Hydroxyl radical inhibits sarcoplasmic reticulum Ca2+-ATPase function by direct attack on the ATP binding site

Oxygen-derived free radicals have been reported to damage the sarcoplasmic reticulum (SR) Ca2+-ATPase, potentially contributing to cellular Ca2+ overload and myocardial damage after ischemia and reperfusion. To determine whether the ATP binding site on Ca2+-ATPase is involved in oxygen radical injury, SR vesicles containing bound Ca2+-ATPase were isolated from rabbit cardiac and skeletal muscle and exposed to a hydroxyl radical (. OH)-generating system consisting of H2O2 and Fe3+-nitrilotriacetic acid in amounts that generate a magnitude of . OH similar to that which occurs in the reperfused heart. . OH exposure completely inhibited Ca2+-ATPase activity and SR Ca-45 uptake for both cardiac and skeletal muscle. In contrast, when the purified vesicles were premixed with 1 mmol/L ATP before exposure to . OH, complete protection was observed: there was no loss of ATPase activity or Ca-45 transport. No significant protection occurred with adenosine, sucrose, AMP, or ADP (1 mmol/L each). SDS-gel electrophoresis indicated that OH did not damage the primary structure of the enzyme. Electron para magnetic resonance spin-trapping experiments demonstrated that ATP did not scavenge OH. These results suggest that . OH denatures the SR Ca2+-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 Ca2+ transport. The depletion of ATP that occurs during ischemia may enhance the toxic effect of OH at the time of reperfusion.