Abstract：
Glutathione-S-transferase enzymes (GSTs) are essential components of the phase II detoxification system and protect organisms from oxidative stress induced by xenobiotics and harmful toxins such as 1-chloro-2,4-dinitrobenzene (CDNB). In Tetrahymena thermophila, the TtGSTm34 gene was previously reported to be one of the most responsive GST genes to CDNB treatment (LD50 = 0.079 mM). This study aimed to determine the kinetic features of recombinantly expressed and purified TtGSTm34 with CDNB and glutathione (GSH). TtGSTm34-8xHis was recombinantly produced in T. thermophila as a 25-kDa protein after the cloning of the 660-bp full-length ORF of TtGSTm34 into the pIGF-1 vector. A three-dimensional model of the TtGSTm34 protein constructed by the AlphaFold and PyMOL programs confirmed that it has structurally conserved and folded GST domains. The recombinant production of TtGSTm34-8xHis was confirmed by SDS‒PAGE and Western blot analysis. A dual-affinity chromatography strategy helped to purify TtGSTm34-8xHis approximately 3166-fold. The purified recombinant TtGSTm34-8xHis exhibited significantly high enzyme activity with CDNB (190 µmol/min/mg) as substrate. Enzyme kinetic analysis revealed Km values of 0.68 mM with GSH and 0.40 mM with CDNB as substrates, confirming its expected high affinity for CDNB. The optimum pH and temperature were determined to be 7.0 and 25 °C, respectively. Ethacrynic acid inhibited fully TtGSTm34-8xHis enzyme activity. These results imply that TtGSTm34 of T. thermophila plays a major role in the detoxification of xenobiotics, such as CDNB, as a first line of defense in aquatic protists against oxidative damage.
摘要：
谷胱甘肽-S-转移酶（GSTs）是II期解毒系统的重要组成部分，可保护生物体免受外源性物质和有害毒素（例如1-氯-2,4-二硝基苯（CDNB））诱导的氧化应激。在嗜热四膜虫中，TtGSTm34基因先前被报道为对CDNB治疗最敏感的GST基因之一(LD50=0.079mM).本研究旨在确定用CDNB和谷胱甘肽（GSH）重组表达和纯化的TtGSTm34的动力学特征。在将TtGSTm34的660-bp全长ORF克隆到pIGF-1载体中后，在嗜热T.thermophila中重组产生TtGSTm34-8xHis为25kDa蛋白。通过AlphaFold和PyMOL程序构建的TtGSTm34蛋白的三维模型证实其具有结构上保守和折叠的GST结构域。通过SDS-PAGE和Western印迹分析证实了TtGSTm34-8xHis的重组产生。双重亲和层析策略有助于纯化TtGSTm34-8xHis约3166倍。纯化的重组TtGSTm34-8xHis以CDNB（190µmol/min/mg）为底物表现出显着的高酶活性。酶动力学分析显示，以GSH为底物的Km值为0.68mM，以CDNB为底物的Km值为0.40mM，证实其对CDNB的预期高亲和力。确定的最佳pH和温度为7.0和25°C，分别。乙丙炔酸完全抑制TtGSTm34-8xHis酶活性。这些结果表明，嗜热T.thermophila的TtGSTm34在外源性物质的解毒中起主要作用，例如CDNB，作为水生原生生物抵御氧化损伤的第一道防线。
