Effects of oxidation, pH, and ionic strength on inhibition of mu- and m-calpain by calpastatin

The objective was to determine the effects of pH, ionic strength, and oxidation on inhibition of mu- and m-calpain by calpastatin. Calcium activated cysteine proteinases, mu- and m-calpain, were evaluated for activity under pH (7.5, 6.5, and 6.0), ionic strength (165 and 295 mM NaCl), and oxidizing (with or without H2O2) conditions in the presence of calpastatin, their inhibitor. Using a fluorogenic peptide, mu-calpain is most active at pH 6.5 and m-calpain is most active at pH 7.5 and 165 mM NaCl. Oxidation with 0.16 muM H2O2 decreased mu- and m-calpain activity. It was proposed that H2O2 oxidizes the calpain active site cysteine residue to inhibit calpain activity. Calpastatin inhibited activity of mu- and m-calpain to a greater extent at 295 mM NaCl. Hydrogen peroxide decreased calpastatin inhibition of mu- and m-calpain. In a separate experiment, mu- and m-calpain activity was determined based on degradation of desmin in purified myofibrils. m-Calpain activity was greater at pH 7.5 and 165 mM NaCl. Oxidation (100 muM) and calpastatin inhibited m-calpain. mu-calpain activity was greatest at pH 6.5 and 165 mM NaCl. Calpastatin and oxidation inhibited mu-calpain singularly, but H2O2 added after mu-calpain exposure to calpastatin caused greater degradation of desmin. These results lead to the hypothesis that calpastatin prevents oxidation of a mu-calpain cysteine residue. Evaluation of effects of cysteine modifier N-ethylmaleimide (NEM) on mu-calpain activity with and without calpastatin indicated that NEM irreversibly inhibits mu-calpain activity and autolysis. Inhibition of mu-calpain activity by H2O 2 was reversible and autolysis did occur. Exposure of mu-calpain to NEM or H2O2 before exposure of calpastatin prevented autolysis indicating calpain was not active. However, when the mu-calpain/calpastatin complex was formed, addition of NEM or H2O2 allowed for activation of mu-calpain. Domain I calpastatin and calpain inhibitor peptide inhibition of mu-calpain were not affected by oxidation. Experiments with cysteine modifier maleimidepolyethyleneglycol indicated that calpastatin, when in complex with mu-calpain, prevents cysteine modification. It was concluded that mu-calpain/calpastatin complex treated with either NEM or H2O2 affects their interaction, thus preventing calpastatin inhibition of mu-calpain. However, calpastatin prevents modification of a calpain cysteine residue thus preventing inactivation.