Characterization of the magnitude and kinetics of xanthine oxidase-catalyzed nitrate reduction: Evaluation of its role in nitrite and nitric oxide generation in anoxic tissues
Characterization of the magnitude and kinetics of xanthine oxidase-catalyzed nitrate reduction: Evaluation of its role in nitrite and nitric oxide generation in anoxic tissues
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DOI：
10.1021/bi026385a
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发表时间：
2003-02-04
期刊：
影响因子：
2.9
通讯作者：
Zweier, JL
中科院分区：
文献类型：
--
作者：
Li, HT;Samouilov, A;Zweier, JL
除了从特定的NO氧化酶产生一氧化氮（NO）外，在缺氧期间也从亚硝酸盐还原形成NO，并且黄嘌呤氧化酶（XO）催化该过程。虽然在组织和血液中存在高硝酸盐水平，但关于硝酸盐是否也是NO的来源以及XO介导的硝酸盐还原是否可以成为生物系统中NO的重要来源的问题仍然存在。为了表征在厌氧条件下XO介导的硝酸盐还原的动力学、幅度和机制，进行了EPR、化学发光NO分析仪和NO电极研究。典型的XO还原底物，黄嘌呤，NADH和2，3-二羟基苯甲醛，触发硝酸盐还原为亚硝酸盐和NO。亚硝酸盐的产生速率遵循Michaelis-Menten动力学，而NO的产生速率随着亚硝酸盐的积累而线性增加，表明硝酸盐逐步还原为亚硝酸盐，然后还原为NO。表明硝酸盐还原发生在钼位点。在较高的黄嘌呤浓度，部分抑制被视为，表明形成一个底物结合的还原酶复合物与黄嘌呤阻断钼网站。亚硝酸盐和NO形成的pH依赖性表明XO介导的硝酸盐还原通过酸催化机制发生。在缺血期间发生的条件下，测定心肌黄嘌呤氧化还原酶和硝酸盐水平，以在10-20分钟内产生高达20 μ M的亚硝酸盐，其可以以与最大活化NOS的速率相当的速率进一步还原为NO。因此，XOR催化的硝酸盐还原为亚硝酸盐和NO发生，并且可以是缺血组织中NO产生的重要来源。
In addition to nitric oxide (NO) generation from specific NO synthases, NO is also formed during anoxia from nitrite reduction, and xanthine oxidase (XO) catalyzes this process. While in tissues and blood high nitrate levels are present, questions remain regarding whether nitrate is also a source of NO and if XO-mediated nitrate reduction can be an important source of NO in biological systems. To characterize the kinetics, magnitude, and mechanism of XO-mediated nitrate reduction under anaerobic conditions, EPR, chemiluminescence NO-analyzer, and NO-electrode studies were performed. Typical XO reducing substrates, xanthine, NADH, and 2,3-dihydroxybenz-aldehyde, triggered nitrate reduction to nitrite and NO. The rate of nitrite production followed Michaelis-Menten kinetics, while NO generation rates increased linearly following the accumulation of nitrite, suggesting stepwise-reduction of nitrate to nitrite then to NO. The molybdenum-binding XO inhibitor, oxypurinol, inhibited both nitrite and NO production, indicating that nitrate reduction occurs at the molybdenum site. At higher xanthine concentrations, partial inhibition was seen, suggesting formation of a substrate-bound reduced enzyme complex with xanthine blocking the molybdenum site. The pH dependence of nitrite and NO formation indicate that XO-mediated nitrate reduction occurs via an acid-catalyzed mechanism. With conditions occurring during ischemia, myocardial xanthine oxidoreductase and nitrate levels were determined to generate up to 20 muM nitrite within 10-20 min that can be further reduced to NO with rates comparable to those of maximally activated NOS. Thus, XOR catalyzed nitrate reduction to nitrite and NO occurs and can be an important source of NO production in ischemic tissues.