Xu K Y, Zweier J L, Becker L C
Department of Medicine, Johns Hopkins Medical Institutions, Baltimore, Md 21224, USA.
Circ Res. 1997 Jan;80(1):76-81. doi: 10.1161/01.res.80.1.76.
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.
据报道，氧衍生的自由基会损伤肌浆网（SR）的Ca(2+)-ATP酶，这可能是导致缺血再灌注后细胞内Ca2+超载和心肌损伤的原因。为了确定Ca(2+)-ATP酶上的ATP结合位点是否参与氧自由基损伤，从兔的心脏和骨骼肌中分离出含有结合态Ca(2+)-ATP酶的SR囊泡，并将其暴露于由H2O2和Fe(3+)-次氮基三乙酸组成的羟基自由基（·OH）生成系统中，该系统产生的·OH量与再灌注心脏中产生的量相似。·OH暴露完全抑制了心脏和骨骼肌的Ca(2+)-ATP酶活性以及SR对45Ca的摄取。相反，当纯化的囊泡在暴露于·OH之前与1 mmol/L ATP预混合时，观察到完全的保护作用：ATP酶活性和45Ca转运没有损失。腺苷、蔗糖、AMP或ADP（各1 mmol/L）均未产生明显的保护作用。SDS凝胶电泳表明·OH没有破坏该酶的一级结构。电子顺磁共振自旋捕获实验表明ATP不会清除·OH。这些结果表明，·OH通过直接攻击ATP结合位点使SR Ca(2+)-ATP酶变性，而ATP占据活性位点可防止·OH诱导的酶活性丧失和SR Ca2+转运。缺血期间发生的ATP耗竭可能会增强再灌注时·OH的毒性作用。
