DETOXICATION OF XENOBIOTICS IN ERYTHROCYTES

谷胱甘肽（GSH）及其相关酶系统，如GSH S- 转移酶（GST）在保护 人体组织中有毒的外源性物质和内源性氧化 应力 GST催化多种有毒物质的结合， 亲电异生物质对GSH的影响。 GSH-异生物质缀合物 在大多数人体内代谢为巯基尿酸 组织，但在红细胞中，它们不能进一步代谢 因为巯基尿酸途径的所有酶都不是 存在于这个组织中。 我们已经证明，共轭 1-氯-2，4-二硝基苯与GSH（S-二硝基苯 谷胱甘肽，缩写为Dnp-SG）被主动转运 红细胞的一种新的ATP酶不相关的任何已知 离子泵ATP酶，现在已经开发出一种方法来分离 该Dnp-SG依赖性ATP酶使用亲和层析 通过与CNBr活化的Sepharose 4 B结合的Dnp-SG柱 使用鲁布罗尔作为洗涤剂。 这种转运蛋白将 从红细胞膜中分离，其特征在于 结构和动力学性质。 的动力学参数 Dnp-SG ATP酶也将使用其他的缀合物测定。 为了检查这个系统是否是一个 GSH-异生素转运的一般机制 共轭 纯化的转运蛋白将被重组 在蛋白脂质体中。 脂质体中的转运动力学， 以及在由红细胞制备的由内而外的囊泡（IOVs）中 将使用3 H Dnp-SG研究重影。 的相互关系 Dnp-SG和氧化型谷胱甘肽（GSSG）之间的转运 系统将通过确定GSSG对 Dnp-SG刺激ATP水解，并通过分离GSSG 使用GSSG-CNBr激活的类似程序的转运蛋白 Sepharose 4 B亲和层析。 产生的抗体 针对红细胞Dnp-SG ATP酶将用于 免疫细胞化学定位的转运蛋白， 红细胞和其他组织。 此外，我们将调查 的结构、动力学和非催化结合性质， 一种新的GST同工酶（GST σ），最近由我们在 红细胞 因为这种同工酶对 环氧化物底物，并且是高度疏水的，我们认为它是 一种膜相关酶，可能在防御中很重要， 防止脂质过氧化。 Dnp-SG的拟议研究 ATP酶和GST sigma将显著增强我们的 了解红细胞的解毒机制。

Glutathione (GSH) and the related enzyme systems such as GSH S- transferase (GST) play an important role in the protection of human tissues from toxic xenobiotics and endogenous oxidative stress. GST catalyzes the conjugation of a wide variety of toxic electrophilic xenobiotics to GSH. The GSH-xenobiotic conjugates are metabolized to mercapturic acids in most of the human tissues, but in erythrocytes, they cannot be metabolized further because all the enzymes of the mercapturic acid pathway are not present in this tissue. We have demonstrated that the conjugate of 1-chloro-2, 4-dini-trobenzene and GSH (S-dinitrophenyl glutathione, abbreviated and Dnp-SG) is actively transported out of the erythrocytes by a novel ATPase not related to any known ion pump ATPase and have now developed a procedure to isolate this Dnp-SG dependent ATPase using affinity chromatography over a column of Dnp-SG bound to CNBr activated sepharose 4B using lubrol as a detergent. This transporter protein will be isolated from erythrocyte membrane and characterized for its structural and kinetic properties. The kinetic parameters of the Dnp-SG ATPase will also be determined using conjugates of other xenobiotics in order to examine whether or not this system is a general mechanism for the transport of GSH-xenobiotic conjugates. The purified transporter protein will be reconstituted in proteoliposomes. The kinetics of transport in the liposomes as well as in the inside-out vesicles (IOVs) prepared from erythrocyte ghosts will be studied using 3H Dnp-SG. The interrelationship between Dnp-SG and oxidized glutathione (GSSG) transport systems will be studied by determining the effect of GSSG on Dnp-SG stimulated ATP hydrolysis and also by isolating the GSSG transporter using analogous procedure of GSSG-CNBr activated sepharose 4B affinity chromatography. The antibodies raised against erythrocyte Dnp-SG ATPase will be used for immunocytochemical localization of transport-protein in the erythrocytes and other tissues. In addition, we will investigate the structural, kinetic, and non-catalytic binding properties of the novel GST isoenzyme (GST sigma) recently described by us in erythrocytes. Because this isoenzyme has high activity towards epoxide substrates and is highly hydrophobic, we believe that it is a membrane associated enzyme and may be important in defense against lipid peroxidation. The proposed studies on Dnp-SG ATPase and GST sigma will significantly enhance our understanding of the detoxification mechanisms in erythrocytes.