Inhibition of erythrocyte Ca²⁺-ATPase by activated oxygen through thiol- and lipid-dependent mechanisms

We have studied erythrocyte Ca²⁺-ATPase as a model target for elucidating effects of activated oxygen on the erythrocyte membrane. Either intracellular or extracellular generation of activated oxygen causes parallel decrements in Ca²⁺-ATPase activity and cytoplasmic GSH, oxidation of membrane protein thiols, and lipid peroxidation. Subsequent incubation with either dithiothreitol or glucose allows only partial recovery of Ca²⁺-ATPase, indicating both reversible and irreversible components which are modeled herein using diamide and t-butyl hydroperoxide. The reversible component reflects thiol oxidation, and its recovery depends upon GSH restoration. The irreversible component is largely due to lipid peroxidation, which appears to act through mechanisms involving neither malondialdehyde nor secondary thiol oxidation. However, some portion of the irreversible component could also reflect oxidation of thiols which are inaccessible for reduction by GSH, since we demonstrate existence of different classes of thiols relevant to Ca²⁺-ATPase activity. Activated oxygen has an exaggerated effect on Ca²⁺-ATPase of GSH-depleted cells. Sickle erythrocytes treated with dithiothreitol show a heterogeneous response of Ca²⁺-ATPase activity. These findings are potentially relevant to oxidant-induced hemolysis. They also may be pertinent to oxidative alteration of functional or structural membrane components in general, since many components share with Ca²⁺-ATPase both free thiols and close proximity to unsaturated lipid.