Gallagher Evan P, Gardner James L, Barber David S
Department of Environmental and Occupational Health Sciences, University of Washington, Seattle, USA.
Biochem Pharmacol. 2006 May 28;71(11):1619-28. doi: 10.1016/j.bcp.2006.02.018. Epub 2006 Apr 17.
The mitochondrial environment is rich in reactive oxygen species (ROS) that may ultimately peroxidize membrane proteins and generate unsaturated aldehydes such as 4-hydroxy-2-nonenal (4HNE). We had previously demonstrated the presence of hGSTA4-4, an efficient catalyst of 4HNE detoxification, in human liver mitochondria to the exclusion of the cytosol. In the present study, GSH-affinity chromatography was used in conjunction with biochemical and proteomic analysis to determine the presence of additional cytosolic glutathione S-transferases (GSTs) in human hepatic mitochondria. HPLC-subunit analysis of GSH affinity-purified liver mitochondrial proteins indicated the presence of several potential mitochondrial GST isoforms. Electrospray ionization-mass spectrometry analysis of eluted mitochondrial GST subunits yielded molecular masses similar to those of hGSTP1, hGSTA1 and hGSTA2. Octagonal matrix-assisted laser desorption/ionization time of flight mass spectrometry and proteomics analysis using MS-FIT confirmed the presence of these three GST subunits in mitochondria, and HPLC analysis indicated that the relative contents of the mitochondrial GST subunits were hGSTA1>hGSTA2>hGSTP1. The mitochondrial localization of the alpha and pi class GST subunits was consistent with immunoblotting analysis of purified mitochondrial GST. Enzymatic studies using GSH-purified mitochondrial GST fractions demonstrated the presence of significant GST activity using the nonspecific GST substrate 1-chloro-2,4-dinitrobenzene (CDNB), as well as 4HNE, delta(5)-androstene-3,17-dione (ADI), and cumene hydroperoxide (CuOOH). Interestingly, the specific mitochondrial GST activities toward 4HNE, a highly toxic alpha,beta-unsaturated aldehyde produced during the breakdown of membrane lipids, exceeded that observed in liver cytosol. These observations are suggestive of a role of GST in protecting against mitochondrial injury during the secondary phase of oxidative stress, or modulation of 4HNE-mediated mitochondrial signaling pathways. However, other properties of mitochondrial GST, such as conjugation of environmental chemicals and binding of lipophilic non-substrate xenobiotics and endogenous compounds, remain to be investigated.
线粒体环境中富含活性氧（ROS），这些活性氧最终可能使膜蛋白发生过氧化反应，并生成不饱和醛，如4-羟基-2-壬烯醛（4HNE）。我们之前已证实在人肝脏线粒体中存在hGSTA4-4，它是4HNE解毒的高效催化剂，而胞质溶胶中则不存在。在本研究中，谷胱甘肽亲和色谱法与生化和蛋白质组学分析相结合，以确定人肝脏线粒体中是否存在其他胞质谷胱甘肽S-转移酶（GST）。对谷胱甘肽亲和纯化的肝脏线粒体蛋白进行高效液相色谱-亚基分析，结果表明存在几种潜在的线粒体GST同工型。对洗脱的线粒体GST亚基进行电喷雾电离-质谱分析，得到的分子量与hGSTP1、hGSTA1和hGSTA2的分子量相似。八极基质辅助激光解吸/电离飞行时间质谱和使用MS-FIT的蛋白质组学分析证实了线粒体中存在这三种GST亚基，高效液相色谱分析表明线粒体GST亚基的相对含量为hGSTA1>hGSTA2>hGSTP1。α类和π类GST亚基的线粒体定位与纯化的线粒体GST的免疫印迹分析结果一致。使用谷胱甘肽纯化的线粒体GST组分进行的酶学研究表明，使用非特异性GST底物1-氯-2,4-二硝基苯（CDNB）以及4HNE、δ⁵-雄烯-3,17-二酮（ADI）和氢过氧化异丙苯（CuOOH）时，存在显著的GST活性。有趣的是，线粒体GST对4HNE（一种在膜脂分解过程中产生的剧毒α,β-不饱和醛）的比活性超过了在肝脏胞质溶胶中观察到的比活性。这些观察结果表明，GST在氧化应激的第二阶段对线粒体损伤具有保护作用，或在调节4HNE介导的线粒体信号通路中发挥作用。然而，线粒体GST的其他特性，如环境化学物质的结合以及亲脂性非底物外源性物质和内源性化合物的结合，仍有待研究。
