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

采用特异性蛋白酶抑制剂研究了红鲷死后肌纤维蛋白（肌球蛋白重链、α-肌动蛋白、β-连接蛋白和肌钙蛋白I）的降解与体内蛋白酶的关系。这些蛋白被内部蛋白酶在25℃下水解3天。我们证实了在Cuvie导管内注射特异性半胱氨酸蛋白酶抑制剂E-64对骨骼肌的半胱氨酸蛋白酶Cathepsin L有抑制作用。丝氨酸蛋白酶抑制剂（leupeptin和DFP）强烈抑制肌球蛋白HC和β-连接蛋白的降解。提示肌球蛋白HC是由丝氨酸蛋白酶（可能是MBP，肌球蛋白结合丝氨酸蛋白酶）水解的。半胱氨酸蛋白酶抑制剂（E-64）抑制α-肌动蛋白的有限降解。结果表明，α-肌动蛋白可被组织蛋白酶B、L或s降解。原肌球蛋白和肌钙蛋白I的水解主要受到丝氨酸蛋白酶抑制剂（lepeptin和DFP）的抑制，其次受到半胱氨酸蛋白酶抑制剂（E-64）的抑制。这些蛋白可能被MBP和组织蛋白酶水解。

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.