Irreversible thiol oxidation in carbonic anhydrase III: Protection by S-glutathiolation and detection in aging rats
Irreversible thiol oxidation in carbonic anhydrase III: Protection by S-glutathiolation and detection in aging rats
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DOI：
10.1515/bc.2002.067
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发表时间：
2002-03-01
影响因子：
3.7
通讯作者：
Thornas, JA
中科院分区：
文献类型：
--
作者：
Mallis, RJ;Hamann, MJ;Thornas, JA
具有反应性巯基的蛋白质是许多重要代谢反应的中心，也是各种信号转导系统的贡献者。在这篇报告中，我们研究了碳酸酐酶III的两个活性巯基的氧化损伤机制。在没有谷胱甘肽(GSH)的情况下，过氧化氢(H_2O_2)、过氧自由基或次氯酸(HOCl)会产生不可逆的氧化形式，主要是半胱氨酸亚磺酸或半胱酸。当谷胱甘肽与蛋白质硫醇的摩尔接近时，不可逆的氧化被阻止。H_2O_2和过氧自由基均可通过部分氧化的蛋白质巯基中间体产生S-谷胱甘肽结合的碳酸氢酶III，而高氯酸不能引起S-谷胱甘肽结合。因此，过氧化氢或碱性磷酸酶的氧化损伤可以通过蛋白质S-谷胱甘肽的氧化损伤来防止，而谷胱甘肽和氧化剂之间的直接反应可能阻止高氯酸盐介导的蛋白质损伤。在培养的大鼠肝细胞中，碳酸酐酶III被甲萘二酮迅速地S-谷胱甘肽氧化。当肝细胞谷胱甘肽耗尽时，甲萘二酮会引起不可逆转的氧化。我们推测，老年动物体内谷胱甘肽的正常消耗也可能导致不可逆氧化的增加。事实上，与年轻动物相比，老年大鼠的总蛋白提取物和碳酸氢酶III都含有明显更多的半胱氨酸亚磺酸。这些实验表明，在缺乏足够的GSH的情况下，氧化反应会导致纯化的蛋白质、细胞系统和整个动物中不可逆的蛋白质巯基损伤。
Proteins with reactive sulfhydryls are central to many important metabolic reactions and also contribute to a variety of signal transduction systems. In this report, we examine the mechanisms of oxidative damage to the two reactive sulfhydryls of carbonic anhydrase III. Hydrogen peroxide (H2O2), peroxy radicals, or hypochlorous acid (HOCl) produced irreversibly oxidized forms, primarily cysteine sulfinic acid or cysteic acid, of carbonic anhydrase III if glutathione (GSH) was not present. When GSH was approximately equimolar to protein thiols, irreversible oxidation was prevented. H2O2 and peroxyl radicals both generated S-glutathiolated carbonic anhydrase III via partially oxidized protein sulfhydryl intermediates, while HOCl did not cause S-glutathiolation. Thus, oxidative damage from H2O2 or AAPH was prevented by protein S-glutathiolation, while a direct reaction between GSH and oxidant likely prevents HOCl-mediated protein damage. In cultured rat hepatocytes, carbonic anhydrase III was rapidly S-glutathiolated by menadione. When hepatocyte glutathione was depleted, menadione instead caused irreversible oxidation. We hypothesized that normal depletion of glutathione in aged animals might also lead to an increase in irreversible oxidation. Indeed, both total protein extracts and carbonic anhydrase III contained significantly more cysteine sulfinic acid in older rats compared to young animals. These experiments show that, in the absence of sufficient GSH, oxidation reactions lead to irreversible protein sulfhydryl damage in purified proteins, cellular systems, and whole animals.