Regulatory Mechanism Of ?-calpain Activity By Protein Phosphorylation

?-Calpain is the key enzyme in postmortem meat tenderization.Studies have shown that?-calpain can be phosphorylated and phosphorylation influences the activity of?-calpain.However,whether?-calpain can be phosphorylated in postmortem muscles and the relative influencing mechanism of phosphorylation on?-calpain activity remains unknown.In this study,the longissimus lumborum?LL?muscles were used.The relationship between phosphorylation level of sarcoplasmic proteins,?-calpain activity and calpastatin degradation in mutton with different tenderness was analyzed.Alkaline phosphatase and phosphatase inhibitor was used to modulate the phosphorylation level of sarcoplasmic proteins,and the effect of phosphorylation of sarcoplasmic proteins on?-calpain activity was studied.The phosphorylation level of?-calpain was regulated in vitro,and the influence of dephosphorylation and phosphorylation of?-calpain on?-calpain activity was investigated.The changes of secondary structure of?-calpain were studied and the phosphorylation sites of?-calpain were identified to illustrate the directly regulatory mechanism of?-calpain activity by phosphorylation.The effects of calpastatin on phosphorylated?-calpain and the effects of phosphorylated calpastatin on?-calpain were also analyzed.The study of the effects of phosphorylation on the interaction between calpastatin and?-calpain clarified the indirectly regulatory mechanism of phosphorylation on?-calpain activity.?1?The relationship between phosphorylation level of sarcoplasmic proteins,?-calpain activity and calpastatin degradation in postmortem mutton with different tenderness was investigated.Muscle with high level of tenderness presented lower phosphorylation level of sarcoplasmic proteins,higher degradation degree and activity of?-calpain and faster degradation rate of calpastatin,which indicated that phosphorylation of sarcoplasmic proteins may influencing postmortem meat tenderness by directly affecting the activity of?-calpain or changing the activity of?-calpain by affecting the inhibitory ability of calpastatin.?2?The effects of phosphorylation of sarcoplasmic proteins on?-calpain activity were studied.Alkaline phosphatase and phosphatase inhibitor was used to broad-spectrum catalyze dephosphorylation or broad-spectrum inhibit dephosphorylation of sarcoplasmic proteins,respectively.The results showed that the degradation rate and activity of?-calpain was higher when the phosphorylation level of sarcoplasmic proteins was lower and vise versa.Higher temperature or Ca²⁺concentration shrink the effects on?-calpain activity induced by phosphorylation.Therefore,the negative regulation of phosphorylation of sarcoplasmic proteins on?-calpain activity was identified.?3?The positive regulation of?-calpain activity by dephosphorylation and protein kinase A phosphorylation was confirmed in the present study.The purified?-calpain was treated with alkaline phosphatase or protein kinase A.Samples were then incubated at controlled freezing point?-1?,4,25and 37°C,respectively.The results showed protein kinase A phosphorylation promotes?-calpain activity.Meanwhile,broad-spectrum dephosphorylation also improves the activity of?-calpain.Low temperature of controlled freezing point prevented dephosphorylation and phosphorylation progression and delayed?-calpain degradation and activation.The effect of phosphorylation on?-calpain activity was weakened by higher temperature due to the accelerated activation and increased degradation.?4?The regulatory mechanism of?-calpain activity by alkaline phosphatase dephosphorylation and protein kinase A phosphorylation was analyzed.?-Calpain was treated with alkaline phosphatase or protein kinase A and then incubated at 0.01,0.05,0.1 and 1 mM Ca²⁺.The positive regulation of?-calpain activity by alkaline phosphatase dephosphorylation and protein kinase A phosphorylation was verified.The content of?-helix structure of?-calpain increased as phosphorylation level rose.Compared to control group,the content of?-sheet structure was lower but the content of random coli structure was higher when?-calpain was treated with alkaline phosphatase and protein kinase A,which indicated that lower content of?-sheet structure and the higher content of random coli structure may contribute to?-calpain activation.Phosphorylation of?-calpain at serine 255,256,476,417 and 420was identified.Protein kinase A catalyzed?-calpain phosphorylation at Ser 255,256 and 476 may positively regulate?-calpain activity by improving its proteolytic activity and promoting auto-activation of?-calpain.?5?The effect of calpastatin on the activity of phosphorylated?-calpain was investigated.25,50,100 and 150 units of alkaline phosphatase or protein kinase A treated?-calpain was mixed with same amounts of heat stable proteins.The results found that although alkaline phosphatase dephosphorylation and protein kinase A phosphorylation promotes the activity of?-calpain,calpastatin presents greater inhibition to protein kinase A phosphorylated?-calpain.Above all,alkaline phosphatase dephosphorylation plays positive roles in regulating?-calpain activity.Although protein kinase A phosphorylation also promotes?-calpain activity,the overall regulation of protein kinase A phosphorylation on?-calpain activity is negative due to the greater inhibition of calpastatin to protein kinase A phosphorylated?-calpain.?6?The regulatory mechanism of calpastatin phosphorylation on the activity of?-calpain was studied.Heat stable proteins,contains calpastatin,were treated with alkaline phosphatase or protein kinase A and then incubated with 25,50,100 and 150 units?-calpain,respectively.Calpastatin with higher phosphorylation level presented much higher inhibitory ability.Two phosphopeptides contain five phosphorylation sites were identified.Protein kinase A catalyzed calpastatin phosphorylate at Ser649 plays positive role in regulating inhibitory ability of calpastatin by promoting the interaction between calpastatin and calpain.