Study On The Regulation Of Actomyosin Dissociation By Protein Phosphorylation And Acetylation

Tenderness is one of the important meat quality indicators,and the binding state of myosin and actin postmortem is a key factor affecting meat tenderness.Studies have shown that phosphorylation regulates the dissociation of actomyosin,and acetylation may affect tenderness by regulating the activity of glycolytic enzymes and the function of sarcomere related proteins.It remains unknown the relationship between protein acetylation level and tenderness at postmortem,whether myosin and actin phosphorylation and acetylation are related and how they affect actomyosin dissociation.This study used the longissimus dorsi muscle of sheep to study the relationship between protein acetylation level and meat quality postmortem and an in vitro model to study the regulation of actomyosin dissociation by phosphorylation and acetylation.(1)The p H,meat color,relative content of myoglobin,myofibril fragmentation index,cooking loss,shear force,total sarcoplasmic protein acetylation level,and total myofibrillar protein acetylation level of the longissimus dorsi muscle were measured at 1 h,6 h,12 h,1 d,2 d,3 d,5 d and 7 d postmortem.The results showed that shear force was positively correlated with the total acetylation level of myofibrillar protein,and myofibrillar protein acetylation negatively regulated tenderness.The p H was negatively correlated with the total sarcoplasmic protein acetylation level in the period of pre-rigor and rigor mortis,and the correlation was weakened in the period of post-rigor.The redness(a~*)and the total sarcoplasmic protein acetylation level were positively correlated in the pre-rigor period,sarcoplasmic protein acetylation positively regulated meat color.(2)The longissimus dorsi muscle of lamb at 30 min postmortem was used in the experiment.An in vitro phosphorylation model was established to study the effect of phosphorylation of myosin heavy chain and actin on its acetylation and actomyosin dissociation through regulating the phosphorylation level of myosin heavy chain and actin.An in vitro acetylation model was established to study the effect of myosin heavy chain and actin acetylation on its phosphorylation and actomyosin dissociation through regulating the acetylation level of myosin heavy chain and actin.The phosphorylation and acetylation level of myosin heavy chain and actin,the degree of actomyosin dissociation,and ATPase activity were measured at 0 min,30 min,4 h,12 h,1 d,2 d,3 d incubation.The results showed that under in vitro conditions,actin phosphorylation promoted the dissociation of actomyosin by inhibiting its acetylation,and phosphorylation of myosin heavy chain directly promoted the dissociation of actomyosin;myosin heavy chain and actin acetylation regulated the interaction between myosin and actin by inhibiting its phosphorylation,and inhibited the dissociation of actomyosin.(3)The myosin heavy chain and actin phosphorylation and acetylation sites of the lamb longissimus dorsi muscle at 30 min and 5 d postmortem were identified by mass spectrometry,and the mechanism of actomyosin phosphorylation and acetylation modification sites regulating the structural stability of actomyosin was explored by molecular dynamics.There were 56 myosin heavy chain phosphorylation sites,20 actin phosphorylation sites,76 myosin heavy chain acetylation sites,and 8 actin acetylation sites identified.The results of molecular dynamics analysis showed that myosin heavy chain and actin phosphorylation increased the total energy of the actomyosin and reduced the actomyosin stability.Myosin heavy chain and actin acetylation reduced the total energy of the actomyosin and increased the actomyosin stability.When the two types of modifications occur at the same time,the stability of the actomyosin system reduced which promoted the dissociation of actomyosin.