FUNCTIONS OF THE HEPATIC GAMMA-GLUTAMYL CYCLE

谷胱甘肽在细胞防御反应性中发挥核心作用 亲电剂和氧化剂，两种最常见的细胞生成物 在化学毒性和疾病方面的伤害。我们的总体目标是 确定谷胱甘肽S结合物和谷胱甘肽的处置机制 新型硫代尿酸(S取代N-乙酰基-L-半胱氨酸)的合成 肝内通路。这条途径最近从我们的 实验室，并被认为在肝脏中发挥重要作用 对亲电体和汞等有毒金属的解毒， 硫代尿酸的生物合成、胆汁形成和谷胱甘肽 动态平衡。 具体目标是：(1)确定肝内伽玛素的作用- 硫代尿酸形成过程中的谷氨酰循环。与当前的情况相反 我们最近的研究证明了硫代尿酸生物合成的模型 这种排毒途径可以完全发生在肝脏内。 此外，与目前的想法相反，具有代表性的硫代尿酸 酸(S.2，4-二硝基苯基-N-乙酰半胱氨酸，或DNP-NAC)由 分离的大鼠肝脏几乎完全进入胆汁，而不是进入 正弦循环。要使该模型达到最佳状态，并确定 涉及的机制，我们计划：A.-检查正弦吸收和 结构不同的半胱氨酸S偶联物和 体内和灌流肝脏中的硫代尿酸(含乙基 碘和1-氯-2，4-二硝基苯作为模型异物)。B.使用 分离的小管和基底外侧膜小泡，定义 肝转运的驱动力和底物特异性 ··· 转移酶III对谷胱甘肽和谷胱甘肽S偶联物的肝清除作用 以及硫代尿酸的生物合成。(2)表达新奇的运河 非依赖三磷酸腺苷的高亲和力谷胱甘肽S结合物载体在非洲爪蛙体内的研究 用从大鼠肝脏中提取的Poly(A)RNA，以及 描述卵母细胞系统中转运蛋白的特性。 以卵母细胞作为转运功能的检测手段，使用表达克隆 识别转运蛋白基因的策略。克隆不仅会 获得有关谷胱甘肽S偶联物的重要结构信息 传输器，但将允许生产分子探针，用于 明确其在肝脏和整个生物体异种代谢中的作用。 预计这些研究最终将提供信息。 对预防和治疗中毒性肝细胞损伤和 反应性亲电刺激引起的肝外并发症 化合物。

Glutathione plays a central role in cellular defense against reactive electrophiles and oxidants, two of the most common producers of cellular injury in both chemical toxicity and disease. Our overall objective is to identify mechanisms for disposition of glutathione S conjugates and mercapturic acids (S-substituted N-acetyl-L-cysteines) via a novel intrahepatic pathway. This pathway was recently described from our laboratory, and is thought to play an important role in hepatic detoxification of electrophiles and toxic metals such as mercury, mercapturic acid biosynthesis, bile formation, and glutathione homeostasis. The specific aims are: (1) Define the role of the intrahepatic gamma- glutamyl cycle in mercapturic acid formation. In contrast to current models of mercapturic acid biosynthesis, our recent studies demonstrated that this detoxification pathway can occur entirely within the liver. Moreover, in contrast to current thinking, a representative mercapturic acid (S.2,4-dinitrophenyl-N-acetylcysteine, or DNP-NAC) was excreted by isolated rat liver almost exclusively into bile, and not into the sinusoidal circulation. To best this model and to identify the mechanisms involved, we plan to: A.- Examine sinusoidal uptake and disposition of structurally distinct cysteine S-conjugates and mercapturic acids, both in vivo and in the perfused liver (with ethyl iodide and 1-chloro-2,4-dinitrobenzene as model xenobiotics). B. Using isolated canalicular and basolateral membrane vesicles, define the driving forces and substrate specificities for hepatic transport of mercapturic acids, and C. Test the role of sinusoidal gamma-glutamyl transferase iii hepatic clearance of GSH and glutathione S-conjugates, and in mercapturic acid biosynthesis. (2) Express the novel canalicular ATP-independent, high affinity glutathione S-conjugate carrier in Xenopus laevis oocytes using poly(A)+ RNA isolated from rat liver, and characterize the properties of the transporter in the oocyte system. With oocytes as a functional assay for transport, use expression cloning strategy to identify the cDNA for the transporter. Cloning will not only yield important structural information on this glutathione S-conjugate transporter, but will allow production of molecular probes with which to define its role in xenobiotic metabolism in liver and the whole organism. It is anticipated that these studies will ultimately provide information critical for the prevention and treatment of toxic liver cell injury and its extrahepatic complications induced by reactive electrophilic compounds.