A comparison of proteins S-thiolated by glutathione to those arylated by acetaminophen.
A comparison of proteins S-thiolated by glutathione to those arylated by acetaminophen.
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谷胱甘肽 S-硫醇化蛋白质与对乙酰氨基酚芳基化蛋白质的比较。
DOI：
10.1016/0006-2952(91)90410-7
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发表时间：
1991
影响因子：
5.8
通讯作者：
Smolin,LA
中科院分区：
文献类型：
--
作者：
Birge,RB;Bartolone,JB;Cohen,SD;Khairallah,EA;Smolin,LA
本研究旨在评估在氧化条件下，与药物反应性亲电体不可逆结合的相同蛋白质是否也与谷胱甘肽（GSH）结合。具体地，将可以被对乙酰氨基酚芳基化的蛋白质与在与二酰胺孵育后形成谷胱甘肽-蛋白质混合二硫化物（PSSG）的蛋白质进行比较。数据表明，谷胱甘肽和对乙酰氨基酚结合到一个子集的N-乙基马来酰亚胺（NEM）反应蛋白硫醇。为了评估结合GSH的蛋白质的模式，在体外通过将胞质蛋白与GSH和二酰胺孵育来形成PSSG。开发了一种灵敏的程序，其中首先用0.1 mM二硫苏糖醇（DTT）还原PSSG，并用[3 H]NEM（用于定量分析）或荧光素-5-马来酰亚胺（用于目视检测）标记新暴露的蛋白质硫醇。对乙酰氨基酚结合是通过将细胞溶质蛋白与对乙酰氨基酚代谢产物N-乙酰基-对-苯醌亚胺（NAPQI）体外孵育实现的。通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳分离来自两种测定的蛋白质，并转移至硝酸纤维素进行Western印迹分析。对乙酰氨基酚结合检测免疫印迹与亲和纯化的抗体对乙酰氨基酚，PSSG可视化使用抗荧光素抗体。在这两种情况下，观察到与蛋白质的结合是选择性的。通过GSH结合修饰的蛋白质与结合对乙酰氨基酚的蛋白质的比较表明，58 kDa的主要胞质对乙酰氨基酚结合蛋白也可以通过氧化条件下的谷胱甘肽化修饰。
This study was designed to evaluate whether the same proteins that irreversibly bind reactive electrophiles of drugs also bind glutathione (GSH) under oxidative conditions. Specifically, proteins that can be arylated by acetaminophen were compared to those that form glutathione-protein mixed disulfides (PSSG) after incubation with diamide. Data are presented which suggest that both GSH and acetaminophen bind to a subset ofN-ethylmaleimide (NEM)-reactive protein thiols. To evaluate the pattern of proteins that bind GSH, PSSGs were formedin vitroby incubating cytosolic proteins with GSH and diamide. A sensitive procedure was developed in which PSSGs were first reduced with 0.1 mM dithiothreitol (DTT), and the newly exposed protein thiols were labeled with either [3H]NEM (for quantitative analysis) or with fluorescein-5-maleimide (for visual detection). Acetaminophen binding was achieved by incubating cytosolic proteinsin vitrowith the reactive acetaminophen metabolite,N-acetyl-P-benzoquinoneimine (NAPQI). Proteins from both assays were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose for Western blot analysis. Acetaminophen binding was detected by immunoblotting with an affinity-purified antibody against acetaminophen, and PSSGs were visualized using anti-fluorescein antibodies. In both instances, binding to proteins was observed to be selective. A comparison of the proteins modified by GSH binding with those that bind acetaminophen indicates that the major cytosolic acetaminophen-binding protein of 58 kDa may also be modified by glutathiolation under oxidative conditions.