Studies on the autolysis of m-calpain from the skeletal muscle of the amphibian Rana ridibunda.
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.
Sargianos
N
N
Department of Zoology, School of Sciences, Aristotle University of Thessaloniki, Greece.
Gaitanaki
C
C
Beis
I
I
D000818:Animals; D001464:Barium / Q000494:pharmacology; D002118:Calcium / Q000494:pharmacology; D002154:Calpain / Q000302:isolation & purification / Q000378:metabolism*; D002413:Cations, Divalent / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D006868:Hydrolysis; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008345:Manganese / Q000494:pharmacology; D008970:Molecular Weight; D018482:Muscle, Skeletal / Q000201:enzymology*; D011480:Protease Inhibitors / Q000494:pharmacology; D011895:Rana ridibunda; D013324:Strontium / Q000494:pharmacology; D013997:Time Factors
D002413:Cations, Divalent; D011480:Protease Inhibitors; D001464:Barium; D008345:Manganese; D002154:Calpain; D008274:Magnesium; D002118:Calcium; D013324:Strontium