Autolysis of mu- and m-calpain from bovine skeletal muscle.
The rate of autolysis of mu- and m-calpain 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 mu-calpain decreased when mu-calpain concentration decreased and when beta-casein, a good substrate for the calpains, was present. Hence, autolysis of both mu-calpain subunits is an intermolecular process at pH 7.5, 0 or 25.0 degrees C, and low ionic strength. The 78 kDa subunit formed in the first step of autolysis of m-calpain was not resolved from the 80 kDa subunit of the native, unautolyzed m-calpain by our densitometer, so autolysis of m-calpain was measured by determining rate of disappearance of the 28 kDa subunit and the 78/80 kDa complex. At Ca2+ concentrations of 1000 microM or higher, neither the m-calpain concentration nor the presence of beta-casein affected the rate of autolysis of m-calpain. Hence, m-calpain autolysis is intramolecular at Ca2+ concentrations of 1000 microM or higher and pH 7.5. At Ca2+ concentrations of 350 microM or less, the rate of m-calpain autolysis decreased with decreasing m-calpain concentration and in the presence of beta-casein. Thus, m-calpain autolysis is an intermolecular process at Ca2+ concentrations of 350 microM or less. If calpain autolysis is an intermolecular process, autolysis of a membrane-bound calpain would require selective participation of a second, cytosolic calpain, making it an inefficient process. By incubating the calpains at Ca2+ concentrations below those required for half-maximal activity, it is possible to show that unautolyzed calpains degrade a beta-casein substrate, proving that unautolyzed calpains are active proteases.
Cottin
P
P
Muscle Biology Group, University of Arizona, Tucson 85721, USA.
Thompson
V F
VF
Sathe
S K
SK
Szpacenko
A
A
Goll
D E
DE
D000818:Animals; D002118:Calcium / Q000494:pharmacology; D002154:Calpain / Q000378:metabolism*; D002364:Caseins / Q000378:metabolism / Q000494:pharmacology; D002417:Cattle; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D004792:Enzyme Precursors / Q000378:metabolism*; D007700:Kinetics; D018482:Muscle, Skeletal / Q000201:enzymology*; D011498:Protein Precursors / Q000378:metabolism*
D002364:Caseins; D004792:Enzyme Precursors; D011498:Protein Precursors; D002154:Calpain; C449001:m-calpain proenzyme; C449002:mu-calpain; C098484:mu-calpain proenzyme; D002118:Calcium