Oxidative inactivation of purified plasma membrane Ca2+-ATPase by hydrogen peroxide and protection by calmodulin

Calmodulin (CaM)-regulated plasma membrane Ca2+-ATPase (PMCA) is critical for the regulation of free intracellular Ca2+ levels. PMCA activity and levels in neuronal membranes are decreased with aging, possibly due to oxidation-induced inactivation. In the present studies, inhibition of PMCA by H2O2 was characterized in enzyme purified from human erythrocyte membranes. Basal and CaM-stimulated PMCA activities were inhibited by exposure to H2O2 (25 - 100 muM). However, neither the concentration-dependent enhancement of PMCA activity by CaM nor the binding of CaM to H2O2-exposed PMCA was disrupted by treatment with H2O2. Rates of inactivation by H2O2 of basal and CaM-stimulated PMCA were nearly identical. The addition of CaM after exposure to H2O2 did not protect enzyme activity, although the binding of CaM to PMCA before exposure to H2O2 protected the enzyme completely, indicating a CaM-induced conformational state resistant to oxidation. H2O2 quenched Trp fluorescence in PMCA, an index of conformational changes, with a rate similar to that observed for enzyme inactivation. H2O2 enhanced the solvent accessibility of Trp residues in PMCA, whereas accessibility of the only Trp residue in the CaM-binding domain peptide was unaltered. Exposure of PMCA to H2O2 led to aggregate formation partially reversible by dithiothreitol (DTT) but not to recovery of activity. Amino acid analysis indicated Cys modification following H2O2 exposure but no Cys oxyacids. Because DTT did not reverse inactivation by H2O2 it appears that the disulfide bond formation led to conformational changes that were not fully reversed when the bonds were reduced. Preincubation of PMCA with CaM protected the enzyme from undergoing this conformational change.