The Mechanism Of Effect Of Titin Phosphorylation On The Muscle Tenderization During Post-mortem

A titin molecule extends half of the sarcomere from the Z line?N-terminus?to the M line?C-terminus?.It keeps the integrity and stability of the myofibril.The degradation of titin could break the structure of Z line to make the myofibrillar fragmentation.The process improved the meat tenderization.Phosphorylation has been shown to regulate protein stability and degradation.Previous studies found that titin was phosphorylated during post-mortem.The objective of this study was to investigate the regulation of tenderization of post-mortem muscle by phosphorylation of titin.Different muscles from sheep were selected as experimental material.Myofibrillar and sarcoplasmic proteins were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis?SDS-PAGE?and stained with Pro-Q Diamond and Ruby stains.The phosphorylation of sarcoplasmic and myofibrillar proteins,the relative activity of?-calpain,the sarcomere length,myofibril fragmentation index?MFI?,the relative phosphorylation level and degradation of titin in three ovine muscles were measured.These results showed that titin played a key role in the tenderization of muscle during pos-mortem.In addition,the study revealed that phosphorylation may influence the reglulation of muscles tenderization.Crude extraction of native titin was incubated with protein kinase A?PKA?and alkaline phosphatase to increase and decrease the phosphorylation of titin in vitro,respectively.These results confirmed that the dephosphorylation accelerated the degradation of titin.The phosphorylated sites and dephosphorylated sites were identified.The change of titin structure was also analyzed.These detailed results were shown as follows:?1?The relationship among the phosphorylation of sarcoplasmic and myofibrillar proteins,pH,the activity of?-calpain and MFI from longissimus lumborum?LL?,semimembranosus?SM?and psoas major?PM?muscles were studied during post-mortem.The phosphorylation proteins of PM muscles were lower within 5 days post-mortem compared with LL and SM muscles?P<0.05?.The pH,the activity of?-calpain and MFI were higher after 1 day post-mortem in PM muscles than those of LL and SM muscles?P<0.05?.These results indicated that the sarcoplasmic proteins phosphorylation may regulate the rate of pH decline to influence the?-calpain activity and then proteolysis of proteins consequently.The phosphorylation level of myofibrillar proteins had the negative relationship with the MFI.?2?The degradation of titin and its phosphorylation level in LL,SM and PM from sheep were investigated during post-mortem.PM muscles had a highest phosphorylation level?P<0.05?at 0.5 h postmortem and showed the greatest degree of titin degradation over 28 days.Two days after exsanguination,the pH and MFI of PM were higher than those of the LL and SM muscles?P<0.05?.This suggested that titin played a key role in the tenderization of muscle during post-mortem and phosphorylation of titin might accelerate its degradation.?3?Effect of muscle ultimate pH?pH_u?on titin degradation in ovine muscle was confirmed.After 50 sheep were slaughtered,LL muscles were stored at 4°C for 7days.Based on muscle the pH at post-mortem 2 d,muscles were classified into three groups:the low pH_u?5.40±0.02?,intermediate pH_u?5.52±0.05?and high pH_u?5.73±0.04?muscles.Muscle pH,MFI and degradation of titin were measured during post-mortem.Titin degraded earlier in the high pH_u group,which had a higher MFI than the low pH_u goup and the intermediate pH_u group?P<0.05?.These results showed that the high pH_u promoted the degradation of titin.?4?Effect of phosphorylation on the degradation of titin was investigated.Crude extract of native titin was treated with protein kinase A?PKA?and alkaline phosphatase?AP?after incubated at 30?for 30 min,while the protein dissolution buffer was added to the control group.Then the mixture added with?-calpain was incubated for 2 d at 4?at the Ca²⁺concentration of 0.01 mM,0.1 mM and1 mM.The phosphorylation of titin from PKA group was significantly higher than that in the control group,which had a higher phosphorylation level than AP group?P<0.05?.With the increase of the Ca²⁺concentration,the rate of titin degradation became faster.At the same of concentration of Ca²⁺,titin degraded in AP groups earlier,compared with PKA group and control group.These results indicated that PKA and AP could obviously alter the phosphorylation level of titin,and the dephosphorylation accelerated its degradation.?5?The mechanism of titin degradation regulated by dephosphorylation catalyzed by AP was analyzed.Crude extract of native titin was treated with PKA and AP after incubated at 30?for 30 min.Then the mixture added with?-calpain was incubated for 2 d at 4?at the Ca²⁺concentration of 0.05 mM.There were 20 phosphorylated sites that were Ser 263,Ser 265,Ser 282,Ser 284,Ser 289,Ser 291,Ser 810,Ser2061,Ser 7478,Ser 10275,Ser 13916,Ser 24795,Ser 24796,Ser 33892,Ser 33897,Ser 34490,Thr 267,Thr 300,Thr 17993 and Tyr 23516.Ser 13916,Ser 24796,Thr17993 and Tyr 23516 of titin were phosphorylated by PKA.Ser 810,Ser 7478,Ser24795 and Ser 33897 were dephosphorylated by AP.The 3D structure of dephosphorylaed titin fragment was simulated using the Discovery Studio^TM.The change in structure of titin caused by AP could be recognized by?-calpain easily and promoted the titin degradation.