Hydroxyl radical inhibits sarcoplasmic reticulum Ca(2+)-ATPase function by direct attack on the ATP binding site.
Oxygen-derived free radicals have been reported to damage the sarcoplasmic reticulum (SR) Ca(2+)-ATPase, potentially contributing to cellular Ca2+ 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 H2O2 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 45Ca 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 45Ca 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 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 Ca2+ transport. The depletion of ATP that occurs during ischemia may enhance the toxic effect of .OH at the time of reperfusion.
Xu
K Y
KY
Department of Medicine, Johns Hopkins Medical Institutions, Baltimore, Md 21224, USA.
Zweier
J L
JL
Becker
L C
LC
D000227:Adenine Nucleotides / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000378:metabolism* / Q000494:pharmacology; D000818:Animals; D001665:Binding Sites / Q000187:drug effects; D002118:Calcium / Q000378:metabolism; D000252:Calcium-Transporting ATPases / Q000737:chemistry / Q000378:metabolism*; D016166:Free Radical Scavengers / Q000494:pharmacology; D017665:Hydroxyl Radical / Q000494:pharmacology*; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000378:metabolism*
D000227:Adenine Nucleotides; D016166:Free Radical Scavengers; D017665:Hydroxyl Radical; D000255:Adenosine Triphosphate; D000252:Calcium-Transporting ATPases; D002118:Calcium