免疫自旋捕获法分析复杂生物基质中谷胱甘肽介导的 S-（去）硝化反应及两种新型底物的鉴定。
Analysis of glutathione mediated S-(de)nitrosylation in complex biological matrices by immuno-spin trapping and identification of two novel substrates.
机构信息
Amity Institute of Biotechnology, Amity University, Kolkata, 700135, West Bengal, India; Department of Biological Sciences and Bioengineering, Indian Institute of Technology, Roorkee, 247667, Uttarakhand, India.
Department of Surgery, University of Pittsburgh, Pittsburgh, PA, 15213, USA.
出版信息
Nitric Oxide. 2022 Jan 1;118:26-30. doi: 10.1016/j.niox.2021.10.008. Epub 2021 Nov 4.
The intracellular concentration of reduced glutathione (GSH) lies in the range of 1-10 mM, thereby indisputably making it the most abundant intracellular thiol. Such a copious amount of GSH makes it the most potent and robust cellular antioxidant that plays a crucial role in cellular defence against redox stress. The role of GSH as a denitrosylating agent is well established; in this study, we demonstrate GSH mediated denitrosylation of HepG2 cell-derived protein nitrosothiols (PSNOs), by a unique spin-trapping mechanism, using 5,5-dimethyl-1-pyrroline N-oxide (DMPO) as the spin trapping agent, followed by a western blot analysis. We also report our findings of two, hitherto unidentified substrates of GSH mediated S-denitrosylation, namely S-nitrosoglutaredoxin 1 (Grx1-SNO) and S-nitrosylated R1 subunit of ribonucleotide reductase (R1-SNO).
细胞内还原型谷胱甘肽（GSH）的浓度范围为 1-10 mM，因此毫无疑问，它是细胞内含量最丰富的巯基化合物。如此大量的 GSH 使其成为最有效和最强的细胞抗氧化剂，在细胞抵御氧化还原应激中发挥关键作用。GSH 作为一种亚硝基化还原剂的作用已得到充分证实；在这项研究中，我们使用 5,5-二甲基-1-吡咯啉 N-氧化物（DMPO）作为自旋捕获剂，通过独特的自旋捕获机制，证明了 GSH 介导的 HepG2 细胞来源的蛋白亚硝硫醇（PSNOs）的亚硝基还原，随后进行了 Western blot 分析。我们还报告了我们的发现，即两种迄今为止尚未鉴定的 GSH 介导 S-亚硝基化的底物，即 S-亚硝基谷胱甘肽还原酶 1（Grx1-SNO）和 S-亚硝基化核糖核苷酸还原酶 R1 亚基（R1-SNO）。
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