Proteolytic degradation of isolated myofibrils and myofibrillar proteins by m-calpain from the skeletal muscle of the amphibianRana ridibunda

The effects of m-calpain isolated from the skeletal muscle of Rana ridibunda on the myofibril and myofibrillar proteins were examined. The results of this study clearly showed that treatment of myofibrils with calpain in the presence of 2 mM Ca2+ results in the complete disappearance of Z-lines within 5 min, whereas prolonged incubation results in the appearance of single sarcomeres or groups of 2-5 sarcomeres. From the other divalent cations examined only Sr2+ at high concentration induces the Z-line removal by calpain. Presence of Mn2+ or Ba2+ reduces the Ca2+ requirement of calpain to the Z-line removal. Protease inhibitors such as endogenous calpastatin, E-64, and leupeptin completely inhibit the Z-line removal by calpain, whereas PMSF and trypsin inhibitor do not inhibit the proteolytic activity of calpain. Studies on the proteolytic degradation of various myofibrillar proteins isolated from the skeletal muscle of Rana ridibunda showed that myosin and G-actin could represent “good” substrates of calpain, whereas F-actin and tropomyosin are not degraded by this protease. Our results also showed that the optimum conditions of calpain action and function on the myofibrillar protein degradation are quite similar to those described for both its maximal caseinolytic activity (Sargianos et al. [1994] J. Exp. Zool., 269:95–105) and autolytic modification (Sargianos et al. [1995] J. Exp. Zool., 271:82–94). © 1996 Wiley-Liss, Inc.