POSTISCHEMIC REPERFUSION INDUCES ALPHA-FODRIN PROTEOLYSIS BY M-CALPAIN IN THE SYNAPTOSOME AND NUCLEUS IN RAT-BRAIN

A membrane cytoskeletal protein, fodrin, is a substrate for a Ca2+-dep endent protease, calpain. It remains unknown whether mu-calpain or m-c alpain is involved in the proteolysis of either alpha- or beta-fodrin and in what subcellular localization during ischemia and reperfusion o f the brain. To address these issues, we examined the distribution of fodrin and calpain and the activities of calpain and calpastatin (endo genous calpain inhibitor) in the same subcellular fractions. Rat foreb rain was subjected to ischemia by a combination of occlusion of both c arotid arteries and systemic hypotension, whereas reperfusion was indu ced by releasing the occlusion. Immunoblotting, activity measurement, and casein zymography did not detect the presence of mu-calpain or a s ignificant change of m-calpain level after ischemia or reperfusion. Ho wever, casein zymography revealed a unique Ca2+-dependent protease tha t was eluted with both 0.18 and 0.40 n/l NaCl from a DEAE-cellulose co lumn, alpha- and beta-fodrins and m-calpain were found to be rich in t he synaptosomal, nuclear, and cytosolic subfractions by immunoblotting analysis. Reperfusion (60 min) following ischemia (30 min) induced se lective proteolysis of alpha-fodrin, which was inhibited by a calpain inhibitor, acetylleucylleucylnorleucinal (400 mu M, 1 ml, i.v.). The m u-calpain-specific fragment of beta-fodrin was not generated during is chemia-reperfusion, supporting the possibility of the involvement of m -calpain rather than mu-calpain in the alpha-fodrin proteolysis.