Hydroxyl radical inhibits sarcoplasmic reticulum Ca (2+) -ATPase function by direct attack on the ATP binding site
Xu, K.Y.; Zweier, J.L.; Becker, L.C.
Circulation Research 80(1): 76-81
1997
ISSN/ISBN: 0009-7330 PMID: 8978325 Document Number: 482963
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 ( cntdot OH)-generating system consisting of H-2O-2 and Fe-3+-nitrilotriacetic acid in amounts that generate a magnitude of cntdot OH similar to that which occurs in the reperfused heart. cntdot OH exposure completely inhibited Ca-2+-ATPase activity and SR 45Ca uptake for both cardiac and skeletal muscle. In contrast, when the purified vesicles were premixed with 1 mmol/L ATP before exposure to cntdot OH, complete protection was observed: there was no loss of ATPase activity or 45Ca transport. No significant protection occurred with adenosine, sucrose, AMP, or ADP (1 mmol/L each). SDS-gel electrophoresis indicated that cntdot OH did not damage the primary structure of the enzyme. Electron paramagnetic resonance spin-trapping experiments demonstrated that ATP did not scavenge cntdot OH. These results suggest that cntdot OH denatures the SR Ca-2+-ATPase by directly attacking the ATP binding site, and occupation of the active site by ATP protects against cntdot 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 cntdot OH at the time of reperfusion.