DETOXICATION OF XENOBIOTICS IN ERYTHROCYTES

红细胞中异生物质的解毒

• 批准号: 6519105
• 负责人: YOGESH Chandra AWASTHI
• 金额: $ 22.27万
• 依托单位: UNIVERSITY OF TEXAS MED BR GALVESTON
• 依托单位国家: 美国
• 财政年份: 1984
• 资助国家: 美国
• 起止时间: 1984-07-01 至 2004-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6519105
• 关键词: Escherichia coli; P glycoprotein; adenosinetriphosphatase; affinity chromatography; chemical conjugate; complementary DNA; detoxification; doxorubicin; enzyme activity; enzyme mechanism; erythrocyte membrane; glutathione; glutathione transferase; human tissue; immunoaffinity chromatography; immunocytochemistry; laboratory rabbit; liposomes; membrane transport proteins; protein purification; protein structure function; recombinant proteins; transfection

DESCRIPTION: (Adapted from the P.I.'s Abstract). In this revised competing renewal application, studies are proposed to characterize a novel xenobiotic/GSH-conjugate transporter, DNP-SG ATPase, present in human erythrocyte membranes. During the prior funding period the P.I. has demonstrated that: 1) Purified DNP-SG ATPase binds 8-azido ATP, catalyze ATP hydrolysis in the presence of an amphiphilic cationic drug, doxorubicin (DOX), as well as anionic GSH-conjugates. 2) It is distinct from the drug efflux pumps, P-glycoprotein (Pgp), and multi-drug resistance associated protein (MRP). 3) Purified DNP-SG ATPase reconstituted in proteoliposomes mediates ATP-dependent, active transport of GSH-conjugates as well as DOX. 4) Using DNP-SG ATPase antibodies, the P.I. has cloned a cDNA from a human cDNA library which yields a recombinant protein (RLip 76) with properties similar to that of DNP-SG ATPase. The P.I. therefore hypothesizes that DNP-SG ATPase, which actively exports from the cell toxic xenobiotics and their metabolites, represents a major detoxication system for structurally diverse xenobiotics in normal cells. The P.I. plans to further characterize the DNP-SG ATPase/Rlip 76, and proposes three Specific Aims. In the first Specific Aim, the P.I. will obtain the recombinant RLip 76 protein from cDNA by heterologous expression in E. coli for use in structural and kinetic studies. The P.I. will transfect H-69 and K-562 cells with RLip 76 cDNA to examine whether the transfected cells are resistant to cytotoxicity mediated by xenobiotics/endobiotics which are substrates for this (RLip76) DNP-SG ATPase. In Specific Aim #2, the P.I. will functionally characterize the DNP-SG ATPase by reconstituting tissue-purified and recombinant (RLip76) DNP-SG ATPase in proteoliposomes to study the kinetics of transport of physiological anionic conjugates (e.g. leukotrienes, 4-HNE GSH conjugates of bilirubin), and drugs (e.g. DOX, daunomycin, etc.). In Specific Aim #3, the P.I. proposes to co-transfect RLip76 into H-69 and K-562 cells with the glutathione S-transferase (GST) isozyme, mGSTA4-4, to test the hypothesis that DNP-SG ATPase (RLip76) in conjunction with GSTs, plays a major role in the detoxication of endogenous and exogenous electrophiles in cells. These studies will provide clinically relevant information on the role of DNP-SG ATPase in cellular detoxication processes and on the mechanisms of multidrug resistance of cancer cells which do not express Pgp and/or MRP.

描述：（改编自P.I.的摘要）。在这个经过修改的竞争中 提出了更新应用，提出了研究以表征新颖的 异种生物/GSH偶联转运蛋白，DNP-SG ATPase，存在于人 红细胞膜。在以前的资金期间有 证明：1）纯化的DNP-SG ATPase结合了8-齐ado ATP，催化ATP 在存在两亲阳离子药物的阿霉素（DOX）的情况下水解 以及阴离子GSH偶联。 2）它与药物外排是不同的 泵，P-糖蛋白（PGP）和多药耐药性相关蛋白 （MRP）。 3）纯化的DNP-SG ATPase在蛋白质脂质体中重构 ATP依赖性，GSH偶联物和DOX的主动运输。 4）使用 DNP-SG ATPase抗体P.I.已经从人cDNA文库克隆了一个cDNA 它产生的重组蛋白（RLIP 76）具有类似于 DNP-SG ATPase。 P.I.因此假设DNP-SG ATPase，这是 积极从细胞有毒异种生物及其代谢产物出口， 代表了结构上多样的异种生物的主要解毒系统 正常细胞。 P.I.计划进一步表征DNP-SG ATPase/RLIP 76， 并提出了三个具体目标。在第一个特定目标中，P.I.将要 通过异源表达从cDNA中获得重组RLIP 76蛋白 大肠杆菌用于结构和动力学研究。 P.I.将转染H-69 带有RLIP 76 cDNA的K-562细胞检查转染的细胞是否为 对异种生物/内生物学介导的细胞毒性的耐药性 此（RLIP76）DNP-SG ATPase的底物。在P.I.的特定目标＃2中将要 通过重组组织纯化的功能表征DNP-SG ATPase 和重组（RLIP76）DNP-SG ATPase在蛋白脂体中研究动力学 生理阴离子结合物的运输（例如白细胞，4-HNE GSH 胆红素的结合物和药物（例如Dox，daunomycin等）。具体 AIM＃3，P.I。建议将RLIPLIP76共同传输到H-69和K-562细胞中 谷胱甘肽S-转移酶（GST）同工酶MGSTA4-4，以检验假设 DNP-SG ATPase（RLIP76）与GST结合 细胞中内源性和外源性亲电的解毒。这些研究 将提供有关DNP-SG ATPase在中的作用的临床相关信息 细胞解毒过程和多药电阻的机制 不表达PGP和/或MRP的癌细胞的。