DETOXICATION OF XENOBIOTICS IN ERYTHROCYTES

描述：(改编自私家侦探S摘要) 续订申请，提出了对小说进行表征的研究 异源生物/谷胱甘肽结合转运蛋白，DNP-SG ATPase，存在于人类 红细胞膜。在之前的资助期内，私家侦探 结果表明：1)纯化的DNP-SG ATPase与8-叠氮基ATP结合，催化ATP 在两亲性阳离子药物阿霉素(DOX)存在下的水解， 以及阴离子GSH偶联物。2)它与药物外排不同。 泵、P-糖蛋白和多药耐药相关蛋白 (MRP)。3)蛋白脂质体中重组的纯化DNP-SG ATPase介导 依赖于ATP的GSH结合物的主动转运以及DOX。4)使用 DNP-SG ATPase抗体，P.I.从一个人的cDNA文库中克隆了一个cDNA 它产生一种重组蛋白(RLip 76)，其性质类似于 DNP-SG ATPase。因此P.I.假设DNP-SG ATPase，这是 从细胞中主动输出有毒的外源物质及其代谢物， 代表了一种主要的解毒系统，用于结构多样化的外来物 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。在第一个具体目标中，私家侦探将 异源表达获得重组RLip 76蛋白 大肠杆菌，用于结构和动力学研究。P.I.将把H-69 并用含RLip 76基因的K-562细胞检测转染后的细胞是否 对外源/内源生物介导的细胞毒性的抗性 该(RLip76)DNP-SG ATPase的底物。在第二个具体目标中，私家侦探将 重组组织纯化的DNP-SG-ATPase的功能研究 和蛋白脂质体中重组(RLip76)DNP-SG ATPase的动力学研究 生理阴离子偶联物(如白三烯、4-HNE-GSH)的运输 胆红素结合物)和药物(例如DOX、柔红霉素等)。具体而言 目的#3，P.I.建议将RLip76共转染H-69和K-562细胞 谷胱甘肽S转移酶同工酶mGSTA4-4来验证这一假说 DNP-SG ATPase(RLip76)与GST一起，在 细胞内源性和外源性亲电体的解毒作用。这些研究 将提供有关DNP-SG ATPase在 细胞解毒过程及多药耐药机制的研究 不表达Pgp和/或MRP的癌细胞。

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.