Kulkarni A P, Sajan M P
Florida Toxicology Research Center, Department of Environmental and Occupational Health, College of Public Health, University of South Florida, Tampa 33612-3805, USA.
Toxicol Appl Pharmacol. 1997 Mar;143(1):179-88. doi: 10.1006/taap.1996.8062.
Ethacrynic acid (EA), a diuretic drug, is known to interact with glutathione transferases in the presence of reduced glutathione (GSH) to yield an EA-SG conjugate. Here we present evidence for a new mechanism for the formation of EA-SG conjugate by a soybean lipoxygenase (SLO)-mediated reaction involving oxidation of GSH to a GS.. Similar to the glutathione transferase-mediated reaction, EA-SG conjugate generated by SLO exhibited an absorbance maximum at 270 nm. The conjugate formation was dependent on the concentration of linoleic acid, EA, GSH, and SLO. The optimal assay conditions to observe a maximal rate of EA-SG formation required the presence of 0.4 mM linoleic acid, 1 mM GSH, 50 nM SLO, and 0.2 mM EA at pH 9.0. Classical inhibitors of lipoxygenase, e.g., nordihydroguaiaretic acid, gossypol, and 5,8,11-eicosatriynoic acid, significantly inhibited EA-SG conjugation. The SLO-generated EA-SG was isolated as a single peak by HPLC. Quantitation of EA-SG by HPLC-coupled radiometry using [3H]GSH yielded a rate of 16.5 mumol/min/mg SLO protein. This rate is up to 1650-fold greater than that reported for different purified isozymes of mammalian glutathione transferase. The structure of EA-SG isolated from HPLC column was confirmed by matrix-assisted laser desorption mass spectroscopy. These results suggest that lipoxygenase, which is primarily known for xenobiotic oxidation, may represent yet another important pathway for GSH conjugate formation that could lead to detoxification of certain chemicals.
依他尼酸（EA）是一种利尿药物，已知在存在还原型谷胱甘肽（GSH）的情况下它会与谷胱甘肽转移酶相互作用，生成EA-SG共轭物。在此，我们提供证据表明大豆脂氧合酶（SLO）介导的反应形成EA-SG共轭物存在一种新机制，该反应涉及将GSH氧化为GS·。与谷胱甘肽转移酶介导的反应类似，SLO生成的EA-SG共轭物在270nm处有最大吸光度。共轭物的形成取决于亚油酸、EA、GSH和SLO的浓度。观察到EA-SG形成最大速率的最佳测定条件是在pH 9.0时存在0.4mM亚油酸、1mM GSH、50nM SLO和0.2mM EA。脂氧合酶的经典抑制剂，如去甲二氢愈创木酸、棉酚和5,8,11-二十碳三烯酸，可显著抑制EA-SG共轭作用。通过高效液相色谱法（HPLC）将SLO生成的EA-SG分离为单一峰。使用[3H]GSH通过HPLC耦合放射测量法定量EA-SG，得到的速率为16.5μmol/min/mg SLO蛋白。该速率比报道的哺乳动物谷胱甘肽转移酶不同纯化同工酶的速率高1650倍。通过基质辅助激光解吸质谱法确认了从HPLC柱分离的EA-SG的结构。这些结果表明，脂氧合酶主要以对外源化合物氧化作用而闻名，它可能代表了GSH共轭物形成的另一条重要途径，这可能导致某些化学物质的解毒。
