Proteolytic degradation of muscllarr proteins of red sea bream by using immuno-electron mycroscope and immunoblot analysis

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We examined the degradation of post-mortem myofibrillar proteins (myosin heavy chain, α-actinin, β-connectin and troponin I) of red sea bream Pagrus major in relation with internal proteases by using specific protease inhibitors. These proteins were considerably hydrolyzed with internal proteinase(s) at 25℃ for 3 days. We confirmed that Cathepsin L which is cysteine protease in skeretal muscle was inhibited by E-64, specific cysteine protease inhibitor, injected to duct of Cuvie. Degradation of myosin HC and β-connectin were strongly inhibited by serine protease inhibitor (leupeptin and DFP). It was suggested that myosin HC is hydrolyzed by serine protease (may be MBP, myosin binding serine proteinase). Limited degradation of α-actinin was inhibited by cysteine protease inhibitor(E-64). The findings suggests that α-actinin is degraded by cathepsins B, L or S. Hydrolysis of tropomyosin and troponin I were mainly inhibited by serine protease inhibitors (leupeptin and DFP) and secondly inhibited by cysteine protease inhibitor(E-64). It is suggested that these proteins are hydrolyzed by MBP and the cathepsins.