Calpain was purified to apparent homogeneity from the skeletal muscle of the amphibian Rana ridibunda. It is composed of two subunits of 78 and 28 kDa, respectively. The enzyme exhibits kinetic properties similar to those of mammalian and avian skeletal muscle m-calpains. Ca2+ requirements for half and maximum activities are 400 microM and 1.5 mM, respectively. It is strongly inhibited by thiol protease inhibitors such as leupeptin, E-64, and antipain and by alkylating thiol group agents such as iodoacetic acid (IAA), iodoacetamide (IAM), and N-ethylmaleimide (NEM). Its activity is enhanced by reduced thiols such as dithiothreitol (DTT), cysteine, and 2-mercaptoethanol. The enzyme is stable in the absence of Ca2+ at 55 degrees C, it displays maximum activity at 25 degrees C, and it shows a broad pH optimum between 6.5 and 7.8. In the absence of Ca2+, various divalent cations such as Sr2+, Mn2+, and Ba2+ strongly activate, while other divalent cations such as Ni2+, Co2+, Cd2+, Zn2+, and Cu2+ have no effect on its activity. In the presence of Ca2+, the cations Sr2+, Mn2+, and Ba2+ show a synergistic effect, while the cations of the other group strongly inhibit the calpain activity. The above data demonstrate that calpain from the skeletal muscle of the amphibian Rana ridibunda is a neutral, Ca(2+)-activated thiol protease and that it belongs to the class of m-calpains.
The autolytic mechanisms responsible for the regulation of m-calpain purified 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 examined for their ability to induce calpain autolysis. The concentrations of these cations required for the complete calpain autolysis were: 500 microM Ca2+, 800 microM Mn2+, 2 mM Sr2+, 10 mM Ba2+, whereas Mg2+, even at 10 mM did not induce any autolysis. Calpain autolysis induced by the above divalent cations is a temperature dependent process. Presence of 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 inhibit the autolysis of calpain; E-64 inhibits irreversibly while leupeptin 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+ sensitivity of the protease for its half maximal and maximal caseinolytic activity. 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.
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