Effects of phosphorylation on μ-calpain activity at different incubation temperature

The study investigated the effects of alkaline phosphatase (AP) dephosphorylation and protein kinase A (PKA) phosphorylation on μ-calpain activity and its sensitivity to temperature. The purified μ-calpain was treated with AP or PKA for 30min at 30°C to modulate its phosphorylation level. Samples were then incubated at controlled freezing point (-1), 4, 25 and 37°C, respectively. The results showed that PKA and AP had no influence on pH values of incubation solution. At -1 and 4°C, the degradation rate of μ-calpain was maximum in AP group and minimum in control group. Low temperature of controlled freezing point prevented dephosphorylation and phosphorylation progression and delayed μ-calpain degradation. Increased incubation temperature of 4, 25 and 37°C increased μ-calpain degradation. Two about 50kDa degradation products from μ-calpain were identified, of which the intensity was also lower in control group than in the other two groups. These observations demonstrated that AP dephosphorylation and PKA phosphorylation of μ-calpain promoted μ-calpain autolysis and activation.