatinolytic enzymes at 55 and 52 kDa were metalloproteinases (Wu et al . 2010a,b).…”

“…Collagen can be hydrolyzed by MMP and MSP (Murphy and Reynolds 2003; Visse and Nagase 2003). Gelatinolytic proteinases are also able to degrade collagen (Wu et al . 2008, 2010a,b), and they are proposed to participate in the postmortem degradation of fish collagen during cold storage (Lødemel and Olsen 2003).…”

“…Gelatinolytic proteinases with properties similar to MMPs have been identified in common carp ( Cyprinius carpio ) muscle (Wu et al . 2008, 2009), red sea bream ( Pagru major ) muscle (Wu et al . 2010b), various organs of yellowtail (Kubota et al .…”

“…Gelatinolytic proteinases have been proposed to participate in the metabolism of collagens physiologically and in the postmortem degradation of fish muscle during cold storage (Bracho and Haard 1995;Saito et al 2000;Lødemel et al 2004), which were possibly derived from MMP and MSP (Kubota et al 2000). Gelatinolytic proteinases with properties similar to MMPs have been identified in common carp (Cyprinius carpio) muscle (Wu et al 2008(Wu et al , 2009, red sea bream (Pagru major) muscle (Wu et al 2010b), various organs of yellowtail (Kubota et al 1998), rainbow trout (Oncorhynchus mykiss) (Saito et al 2000), Pacific rockfish (Sebastes sp.) (Bracho and Haard 1995), muscles of ayu (Plecoglossus altivelis) (Kubota et al 2000), Atlantic cod (Gadus morhua), spotted wolfish (Anarhichas minor) and Atlantic salmon (Salmo salar) (Lødemel and Olsen 2003).…”