The objective of this study was to investigate the effects of myosin heavy chain and actin phosphorylation on their acetylation levels, actomyosin dissociation, and ATPase activity, so as to provide a theoretical basis for improving meat tenderness by regulating protein phosphorylation level.

The homogenate of sheep longissimus dorsi muscle was incubated with alkaline phosphatase inhibitor (inhibiting dephosphorylation) and protein kinase inhibitor (inhibiting phosphorylation) at 4℃ for 0, 0.5, 4, 12, 24, 48, and 72 h to regulate the phosphorylation levels of myosin heavy chain and actin. The protein phosphorylation level was measured by SDS-PAGE and fluorescent staining, and the acetylation level and actomyosin dissociation degree were measured by Western blotting. The ATPase activity was measured using an assay kit. The influence of myosin heavy chain and actin phosphorylation on the structure of actomyosin was analyzed by molecular dynamics simulation.

The phosphorylation level of myosin heavy chain in the alkaline phosphatase inhibitor treatment group was significantly higher than that in the control and protein kinase inhibitor treatment groups at 4, 12, and 72 h of incubation (P<0.05). The phosphorylation level of actin was significantly higher than that in the control and protein kinase inhibition treatment groups at 4, 12, 24, 48, and 72 h of incubation (P<0.05), which indicated that alkaline phosphatase inhibitors could inhibit the dephosphorylation of myosin heavy chain and actin during incubation in vitro. The acetylation level of actin in the alkaline phosphatase inhibitor treatment group was significantly lower than that in the protein kinase inhibitor treatment group after incubation for 4, 12, 24, 48, and 72 h (P<0.05), while the acetylation level of myosin heavy chain changed irregularly. The results indicated that the phosphorylation of actin inhibited its acetylation, while the phosphorylation of myosin heavy chain had no obvious regularity on its acetylation. The results of molecular dynamics showed that the phosphorylation of the 2nd, 3rd and 54th serine positions of the myosin heavy chain and the 54th and 55th tyrosine positions of actin increased the total energy, potential energy, and kinetic energy of actomyosin. However, the bond energy of actomyosin was reduced, which caused the unstable structure of actomyosin. The dissociation degree of actomyosin in the alkaline phosphatase inhibitor treatment group was always higher than that of the protein kinase inhibitor treatment group during 0-72 h incubation (P<0.05). The ATPase activity was always lower than that in the protein kinase inhibitor treatment group during 0-72 h incubation (P<0.05). The myosin heavy chain and actin phosphorylation promoted actomyosin dissociation.

The phosphorylation of myosin heavy chain directly promoted the dissociation of actomyosin, while the phosphorylation of actin promoted the dissociation of actomyosin by inhibiting its acetylation.