PROTEOLYTIC DEGRADATION OF ISOLATED MYOFIBRILS AND MYOFIBRILLAR PROTEINS BY M-CALPAIN FROM THE SKELETAL-MUSCLE OF THE AMPHIBIAN RANA-RIDIBUNDA

The effects of m-calpain isolated from the skeletal muscle of Rana rid ibunda on the myofibril and myofibrillar proteins were examined. The r esults of this study clearly showed that treatment of myofibrils with calpain in the presence of 2 mM Ca2+ results in the complete disappear ance 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 in duces the Z-line removal by calpain. Presence of Mn2+ or Ba2+ reduces the Ca2+ requirement of calpain to the Z-line removal. Protease inhibi tors such as endogenous calpastatin, E-64, and leupeptin completely in hibit the Z-line removal by calpain, whereas PMSF and trypsin inhibito r do not inhibit the proteolytic activity of calpain. Studies on the p roteolytic degradation of various myofibrillar proteins isolated from the skeletal muscle of Rana ridibunda showed that myosin and G-actin c ould represent ''good'' substrates of calpain, whereas F-actin and tro pomyosin are not degraded by this protease. Our results also showed th at the optimum conditions of calpain action and function on the myofib rillar protein degradation are quite similar to those described for bo th 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). (C) 1996 Wiley-Liss, Inc.