The Mechanism Of Myosin Phosphorylation To Actin-myosin Interaction

The objective of this study was to investigate the regulation of myosin phosphorylation to actin-myosin interaction. The sheep longissimus dorsi muscles were used as experimental material. The phosphorylation level of myosin and actin-myosin interaction during postmortem were measured by SDS-PAGE, Pro-Q, Ruby fluorescence staining and Western blot. The effect of myosin regulatory light chain(MRLC) phosphorylation and dephosphorylation to actin-myosin interaction was validated by changing the protein phosphorylation level of sheep longissimus dorsi muscleshomogenate and actomyosin crude extract. The detailed results are list as follows:(1) The change of myosin phosphorylation levels, actin-myosin interaction and sarcomere length during postmortem was investigated by a combination of SDS-PAGE coupled with ProQ Diamond-SYPRO Ruby staining, western blot, ATPase activity assay kit and transmission electron microscope, respectively. The results indicated that the phosphorylation levels of MRLC decreased significantly from 6 to 24 h postmortem time, while the actomyosin dissociation degree, actomyosin ATPase activity increased significantly and the sarcomere length exhibited significant decrease during the same postmortem time. The actomyosin ATPase activity has significant negative correlation with sarcomere length(r=-0.890, p < 0.05), the MRLC phosphorylation level was highly negative relevant to actomyosin dissociation degree(r=-0.823).(2) The regulation of myofibrillar proteins phosphorylation to actin-myosin interaction was investigated when the muscle homogenization solution was treated with alkaline phosphatase and phosphatase inhibitorafter incubated at 4℃ for 72 h. The dephosphorylation of MRLC was significant higher in alkaline phosphatase group compared with control group,the actomyosin dissociation degree decreased significantly, while the actomyosin ATPase activity increased significantly. However, the dephosphorylation of MRLC was significant lower in alkaline phosphatase group compared with control group, the actomyosin dissociation degree increased significantly, while the actomyosin ATPase activity decreased significantly. The results indicate that the dephosphorylation of MRLC enhances the force and the numbers of binding that interact between myosin and actin, but the regulate approaches and tensity are different, it has a more direct regulation to myosin-actin force binding ratherthan actomyosin ATPase activity.(3) The regulation of actomyosin phosphorylation to actin-myosin interaction was investigated when the actomyosin crude extract was treated with protein kinase A and alkaline phosphatase after incubated at 4℃ for 48 h. The phosphorylation of troponin T(TnT) and MRLC were significant higher in protein kinase A group compared with control group, the actomyosin dissociation degree increased significantly, while the actomyosin ATPase activity decreased significantly. However, the phosphorylation of TnT and MRLC was significant lower in alkaline phosphatase group compared with control group, the actomyosin dissociation degree decreased significantly, while the actomyosin ATPase activity increased significantly. The results indicate that the phosphorylation of TnT and MRLC reduce actomyosin ATPase activity(the force of binding that interact between myosin and actin),thereby enhance actomyosin dissociation.