Regulatory Mechanism Of μ-calpain Activity By Protein Phosphorylation

μ-Calpain is the key enzyme in postmortem meat tenderization.Studies have shown thatμ-calpain can be phosphorylated and phosphorylation influences the activity ofμ-calpain.However,whetherμ-calpain can be phosphorylated in postmortem muscles and the relative influencing mechanism of phosphorylation onμ-calpain activity remains unknown.In this study,the longissimus lumborum（LL）muscles were used.The relationship between phosphorylation level of sarcoplasmic proteins,μ-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 onμ-calpain activity was studied.The phosphorylation level ofμ-calpain was regulated in vitro,and the influence of dephosphorylation and phosphorylation ofμ-calpain onμ-calpain activity was investigated.The changes of secondary structure ofμ-calpain were studied and the phosphorylation sites ofμ-calpain were identified to illustrate the directly regulatory mechanism ofμ-calpain activity by phosphorylation.The effects of calpastatin on phosphorylatedμ-calpain and the effects of phosphorylated calpastatin onμ-calpain were also analyzed.The study of the effects of phosphorylation on the interaction between calpastatin andμ-calpain clarified the indirectly regulatory mechanism of phosphorylation onμ-calpain activity.（1）The relationship between phosphorylation level of sarcoplasmic proteins,μ-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 ofμ-calpain and faster degradation rate of calpastatin,which indicated that phosphorylation of sarcoplasmic proteins may influencing postmortem meat tenderness by directly affecting the activity ofμ-calpain or changing the activity ofμ-calpain by affecting the inhibitory ability of calpastatin.（2）The effects of phosphorylation of sarcoplasmic proteins onμ-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 ofμ-calpain was higher when the phosphorylation level of sarcoplasmic proteins was lower and vise versa.Higher temperature or Ca²⁺concentration shrink the effects onμ-calpain activity induced by phosphorylation.Therefore,the negative regulation of phosphorylation of sarcoplasmic proteins onμ-calpain activity was identified.（3）The positive regulation ofμ-calpain activity by dephosphorylation and protein kinase A phosphorylation was confirmed in the present study.The purifiedμ-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 promotesμ-calpain activity.Meanwhile,broad-spectrum dephosphorylation also improves the activity ofμ-calpain.Low temperature of controlled freezing point prevented dephosphorylation and phosphorylation progression and delayedμ-calpain degradation and activation.The effect of phosphorylation onμ-calpain activity was weakened by higher temperature due to the accelerated activation and increased degradation.（4）The regulatory mechanism ofμ-calpain activity by alkaline phosphatase dephosphorylation and protein kinase A phosphorylation was analyzed.μ-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 ofμ-calpain activity by alkaline phosphatase dephosphorylation and protein kinase A phosphorylation was verified.The content ofα-helix structure ofμ-calpain increased as phosphorylation level rose.Compared to control group,the content ofβ-sheet structure was lower but the content of random coli structure was higher whenμ-calpain was treated with alkaline phosphatase and protein kinase A,which indicated that lower content ofβ-sheet structure and the higher content of random coli structure may contribute toμ-calpain activation.Phosphorylation ofμ-calpain at serine 255,256,476,417 and 420was identified.Protein kinase A catalyzedμ-calpain phosphorylation at Ser 255,256 and 476 may positively regulateμ-calpain activity by improving its proteolytic activity and promoting auto-activation ofμ-calpain.（5）The effect of calpastatin on the activity of phosphorylatedμ-calpain was investigated.25,50,100 and 150 units of alkaline phosphatase or protein kinase A treatedμ-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 ofμ-calpain,calpastatin presents greater inhibition to protein kinase A phosphorylatedμ-calpain.Above all,alkaline phosphatase dephosphorylation plays positive roles in regulatingμ-calpain activity.Although protein kinase A phosphorylation also promotesμ-calpain activity,the overall regulation of protein kinase A phosphorylation onμ-calpain activity is negative due to the greater inhibition of calpastatin to protein kinase A phosphorylatedμ-calpain.（6）The regulatory mechanism of calpastatin phosphorylation on the activity ofμ-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 unitsμ-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.