Sies H, Sharov V S, Klotz L O, Briviba K
Institut für Physiologische Chemie I, Heinrich-Heine-Universität Düsseldorf, Postfach 101007, D-40001 Düsseldorf, Germany.
J Biol Chem. 1997 Oct 31;272(44):27812-7. doi: 10.1074/jbc.272.44.27812.
There is a requirement for cellular defense against excessive peroxynitrite generation to protect against DNA strand breaks and mutations and against interference with protein tyrosine-based signaling and other protein functions due to formation of 3-nitrotyrosine. Here, we demonstrate a role of selenium-containing enzymes catalyzing peroxynitrite reduction using glutathione peroxidase (GPx) as an example. GPx protected against the oxidation of dihydrorhodamine 123 by peroxynitrite more effectively than ebselen (2-phenyl-1,2-benzisoselenazol-3(2H)-one), a selenoorganic compound exhibiting a high second-order rate constant for the reaction with peroxynitrite, 2 x 10(6) M-1 s-1. Carboxymethylation of selenocysteine in GPx by iodoacetate led to the loss of "classical" glutathione peroxidase activity but maintained protection against peroxynitrite-mediated oxidation. The maintenance of protection by GPx against peroxynitrite requires GSH as reductant. When peroxynitrite was infused to maintain a 0.2 microM steady-state concentration, GPx in the presence of GSH, but neither GPx nor GSH alone, effectively inhibited the hydroxylation of benzoate by peroxynitrite. Under these steady-state conditions peroxynitrite did not cause the loss of classical GPx activity. GPx, like selenomethionine, protected against protein 3-nitrotyrosine formation in human fibroblast lysates, shown in Western blots. The formation of nitrite rather than nitrate from peroxynitrite was enhanced by GPx or by selenomethionine. The results demonstrate a novel function of GPx and potentially of other selenoproteins containing selenocysteine or selenomethionine, in the GSH-dependent maintenance of a defense line against peroxynitrite-mediated oxidations, as a peroxynitrite reductase.
细胞需要防御过氧亚硝酸盐的过度生成，以防止DNA链断裂和突变，并防止由于3-硝基酪氨酸的形成而干扰基于蛋白质酪氨酸的信号传导和其他蛋白质功能。在此，我们以谷胱甘肽过氧化物酶（GPx）为例，证明了含硒酶催化过氧亚硝酸盐还原的作用。与依布硒啉（2-苯基-1,2-苯并异硒唑-3(2H)-酮）相比，GPx能更有效地保护二氢罗丹明123不被过氧亚硝酸盐氧化，依布硒啉是一种硒有机化合物，与过氧亚硝酸盐反应的二级速率常数很高，为2×10⁶ M⁻¹ s⁻¹。碘乙酸对GPx中硒代半胱氨酸的羧甲基化导致“经典”谷胱甘肽过氧化物酶活性丧失，但仍保持对过氧亚硝酸盐介导氧化的保护作用。GPx对过氧亚硝酸盐的保护作用的维持需要谷胱甘肽作为还原剂。当过氧亚硝酸盐被注入以维持0.2微摩尔的稳态浓度时，在谷胱甘肽存在下的GPx，但单独的GPx或谷胱甘肽都不能有效抑制过氧亚硝酸盐对苯甲酸盐的羟基化作用。在这些稳态条件下，过氧亚硝酸盐不会导致经典GPx活性的丧失。如蛋白质印迹所示，GPx与硒代蛋氨酸一样，能保护人成纤维细胞裂解物中蛋白质3-硝基酪氨酸的形成。GPx或硒代蛋氨酸可增强过氧亚硝酸盐形成亚硝酸盐而非硝酸盐的过程。结果表明，GPx以及其他含有硒代半胱氨酸或硒代蛋氨酸的硒蛋白具有一种新功能，即在谷胱甘肽依赖性维持针对过氧亚硝酸盐介导氧化的防御防线中作为过氧亚硝酸盐还原酶。
