Hydroxyl Radical Inhibits Sarcoplasmic Reticulum Ca2+-ATPase Function by Direct Attack on the ATP Binding Site

Abstract

Oxygen-derived free radicals have been reported to damage the sarcoplasmic reticulum (SR) Ca 2+-ATPase, potentially contributing to cellular Ca 2+overload and myocardial damage after ischemia and reperfusion. To determine whether the ATP binding site on Ca 2+-ATPase is involved in oxygen radical injury, SR vesicles containing bound Ca 2+-ATPase were isolated from rabbit cardiac and skeletal muscle and exposed to a hydroxyl radical (·OH)–generating system consisting of H 2 O 2 and Fe 3+-nitrilotriacetic acid in amounts that generate a magnitude of ·OH similar to that which occurs in the reperfused heart. ·OH exposure completely inhibited Ca 2+-ATPase activity and SR 45 Ca uptake for both cardiac and skeletal muscle. In contrast, when the purified vesicles were premixed with l mmol/L ATP before exposure to ·OH, complete protection was observed: there was no loss of ATPase activity or 45 Ca transport. No significant protection occurred with adenosine, sucrose, AMP, or ADP (l mmol/L each). SDS–gel electrophoresis indicated that ·OH did not damage the primary structure of the enzyme. Electron paramagnetic resonance spin-trapping experiments demonstrated that ATP did not scavenge ·OH. These results suggest that ·OH denatures the SR Ca 2+-ATPase by directly attacking the ATP binding site, and occupation of the active site by ATP protects against ·OH-induced loss of enzymatic activity and SR Ca 2+transport. The depletion of ATP that occurs during ischemia may enhance the toxic effect of ·OH at the time of reperfusion.