DETOXICATION OF XENOBIOTICS IN ERYTHROCYTES

在该项目的资助年期间，我们描述了一个 参与ATP依赖的初级主动转运的转运蛋白 人红细胞膜谷胱甘肽（GSH）结合物， 命名为二硝基苯基S-谷胱甘肽（Dnp-SG）ATP酶 因为使用Dnp-SG作为模型底物。而后 表明Dnp-SG ATP酶在人细胞浆中普遍存在 膜，并且除了GSH-缀合物之外， 参与红细胞结合物的ATP依赖性转运， 白三烯和结构无关的化合物， 阿霉素和其他底物的P-糖蛋白，以及 特征性ATP依赖性泵在多药耐药细胞中过表达 抵抗癌细胞。这些研究首次 证明了一种极其通用的传送器的存在 （与P-糖蛋白不同）在人类细胞中， 运输不同种类的外源性物质、药物及其第一阶段 和II相代谢物。 为建立一个统一的主题 这种结构不同的化合物的运输机制， Dnp-SG ATP酶，在此应用中提出了研究，因为其 结构和功能表征。 Dnp-SG ATP酶将是 通过Dnp-SG亲和性从人红细胞和其他组织中纯化 层析和免疫亲和层析，以确定其 结构和功能特性。 的氨基酸序列 CNBr裂解产生的Dnp-SG ATP酶肽段 并通过HPLC和/或在SDS凝胶中分离，然后进行transblotting P-PVDF膜的性能。这些序列将是 用于设计和合成核苷酸探针， 对Dnp-SG ATP酶的cDNA进行测序，推测其一级结构。 重组Dnp-SG ATP酶将通过在E. 大肠杆菌和/或其它合适的载体来获得足够的蛋白质， 其结构和功能特征。 抗体 Dnp-SG ATP酶将用于替代方法， 克隆。Dnp-SG ATP酶基因座可能存在的基因组遗传异质性， 调查是否存在其他相关的 在这个位置上的转运蛋白。ATP水解动力学 谷胱甘肽结合物刺激的Dnp-SG ATP酶活性 外源性物质和脂质过氧化的有毒产物，如4- 羟基壬烯醛（4-HNE）和P-糖蛋白的底物（例如， 阿霉素、长春新碱）进行研究。 此外，动力学和 这些化合物的ATP依赖性转运机制 由红细胞膜制备的内而外囊泡（IOV）， 在具有天然重组Dnp-SG的重构蛋白脂质体中 ATP酶将被研究。我们将检验Dnp-SG是否 ATP酶是多柔比星转运的介质，因此是耐药性的介质 P糖蛋白阴性、阿霉素耐药的小细胞肺 癌细胞系由我们从亲本NCI H-69细胞系开发。 本项目中提出的研究将确定DNP-SG的作用 ATP酶在结构多样性免疫保护机制中的作用 外源性物质和有毒的内源性物质（如4-HNE），并将测试 Dnp-SG ATP酶可能参与了这一机制 癌细胞的抗药性，特别是那些没有 表达P糖蛋白。

During the funded year of this project, we have characterized a transporter involved in ATP-dependent primary active transport of glutathione (GSH)-conjugates in human erythrocyte membrane and designated it as dinitrophenyl S-glutathione (Dnp-SG) ATPase because the use of Dnp-SG as a model substrate. Subsequently we showed that Dnp-SG ATPase was ubiquitous in human cell plasma membranes and that in addition to GSH-conjugates it was also involved in the ATP-dependent transport of bilirubin-conjugates, leukotrienes, and structurally unrelated compounds such as doxorubicin and other substrates of P-glycoprotein, a well characterized ATP-dependent pump overexpresed in multidrug resistance cancer cells. These studies, for the first time demonstrated the presence of an extremely versatile transporter (distinct from P-glycoprotein) in human cells which could actively transport diverse group of xenobiotics, drugs, and their phase I and phase II metabolites. To establish a unifying theme for the mechanisms of transport of such structually diverse compounds by Dnp-SG ATPase, studies are proposed in this application for its structural and functional characterization. Dnp-SG ATPase will be purified from human erytocytes and other tissues by Dnp-SG affinity chromatography and immunoaffinity chromatography to determine its structural and functional properties. The amino acid sequences of the peptide fragments of Dnp-SG ATPase generated by CNBr cleavage and isolated by HPLC and/or in SDS gels followed by transblotting on P-PVDF membranes will be determined. These sequences will be used to design and synthesize nucleotide probes to clone and sequence the cDNA of Dnp-SG ATPase to deduce its primary structure. Recombinant Dnp-SG ATPase will be prepared by expressing it in E. coli and/or other suitable vectors to get sufficient protein for its structural and functional characterization. Antibodies against Dnp-SG ATPase will be usssl in the alternate approaches for cloning. Possible genomic heterdgereity at Dnp-SG ATPase locus will be investigated to examine the existence of other related transporters at this locus. The kinetics of the ATP hydrolyzing activity of Dnp-SG ATPase stimulated by GSH-conjugates of xenobiotics and toxic products of lipid peroxidation such as 4- hydroxynonenal (4-HNE), and the substrates of P-glycoprotein (e.g. doxorubicin, vincristine) will be studied. Also the kinetics and mechanisms of the ATP-dependent transport of these compounds in the inside out vesicles (IOVs) prepared from erythrocyte membranes and in reconstituted proteoliposomes with native recombinant Dnp-SG ATPase will be studied. We will test the hypothesis whether Dnp-SG ATPase is a mediator of doxorubicin transport and hence resistance of P glycoprotein negative, doxorubicin resistant small cell lung cancer cell lines developed by us from parental NCI H-69 cell line. Studies proposed in this project will define the role of Dnp-SG ATPase in the protection mechanisms against structurally diverse xenobiotics and toxic endobiotics (such as 4-HNE), and will test the hypothesis that Dnp-SG ATPase may be involved in the mechanisms of drug resistance of cancer cells, particularly those which do not express P glycoprotein.