Purification And Characterization Of Metalloproteinases From Common Carp Muscle

Proteinases are enzymes that can hydrolyze polypeptides and proteins. Proteinases can be divided into serine proteinases, cysteine proteinases, aspartic proteinases and metalloproteinases based on their catalytic properties and inhibitor sensitivities. Metalloproteinases are enzymes that their active sites are composed of metal ions and animo acid residues.Matrix metalloproteinases (MMPs) are zinc-dependent enzymes that are largely responsible for degrading ECM proteins. MMPs have been proposed to participate in the post-mortem degradation of fish muscle. However, there have no reporty about MMPs from freshwater fish muscle at home. In the present study, we studied the characterization of metalloproteinases from the sarcoplasmic fraction of common carp muscle and purified these enzymes, then further studied the characterization of these enymes. This study is helpful for understanding the biochemical role in fish muscle and during the post-mortem tenderization of fish muscle. And while this study also firstly carried out at home.Using gelatin zymography, MMPs from the sarcoplasmic fraction of common carp were studied. We found gelatinolytic proteinases between 60kDa and 97kDa and these geltinoltic activities in dark muscle are higher than white muscle. After treated by APMA, the active forms were burn out. This result suggested that thses enzymes are proMMPs and active MMPs. Metal ions chelating agent EDTA, EGTA and 1, 10-phenanthroline can effectively inhibit gelatinolytic activity, but other inhibitors didn't show any inhibition effects, and high gelatinolytic activity needs Ca2+, these results indicated that these enymes are metalloproteinases. These proteinases from common carp dark muscle were purified by 30 % - 60 % ammonium sulfate fractionation and a combination of chromatographic steps including ion exchange on DEAE-Sephacel, gel filtration on Sephacryl S-200, ion exchange on High-Q and affinity on gelatin-Sepharose. The molecular weights of these proteinases as estimated by SDS-PAGE were 75 kDa, 67 kDa and 64 kDa under non-reducing conditions. The optimum temperatures of these proteinases are 40℃, and revealed high activity at pH 8.0. Metalloproteinase inhibitors, EDTA, EGTA and 1, 10- phenanthroline almost completely suppressed the gelatinolytic activity, while other proteinase inhibitors did not show any inhibitory effect. Divalent metal ion Ca2+ is essential for the gelatinolytic activity. Furthermore, these gelatinolytic proteinases effectively hydrolyze native type I collagen, but they cann't diggest myofibril proteins.While we found Myofibril-bound metalloproteinase, this supplies a new way about MHC degradation and is helpful for studying the diggestion myofibril proteins. Through one whole year experiments, we found that MHC degradated in dark muscle much faster than that in white muscle. And only metalloproteinase inhibitors EDTA and EGTA can effectively inhibit the MHC degradation, the Cu2+ and Mn2+ could accelerate myofibril degradation,but Zn2+ and Ge2+ showed some inhibition for myofibril degradation. Therefore the proteinase for myofibril degradation may be metalloproteinase, that is to say, there has MBMP in common carp muscle. And while, the seasonal change have a significant effect on activity of MBMP.On a whole, the innovations of this study are laid on the following. 1, Common carp dark muscle and white muscle were separately detached and studied, the metalloproteinases activity in dark muscle and white muscle were compared, and while metalloproteinases activity in dark muscle are higher than that in white muscle; 2, MMPs from dark muscle were firstly purified and characterized, which can digest collegen; 3, MBMP from common carp muscle were firstly found from myofibril protein, which seasonal changes were worked out.