Abstract

The rate of autolysis of and mcalpain from bovine skeletal muscle was measured by using densitometry of SDS polyacrylamide gels and determining the rate of disappearance of the 28 and 80 kDa subunits of the native, unautolyzed calpain molecules. Rate of autolysis of both the 28 and 80 kDa subunits of calpain decreased when calpain concentration decreased and when ?casein, a good substrate for the calpains, was present. Hence, autolysis of both calpain subunits is an intermolecular process at pH 7.5, 0 or 25.0 C, and low ionic strength. The 78 kDa subunit formed in the first step of autolysis of mcalpain was not resolved from the 80 kDa subunit of the native, unautolyzed mcalpain by our densitometer, so autolysis of mcalpain was measured by determining rate of disappearance of the 28 kDa subunit and the 78/80 kDa complex. At Ca2+ concentrations of 1000 mM or higher, neither the mcalpain concentration nor the presence of bcasein affected the rate of autolysis of mcalpain. Hence, mcalpain autolysis is intramolecular at Ca[2+] concentrations of 1000 M or higher and pH 7.5. At Ca[2+] concentrations of 350 M or less, the rate of mcalpain autolysis decreased with decreasing mcalpain concentration and in the presence of bcasein. Thus, mcalpain autolysis is an intermolecular process at Ca[2+] concentrations of 350 M or less. If calpain autolysis is an intermolecular process, autolysis of a membranebound calpain would require selective participation of a second, cytosolic calpain, making it an inefficient process. By incubating the calpains at Ca[2+] concentrations below those required for halfmaximal activity, it is possible to show that unautolyzed calpains degrade a ?casein substrate, proving that unautolyzed calpains are active proteases.