STUDIES ON THE AUTOLYSIS OF M-CALPAIN FROM THE SKELETAL-MUSCLE OF THEAMPHIBIAN RANA-RIDIBUNDA

The autolytic mechanisms responsible for the regulation of m-calpain p urified from the skeletal muscle of the amphibian Rana ridibunda were examined. Both subunits of the calpain molecule were found to undergo autolysis in the presence of Ca2+. Various divalent cations were exami ned for their ability to induce calpain autolysis. The concentrations of these cations required for the complete calpain autolysis were: 500 mu M Ca2+, 800 mu M Mn2+, 2 mM Sr2+, 10 mM Ba2+, whereas Mg2+, even a t 10 mM did not induce any autolysis. Calpain autolysis induced by the above divalent cations is a temperature dependent process. Presence o f Mn2+ or Sr2+ reduces the Ca2+ requirement of calpain for autolysis. The rate of autolysis depends on the protease concentration; protease inhibitors such as E-64, leupeptin, antipain, and iodoacetic acid inhi bit the autolysis of calpain; E-64 inhibits irreversibly while leupept in inhibits reversibly the autolysis; and irreversibly inactivated by E-64 calpain is fully digested by native calpain. Autolysis of calpain in the presence of alkali denatured casein increases the Ca2+ sensiti vity of the protease for its half maximal and maximal caseinolytic act ivity. Limited autolysis of calpain is also induced in the presence of the endogenous substrate G-actin, and the rate of autolysis is slower than that obtained in the absence of substrates. (C) 1995 Wiley-Liss, Inc.