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Notes: Abstract: Calcium-activated neutral proteases (calpains) are regulated by specific endogenous protein inhibitors, the calpastatins, which are widely distributed in mammalian tissues. Calpastatins from different species or in various tissues from the same species exhibit considerable size heterogeneity on sodium dodecyl sulfate (SDS) gels, reflecting both transcriptional and posttranslational regulation. This heterogeneity has complicated previous biochemical characterizations. In this study, we purified bovine brain calpastatin to homogeneity. The inhibitor was purified 2,463-fold from a cytosolic fraction of fresh bovine cerebral cortex by chromatographies on diethylaminoethyl cellulose, Ultrogel AcA44, phenyl-Sepharose, concanavalin A-Sepharose, and Q-Sepharose. The major calpastatin displayed a native molecular mass of 250–300 kDa by gel filtration and was composed of 125-kDa polypeptide chains by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Small amounts of a 68-kDa calpastatin fragment were detected particularly in molecules exhibiting smaller native molecular mass (250 kDa). When electroeluted from SDS gels, the 125- and 68-kDa polypeptides each inhibited calpain. The purified protein was strongly immunoreactive toward antibodies raised against a synthetic peptide, CEKLGEKEETIPPDYR, shown to be a conserved repetitive motif in the calpastatin gene or a recombinant polypeptide corresponding to domains L and 1 of human calpastatin. Calpastatins purified from bovine and human erythrocytes exhibited molecular masses of 78 and 68 kDa, respectively, by SDS-PAGE. Both erythrocyte calpastatins reacted strongly with antibodies against the conserved sequence but not with antibodies raised against domains L and 1 of human calpastatin, indicating that the erythrocyte inhibitors lack these two domains. The heat-stable inhibitor was effective toward calpain I and calpain II and showed no inhibitory activity toward other proteases, including trypsin, chymotrypsin, cathepsin D, bromelain, and papain. Brain calpastatin therefore shares molecular properties with calpastatins from other tissues but is distinct from the erythrocyte form.