S-Glutathionylation of the Na,K-ATPase Catalytic α Subunit Is a Determinant of the Enzyme Redox Sensitivity
S-Glutathionylation of the Na,K-ATPase Catalytic α Subunit Is a Determinant of the Enzyme Redox Sensitivity
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DOI：
10.1074/jbc.m112.391094
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发表时间：
2012-09-14
影响因子：
4.8
通讯作者：
Bogdanova, Anna
中科院分区：
文献类型：
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作者：
Petrushanko, Irina Yu.;Yakushev, Sergej;Bogdanova, Anna
Na，K-ATPase对氧化还原状态的变化高度敏感，但其氧化还原敏感性的机制尚不清楚。我们探索了催化α亚基的S谷胱甘肽基化可能参与氧化还原诱导的反应。首次在鸭盐腺、兔肾和大鼠心肌的α亚基中发现了S谷胱甘肽修饰的半胱氨酸残基。Na，K-ATPase暴露于氧化谷胱甘肽(GSSG)导致S谷胱甘肽基化半胱氨酸残基增加。S谷胱甘肽基化的增加与该酶功能的剂量和时间依赖性抑制有关，直至其完全抑制。酶抑制与半胱氨酸-454、-458、-459和-244的S-谷胱甘肽基化一致。当谷胱甘肽与这些半胱氨酸结合时，该酶不能与腺嘌呤核苷酸相互作用。当ATP浓度大于0.5 mM时，GSSG对Na，K-ATPase无抑制作用。在谷氧还蛋白或二硫苏糖醇的催化下，α亚基发生脱谷硫化反应，导致Na，K-ATPase活性恢复。调节半胱氨酸的氧化使它们不能进行谷胱甘肽基化，但对酶活性没有深刻影响。低氧诱导大鼠心肌细胞α亚基的调节性S谷胱甘肽基化，并与氧化应激和三磷酸腺苷耗竭有关。S谷胱甘肽基化后，Na，K-ATPase活性受到抑制。大鼠α2亚型比α1亚型对GSSG更敏感。我们的发现表明，催化亚基的S谷胱甘肽调节在氧化还原诱导的Na，K-ATPase活性调节中起关键作用。
Na,K-ATPase is highly sensitive to changes in the redox state, and yet the mechanisms of its redox sensitivity remain unclear. We have explored the possible involvement of S-glutathionylation of the catalytic alpha subunit in redox-induced responses. For the first time, the presence of S-glutathionylated cysteine residues was shown in the alpha subunit in duck salt glands, rabbit kidneys, and rat myocardium. Exposure of the Na, K-ATPase to oxidized glutathione (GSSG) resulted in an increase in the number of S-glutathionylated cysteine residues. Increase in S-glutathionylation was associated with dose- and time-dependent suppression of the enzyme function up to its complete inhibition. The enzyme inhibition concurred with S-glutathionylation of the Cys-454, -458, -459, and -244. Upon binding of glutathione to these cysteines, the enzyme was unable to interact with adenine nucleotides. Inhibition of the Na, K-ATPase by GSSG did not occur in the presence of ATP at concentrations above 0.5 mM. Deglutathionylation of the alpha subunit catalyzed by glutaredoxin or dithiothreitol resulted in restoration of the Na,K-ATPase activity. Oxidation of regulatory cysteines made them inaccessible for glutathionylation but had no profound effect on the enzyme activity. Regulatory S-glutathionylation of the alpha subunit was induced in rat myocardium in response to hypoxia and was associated with oxidative stress and ATP depletion. S-Glutathionylation was followed by suppression of the Na, K-ATPase activity. The rat alpha 2 isoform was more sensitive to GSSG than the alpha 1 isoform. Our findings imply that regulatory S-glutathionylation of the catalytic subunit plays a key role in the redox-induced regulation of Na,K-ATPase activity.