宰后肌肉中肌球蛋白磷酸化调控肌动球蛋白解离作用机制

【目的】研究宰后肌肉中肌球蛋白磷酸化与肌动球蛋白解离之间的关系,分析其磷酸化水平的变化对肌动球蛋白解离的影响,探究肌球蛋白磷酸化对宰后肌肉肌节长度与嫩度的作用。【方法】取宰后30 min内的羊背最长肌,在4℃条件下分别成熟6、24、48和72 h,通过SDS-PAGE电泳、Pro-Q染色和蛋白质免疫印迹测定肌球蛋白的磷酸化水平和肌动球蛋白解离程度随宰后时间的变化;测定肌动球蛋白ATP酶的活性,分析宰后不同时间点肌球蛋白与肌动蛋白结合作用力的强弱;采用透射电镜分析宰后肌节长度随时间的变化。【结果】研究发现宰后肌肉中肌球蛋白轻链2的磷酸化水平在0.5—48 h快速降低(P0.05),并在48 h达到最低点,在48—72 h有所升高(P0.05),但其最终磷酸化水平明显低于初始值。肌动球蛋白的解离程度在宰后初期(0.5—6 h)显著降低(P0.05),在6—48 h显著升高(P0.05),并于48—72 h维持稳定,其最终解离程度显著高于宰后0.5 h的初始值。肌动球蛋白ATPase活性在宰后初期(0.5—6 h)略有升高,6—24 h快速上升(P0.05),并在24 h达到最高点,24—72 h逐渐降低;而肌节长度的变化则与之相反,呈先下降后上升的趋势,并在24 h达到肌节最短点。【结论】羊宰后肌肉中的肌球蛋白轻链2磷酸化水平的变化对肌球蛋白与肌动蛋白的相互作用有较大的影响,且肌节收缩(肌球蛋白与肌动蛋白的相互作用力)与肌动球蛋白的解离(肌球蛋白与肌动蛋白的相互作用量)并不是一个同步的进程。肌球蛋白轻链2的磷酸化修饰负向调控肌动球蛋白解离和肌动球蛋白ATPase活性,导致肌节的收缩与舒张,进而调控肉品最终的嫩度。

The Mechanism of Myosin Phosphorylation Regulating Actomyosin Dissociation of Skeletal Muscle During Postmortem

【Objective】The aim of this study was to investigate the effect of myosin phosphorylation modification on actomyosin dissociation by analyzing the correlation between myosin phosphorylation and actomyosin dissociation, and then reveal the function of myosin phosphorylation to sarcomere length and tenderness during postmortem.【Method】Samples of ovine longissimus dorsi after storage at 4℃ for 6, 24, 48, 72 h were collected for myosin phosphorylation and actomyosin dissociation by SDS-PAGE, Pro-Q Diamond staining and western blotting, actomyosin ATPase activity. And sarcomere length was measured by transmission electron microscope.【Result】The phosphorylation level of myosin light chain 2 decreased sharply from 0.5-48 h (P＜0.05), then it increased from 48-72 h, and the final value was lower than the initial phosphorylation value. While to the dissociation of actomyosin, it displayed a gradual decrease from 0.5-6 h and then increased from 6-48 h (P＜0.05), finally kept stable during 48-72 h postmortem, and the final dissociation degree was significantly higher than 0.5 h. The actomyosin ATPase activity increased to the highest value at 24 h, followed by gradual decrement from 24 to 72 h. On the contrary, the sarcomere length decreased to the shortest value at 24 h, followed by gradual sarcomere relaxation from 24 to 72 h.【Conclusion】The phosphorylation level of myosin light chain 2 had a great influence on myosin and actin interaction, in addition, the sarcomere contraction (myosin and actin interaction force) and the dissociation of actomyosin (myosin and actin interaction amount) happened out of sync during postmortem. Phosphorylation modification of myosin light chain 2 caused the sarcomere shrinkage and relaxation by negative regulating actomyosin dissociation and actomyosin ATPase activity negatively, which could control the final meat tenderness.