Effects of oxidation, pH, and ionic strength on inhibition of [mu]- and m-calpain by calpastatin /
Abstract (Summary)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 H?O?) 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 [Mu]M H?O? decreased [Mu]- and m-calpain activity. It was proposed that H?O? 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 [Mu]M) 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 H?O? 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 H?O? was reversible and autolysis did occur. Exposure of [Mu]-calpain to NEM or H?O? before exposure of calpastatin prevented autolysis indicating calpain was not active. However, when the [Mu]-calpain/calpastatin complex was formed, addition of NEM or H?O? 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 maleimide-polyethyleneglycol 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 H?O? affects their interaction, thus preventing calpastatin inhibition of [Mu]-calpain. However, calpastatin prevents modification of a calpain cysteine residue thus preventing inactivation.
School:Iowa State University
School Location:USA - Iowa
Source Type:Master's Thesis
Date of Publication: