Studies on the autolysis of m‐calpain from the skeletal muscle of the amphibian Rana ridibunda

Abstract

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.

Disruption of excitation–contraction coupling and titin by endogenous Ca2+‐activated proteases in toad muscle fibres

“…It seems very likely that this effect is also the result of the activation of endogenous calpains because of the following: (i) the evidence given above shows that calpains are indeed activated within the fibre over this exact [Ca 2+ ] range; (ii) the progression of Ca 2+ -dependent uncoupling can be stopped by lowering the [Ca 2+ ] and restarted again by raising the [Ca 2+ ] (data not shown), as found with titin proteolysis by calpains; (iii) the uncoupling can be inhibited by leupeptin, provided the activating [Ca 2+ ] is relatively low (e.g. ∼2 µm); (iv) the uncoupling is slowed by more than 10-fold by lowering the temperature from ∼23 to ∼3 • C, and by more than threefold by reducing the pH from 7.1 to 5.8 (Lamb et al 1995), consistent with effects of such changes on calpains (Ono et al 2004); and (v) the uncoupling is triggered by Sr 2+ at ∼10-fold higher concentration than by Ca 2+ (Lamb et al 1995), consistent with the known Sr 2+ activation characteristics of calpains (Inomata et al 1984;Sargianos et al 1995).…”

Proteinases of the calpain family in water invertebrates and fishes

“…Ability of different ions to activate calpains from fishes, crustaceans, and mollusks (data about calpain 110 kDa from whitefish is presented as an example; The established calpain activating ability of Sr 2+ and Ba 2+ extends more or less to enzymes isolated from different sources [33][34][35][36][37][38]. Previously, data about the ability of these elements belonging to common group of the periodic table to replace Ca 2+ during var ious biochemical processes was obtained [39][40][41].…”