肌原纤维蛋白磷酸化对其被μ-钙蛋白酶降解的影响

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(P0.05);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(P0.05);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。

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

Objective: The objective of this study was to investigate the effect of phosphorylation on the degradation of myofibrillar proteins by μ-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 by μ-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 of μ-calpain were suppressed by PKA treatment, thereby leading to prolonged hydrolysis of myofibrillar proteins by μ-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 and μ-calpain activity and therefore the degradation of myofibrillar proteins by μ-calpain.