CALPAIN INHIBITOR IN RABBIT SKELETAL-MUSCLE - AN IMMUNOCHEMICAL AND HISTOCHEMICAL-STUDY

Calpastatin, the endogenous inhibitor of calcium-activated neutral pro teases (calpains; EC 3.4.22.17), was studied in the rabbit vastus late ralis muscle by means of immunochemical and immunohistochemical techni ques. Immunoaffinity chromatography using an antibody raised against t he 34-kDa monomer of the 68-kDa dimeric inhibitor allowed us to isolat e three main proteins (130-, 100- and 80-kDa). These proteins strongly inhibited calpain activity in muscle homogenate (I50 at about 50-mu-g /ml). Immunohistochemical experiments showed that calpastatin-related immunoreactivity was present in all fibre types (oxidative, glycolytic , oxidative-glycolytic) at both surface and cytoplasmic level. However , a few (20%) of the slow-twitch, oxidative fibres (5% of the total fi bres), did not contain the cytoplasmic inhibitor. Specific immunoreact ivity for calpastatin was also associated with the interstitial connec tive tissue. These results suggest that (i) calpastatin in skeletal mu scle, as in other tissues, is present as a mixture of proteins of vari ous molecular weights and (ii) the inhibitor may act not only in the c ytoplasm but also at the surface or extracellular level.