Lee C I, Liu X, Zweier J L
Molecular and Cellular Biophysics Laboratories, Department of Medicine, Division of Cardiology and the Electron Paramagnetic Resonance Center, The Johns Hopkins Medical Institutions, Baltimore, Maryland 21224, USA.
J Biol Chem. 2000 Mar 31;275(13):9369-76. doi: 10.1074/jbc.275.13.9369.
Xanthine oxidase (XO) is a central mechanism of oxidative injury as occurs following ischemia. During the early period of reperfusion, both nitric oxide (NO()) and superoxide (O-(2)) generation are increased leading to the formation of peroxynitrite (ONOO(-)); however, questions remain regarding the presence and nature of the interactions of NO() or ONOO(-) with XO and the role of this process in regulating oxidant generation. Therefore, we determined the dose-dependent effects of NO() and ONOO(-) on the O-(2) generation and enzyme activity of XO, respectively, by EPR spin trapping of O-(2) using 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline-N-oxide and spectrophotometric assay. ONOO(-) markedly inhibited both O-(2) generation and XO activity in dose-dependent manner, while NO() from NO() gas in concentrations up to 200 microM had no effect. Furthermore, we observed that NO() donors such as NOR-1 also inhibited O-(2) generation and XO activity; however, these effects were O-(2)-dependent and blocked by superoxide dismutase or ONOO(-) scavengers. Finally, we found that ONOO(-) totally abolished the Mo(V) EPR spectrum. These changes were irreversible, suggesting oxidative disruption of the critical molybdenum center of the catalytic site. Thus, ONOO(-) formed in biological systems can feedback and down-regulate XO activity and O-*(2) generation, which in turn may serve to limit further ONOO(-) formation.
黄嘌呤氧化酶（XO）是缺血后发生氧化损伤的核心机制。在再灌注早期，一氧化氮（NO*）和超氧阴离子（O-₂）的生成均增加，导致过氧亚硝酸根（ONOO⁻）的形成；然而，关于NO或ONOO⁻与XO相互作用的存在及性质以及该过程在调节氧化剂生成中的作用仍存在疑问。因此，我们分别通过使用5-（二乙氧基磷酰基）-5-甲基-1-吡咯啉-N-氧化物对O-₂进行电子顺磁共振自旋捕获以及分光光度法测定，确定了NO和ONOO⁻对XO的O-₂生成和酶活性的剂量依赖性影响。ONOO⁻以剂量依赖性方式显著抑制O-₂生成和XO活性，而浓度高达200 μM的NO气体中的NO则无影响。此外，我们观察到诸如NOR-1等NO*供体也抑制O-*₂生成和XO活性；然而，这些作用依赖于O-*₂，并被超氧化物歧化酶或ONOO⁻清除剂阻断。最后，我们发现ONOO⁻完全消除了Mo（V）电子顺磁共振谱。这些变化是不可逆的，表明催化位点关键钼中心的氧化破坏。因此，生物系统中形成的ONOO⁻可反馈并下调XO活性和O-*₂生成，这反过来可能有助于限制进一步的ONOO⁻形成。
