Effect of calpastatin on degradation of myofibrillar proteins by mu-calpain under postmortem conditions

To improve our understanding of the regulation of mu-calpain activity in si tu during postmortem storage of muscle, the effect of different calpastatin levels on proteolysis of myofibrillar proteins by mu-calpain in a system c losely mimicking postmortem conditions was studied. Increasing the amount o f calpastatin in the incubations limited both the rate and extent of proteo lysis of myofibrillar proteins and autolysis of mu-calpain. Excess calpasta tin (i.e., a mu-calpain:calpastatin ratio of 1:4) did not inhibit proteolys is completely. Western blot analysis revealed that proteolysis of myofibril lar proteins virtually ceased after 7 d of incubation, despite the presence of partly autolyzed, therefore seemingly active, mu-calpain. A series of i ncubations of autolyzed mu-calpain revealed that the autolyzed form of this enzyme is unstable at an ionic strength observed in postmortem muscle. The possible significance of these results in terms of the regulation of mu-ca lpain activity in postmortem muscle is discussed.