Molecular cloning and characterization of a glutathione S-transferase from largemouth bass (Micropterus salmoides) liver that is involved in the detoxification of 4-hydroxynonenal
Molecular cloning and characterization of a glutathione S-transferase from largemouth bass (Micropterus salmoides) liver that is involved in the detoxification of 4-hydroxynonenal
复制标题
DOI：
10.1016/j.bcp.2004.01.024
复制
发表时间：
2004-06-01
影响因子：
5.8
通讯作者：
Gallagher, EP
中科院分区：
文献类型：
--
作者：
Doi, AM;Pham, RT;Gallagher, EP
我们目前正在研究解毒途径在保护大口黑鱼(Micropterus Salmoides)免受化学物质亚致死影响方面的作用。为此，我们实验室以前的工作表明，鲈鱼肝脏谷胱甘肽转移酶(GSTs)具有显著的解毒4-羟基壬烯醛(4HNE)的能力，4HNE是一种常见的诱变剂，在脂质过氧化过程中产生具有细胞毒性的α，β-不饱和醛。在目前的研究中，我们观察到GST介导的4HNE在鲈鱼肝脏中的结合遵循高效的单酶Michaelis-Menten动力学，表明单个GST亚型参与了4HNE的解毒。利用5‘和3’端快速扩增技术(RACE)，克隆了全长957个碱基对的GST全长cDNA678个碱基对，编码225个氨基酸的多肽。有趣的是，对BLAST蛋白数据库的搜索发现，在白斑侧耳鱼(Pleuronectes Plessa)、欧洲比目鱼(Platichhysflesus)和黑头鱼(Pimephales Proelas)中存在同源GST蛋白，但在其他鱼种中没有。此外，bass GST蛋白与哺乳动物快速代谢4HNE的GSTA4亚类蛋白几乎没有同源性。经6xHis标记的重组表达的GST蛋白对4HNE具有较高的催化活性，而对其他类特异性GST底物具有中等或较低的催化活性。HPLC-GST亚基分析和测序结果表明，分离得到的黑鱼肝脏GST亚基是黑鱼肝脏中主要的GST蛋白，其相对分子质量为26.4 kDa。综上所述，GST同工酶在鲈鱼和几个进化上不同的鱼种中的存在表明，在某些水生生物中，一种重要的和独特的解毒蛋白被保存下来，以保护其免受氧化损伤。(C)2004 Elsevier Inc.保留所有权利。
We are currently investigating the role of detoxification pathways in protecting against the sublethal effects of chemicals in largemouth bass (Micropterus salmoides). To this end, previous work in our laboratory indicated a remarkable ability of bass liver glutathione Stransferases (GSTs) to detoxify 4-hydroxynonenal (4HNE), a common mutagenic and cytotoxic alpha,beta- unsaturated aldehyde produced during the peroxidation of lipids. In the current study, we observed that GST-mediated 4HNE conjugation in bass liver follows high efficiency single-enzyme Michaelis-Menten kinetics, suggesting that an individual GST isoform is involved in 4HNE detoxification. Using 5' and 3' rapid amplification of cDNA ends (RACE), a full-length GST cDNA of 957 base pairs (bp) in length, containing an open reading frame of 678 bp and encoding a polypeptide of 225 amino acids, has been cloned. Interestingly, a search of the BLAST protein database revealed the presence of homologous GST proteins in the plaice (Pleuronectes platessa), European flounder (Platichthysflesus) and fathead minnow (Pimephales promelas), but not in other fish species. Furthermore, the bass GST protein exhibited little homology with the mammalian GSTA4 subclass of proteins which rapidly metabolize 4HNE. The recombinant 6xHis-tagged expressed GST protein showed high catalytic activity towards 4HNE, while showing moderate or low activity toward other class specific GST substrates. HPLC-GST subunit analysis, followed by sequencing, demonstrated that the isolated bass liver GST subunit constitutes the major GST protein in bass liver, with a molecular mass of 26.4 kDa. In summary, the presence of a highly expressed GST isozyme in bass and several evolutionarily divergent fish species indicates the conservation of an important and distinct detoxification protein that protects against oxidative damage in certain aquatic organisms. (C) 2004 Elsevier Inc. All rights reserved.