肌原纤维蛋白磷酸化对其被μ-钙蛋白酶降解的影响

Effect of Phosphorylation on the Degradation of Myofibrillar Proteins by μ-Calpain

目的:研究肌原纤维蛋白磷酸化对其被μ-钙蛋白酶降解的影响。方法:以羊背最长肌肌原纤维蛋白为原料,添加蛋白激酶A和碱性磷酸酶催化磷酸化和去磷酸化反应,反应后加入μ-钙蛋白酶,通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(sodium dodecyl sulfate-polyacrylamide gel electrophoresis,SDS-PAGE)和荧光染色、蛋白质免疫印迹测定肌原纤维蛋白的磷酸化水平、蛋白降解程度。结果:蛋白激酶A处理组肌原纤维蛋白磷酸化水平显著高于对照组(P<0.05);碱性磷酸酶处理组肌原纤维蛋白磷酸化水平显著低于对照组(P<0.05);蛋白激酶A处理抑制μ-钙蛋白酶自溶,降低其活性,延长其发挥活性的时间;蛋白激酶A处理促进肌球蛋白重链及肌钙蛋白T降解;碱性磷酸酶处理促进肌动蛋白及肌间线蛋白降解。结论:磷酸化通过作用于μ-钙蛋白酶活性及肌原纤维蛋白进而影响μ-钙蛋白酶降解肌原纤维蛋白。

Objective:The objective of this study was to investigate the effect of phosphorylation on the degradation of myofibrillar proteins byμ-calpain.Methods:Protein kinase A(PKA)and alkaline phosphatase(AP)were used to catalyze the phosphorylation and dephosphorylation of myofibrillar proteins of mutton longissimus dorsi muscle,followed by hydrolysis byμ-calpain.The levels of protein phosphorylation and degradation were measured by SDS-PAGE,Pro-Q Diamond fluorescent staining,and Western blotting,respectively.Results:The phosphorylation level of myofibrillar proteins in the PKA group was significantly higher than that of the control group(P<0.05),while the phosphorylation level of myofibrillar proteins in the AP group was significantly lower than that of the control group(P<0.05).The autolysis and activity ofμ-calpain were suppressed by PKA treatment,thereby leading to prolonged hydrolysis of myofibrillar proteins byμ-calpain.Phosphorylation enhanced the degradation of myosin heavy chain and troponin T,while dephosphorylation enhanced the degradation of actin and desmin.Conclusion:Phosphorylation affected both myofibrillar proteins andμ-calpain activity and therefore the degradation of myofibrillar proteins byμ-calpain.