FUNCTIONS OF THE HEPATIC GAMMA-GLUTAMYL CYCLE

谷氨酸在细胞防御反应性 亲电体和氧化剂，两个最常见的生产商的细胞 化学毒性和疾病的伤害。 我们的总体目标是 确定谷胱甘肽S结合物的处置机制， 巯基尿酸（S-取代的N-乙酰基-L-半胱氨酸）通过一种新的 肝内途径 这条途径最近在我们的 实验室，并被认为在肝脏中发挥重要作用 亲电试剂和有毒金属如汞的解毒， 巯基尿酸生物合成、胆汁形成和谷胱甘肽 体内平衡 具体目的是：（1）明确肝内γ-淀粉酶的作用; 巯基尿酸形成中的谷氨酰循环。与当前相比， 巯基尿酸生物合成的模型，我们最近的研究表明， 这种解毒途径可以完全发生在肝脏内。 此外，与目前的想法相反， 酸（S. 2，4-二硝基苯-N-乙酰半胱氨酸，或DNP-NAC）由 离体大鼠肝脏几乎完全进入胆汁，而不是进入 正弦循环 为了优化这个模型， 涉及的机制，我们计划：A.-检查正弦摄取， 结构上不同的半胱氨酸S-缀合物的处置， 巯基尿酸，无论是在体内和灌注的肝脏（与乙基 碘化物和1-氯-2，4-二硝基苯作为模型异生物质）。 B。使用 孤立的小管和基底外侧膜囊泡，定义了 肝脏转运的驱动力和底物特异性 巯基尿酸和C.测试正弦γ-谷氨酰的作用 GSH和谷胱甘肽S-缀合物的转移酶iii肝清除率， 和巯基尿酸生物合成。 (2)表达新颖的泪小管 非ATP依赖性、高亲和力谷胱甘肽S-结合物在非洲爪蟾中的载体 使用从大鼠肝脏分离的poly（A）+ RNA， 表征卵母细胞系统中转运蛋白的性质。 用卵母细胞作为转运的功能测定，使用表达克隆 策略来鉴定转运蛋白的cDNA。 克隆不仅 在谷胱甘肽S-结合物上产生重要的结构信息 转运蛋白，但将允许生产分子探针， 确定其在肝脏和整个生物体中的异生物质代谢中的作用。 预计这些研究将最终提供信息， 对于预防和治疗毒性肝细胞损伤至关重要， 反应性亲电性肝外并发症 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.