Autolysis of mu- and m-calpain from bovine skeletal muscle

The rate of autolysis of mu- and m-calpain from bovine skeletal muscle was measured by using densitometry of SDS polyacrylamide gels and determining t he rate of disappearance of the 28 and 80 kDa subunits of the native, unaut olyzed calpain molecules. Rate of autolysis of both the 28 and 80 kDa subun its of mu -calpain decreased when mu -calpain concentration decreased and w hen beta -casein, a good substrate for the calpains, was present. Hence, au tolysis of both mu -calpain subunits is an intermolecular process at pH 7.5 , 0 or 25.0 degreesC, and low ionic strength. The 78 kDa subunit formed in the first step of autolysis of m-calpain was not resolved from the 80 kDa s ubunit of the native, unautolyzed m-calpain by our densitometer, so autolys is of m-calpain was measured by determining rate of disappearance of the 28 kDa subunit and the 78/80 kDa complex. At Ca2+ concentrations of 1000 muM or higher, neither the m-calpain concentration nor the presence of beta -ca sein affected the rate of autolysis of m-calpain. Hence, m-calpain autolysi s is intramolecular at Ca2+ concentrations of 1000 muM or higher and pH 7.5 . At Ca2+ concentrations of 350 muM or less, the rate of m-calpain autolysi s decreased with decreasing m-calpain concentration and in the presence of beta -casein. Thus, m-calpain autolysis is an intermolecular process at Ca2 + concentrations of 350 muM or less. If calpain autolysis is an intermolecu lar process, autolysis of a membrane-bound calpain would require selective participation of a second, cytosolic calpain, making it an inefficient proc ess. By incubating the calpains at Ca2+ concentrations below those required for half-maximal activity, it is possible to show that unautolyzed calpain s degrade a beta -casein substrate, proving that unautolyzed calpains are a ctive proteases.