Identification of two calpastatin forms in rat skeletal muscle and their susceptibility to digestion by homologous calpains

Abstract Two forms of calpastatin, differing in their specificity for the homologous calpain isozymes I and II, have been separated from rat skeletal muscle extracts and purified to homogeneity. Calpastatin I, the first form to elute in chromatography on DE32, is more effective against calpain I, while calpastatin II is more effective as an inhibitor of calpain II. Based on their molecular mass (~105 kDa) both calpastatin forms belong to the high molecular mass class found in muscles of other animal species ( Murachi, T., 1989, Biochem. Int. 18, 263–294 ). For calpain I, which is active with low (μ m ) concentrations of Ca 2+ , maximum inhibition with either calpastatin form was observed over a wide range of Ca 2+ concentrations. With calpain II, which requires high (m m ) concentrations of Ca 2+ for activity, maximum inhibition required Ca 2+ concentrations above 1 m m . Both calpastatin forms were found to be highly sensitive to degradation by calpain II, but almost completely resistant to degradation by calpain I. Degradation of calpastatin by calpain II is competitively inhibited by the addition of a calpain substrate. Isovaleryl carnitine (IVC), an intermediate product of l -leucine catabolism, previously demonstrated to be a potent and specific activator of rat skeletal muscle calpain II ( Pontremoli, S., Melloni, E., Viotti, P. L., Michetti, M., Di Lisa, F., and Siliprandi, N., 1990, Biochem. Biophys. Res. Commun. 167, 373–380 ) greatly enhances the rate of degradation of calpastatins by calpain II. IVC, which decreases the Ca 2+ requirement for maximal calpain II activity, also decreases the concentration of Ca 2+ required for digestion of the inhibitor. For calpain II, regulation by either calpastatins may occur only in the presence of high [Ca 2+ ].