Modulation of the redox state of tubulin by the glutathione/glutaredoxin reductase system
Modulation of the redox state of tubulin by the glutathione/glutaredoxin reductase system
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DOI：
10.1016/j.bbrc.2003.12.126
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发表时间：
2004-02-06
影响因子：
3.1
通讯作者：
Kennett, KL
中科院分区：
文献类型：
--
作者：
Landino, LM;Moynihan, KL;Kennett, KL
蛋白质氧化还原状态的改变与几种神经退行性疾病的病理学有关。我们报告过亚硝酸盐诱导的猪脑微管蛋白中的二硫化物修复由谷胱甘肽还原酶，人或大肠杆菌谷氧还蛋白，还原型谷胱甘肽和NADPH组成的谷氧还蛋白还原酶系统。通过蛋白质印迹法评估谷胱甘肽还原酶系统对α-和β-微管蛋白亚基之间二硫键的还原。微管蛋白半胱氨酸的氧化和还原通过监测5-碘乙酰胺-荧光素（一种巯基特异性标记试剂）的掺入来定量。微管蛋白二硫键还原谷氧还蛋白还原酶系统恢复微管蛋白聚合活性，失去了以下过氧亚硝酸盐添加。为了支持谷胱甘肽对微管蛋白的氧化还原调节，通过HPLC检测并定量微管蛋白和氧化型谷胱甘肽之间的巯基-二硫键交换。此外，微管蛋白的谷胱甘肽化通过使用抗GSH抗体的斑点印迹检测。(C)2003年爱思唯尔公司All rights reserved.
Alterations in the redox status of proteins have been implicated in the pathology of several neurodegenerative diseases. We report that peroxynitrite-induced disulfides in porcine brain tubulin are repaired by the glutaredoxin reductase system composed of glutathione reductase, human or Escherichia coli glutaredoxin, reduced glutathione, and NADPH. Reduction of disulfide bonds between the alpha- and beta-tubulin subunits by the glutathione reductase system was assessed by Western blot. Tubulin cysteine oxidation and reduction was quantitated by monitoring the incorporation of 5-iodoacetamido-fluorescein, a thiol-specific labeling reagent. Tubulin disulfide bond reduction by the glutaredoxin reductase system restored tubulin polymerization activity that was lost following peroxynitrite addition. In support of redox modulations of tubulin by glutathione, thiol-disulfide exchange between tubulin and oxidized glutathione was detected and quantitated by HPLC. In addition, glutathionylation of tubulin was detected by dot blot using an anti-GSH antibody. (C) 2003 Elsevier Inc. All rights reserved.