Characterization Of Metalloproteases From Body Wall Of Sea Cucumber(Stichopus Japonicus) And Their Relationship With Autolysis

Sea cucumbers are extremely sensitive to the external environment.Autolysis is always induced by change of environment physiochemical factors,which cause degradation of extracellular matrix of sea cucumber body.MMPs(Matrix metalloproteinases,MMPs)are a big family of extracellular proteolytic enzymes,can almost degrade all the extracellular matrix components.Endogenous proteases are the main reason for autolysis of aquatic animals.MMPs were reported to be involved in autolysis of fish postmortem.So,in this presentation,the presence of MMPs in body wall and intestine of sea cucumber and their characterization were analyzed.The relationship between MMPs and autolysis of sea cucumber was further investigated to clarify the effect of MMPs on autolysis of sea cucumber.First,the MMPs were extracted from body wall and intestine by Tris-HCl buffer.The activity of the obtained MMPs was assayed by gelatin zymography.Results showed that there existed several kinds of MMPs in the body wall and intestine of sea cucumber.The molecular weight of MMPs distributed widely,ranging from about 35.32kDa to more than 257kDa?A maximum activity in body wall was observed for a protein band with 67 kDa.Second,the effect of MMPs on autolysis of out-layer and inner-layer of sea cucunmber was investigated by EDTANa2,a specific inhibitors of MMPs.The change of MMPs activities,total soluble protein and protein profile was evaluated in the presence of EDTANa2.Results showed that the activity of MMPs and total soluble protein increased significantly during a 6h's autolysis,which could be inhibited or lowered by EDTANa2.Reducing and non-reducing SDS-PAGE demonstrated obvious protein degradation during autolysis,which could be inhibited almost completely by EDTANa2.Above results demonstrated that there existed various kinds of MMPs in body wall and intestine of sea cucumber.The MMPs in the body wall were involved in autolysis of sea cucumber,causing protein degradation.So,MMPs were critical autolytic protease in sea cucumber.