PURIFICATION AND PROPERTIES OF HIGH-MOLECULAR-WEIGHT CALPASTATIN FROMBOVINE BRAIN

Calcium-activated neutral proteases (calpains) are regulated by specif ic endogenous protein inhibitors, the calpastatins, which are widely d istributed in mammalian tissues. Calpastatins from different species o r in various tissues from the same species exhibit considerable size h eterogeneity on sodium dodecyl sulfate (SDS) gels, reflecting both tra nscriptional and posttranslational regulation. This heterogeneity has complicated previous biochemical characterizations. In this study, we purified bovine brain calpastatin to homogeneity. The inhibitor was pu rified 2,463-fold from a cytosolic fraction of fresh bovine cerebral c ortex by chromatographies on diethylaminoethyl cellulose, Ultrogel AcA 44, phenyl-Sepharose, concanavalin A-Sepharose, and Q-Sepharose. The m ajor calpastatin displayed a native molecular mass of 250-300 kDa by g el filtration and was composed of 125-kDa polypeptide chains by SDS-po lyacrylamide gel electrophoresis (SDS-PAGE). Small amounts of a 68-kDa calpastatin fragment were detected particularly in molecules exhibiti ng smaller native molecular mass (250 kDa). When electroeluted from SD S gels, the 125- and 68-kDa polypeptides each inhibited calpain. The p urified protein was strongly immunoreactive toward antibodies raised a gainst 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 pu rified from bovine and human erythrocytes exhibited molecular masses o f 78 and 68 kDa, respectively, by SDS-PAGE. Both erythrocyte calpastat ins reacted strongly with antibodies against the conserved sequence bu t not with antibodies raised against domains L and 1 of human calpasta tin, indicating that the erythrocyte inhibitors lack these two domains . The heat-stable inhibi tor was effective toward calpain I and calpai n II and showed no inhibitory activity toward other proteases, includi ng trypsin, chymotrypsin, cathepsin D, bromelain, and papain. Brain ca lpastatin therefore shares molecular properties with calpastatins from other tissues but is distinct from the erythrocyte form.