DETOXICATION OF XENOBIOTICS IN ERYTHROCYTES

在这项计划的资助年度内，我们的特点是 转运体参与ATP依赖的初级主动转运 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ATPase 因为使用了DNP-SG作为模型衬底。随后，我们 结果表明，DNP-SG ATPase在人细胞质中普遍存在 膜，除了GSH偶联物外，它还 参与依赖于ATP的胆红素结合物的运输， 白三烯和结构上无关的化合物，如 阿霉素等P-糖蛋白底物，一井 特征性的ATP依赖泵在多药中过度表达 耐药癌细胞。这些研究是第一次 展示了一种非常多功能的运输机的存在 (有别于P-糖蛋白)在人类细胞中可以活跃地 运输不同种类的外源物质、药物及其I期 和第二相代谢物。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 ATPase，建议在这一应用中对其进行研究 结构和功能表征。DNP-SG ATPase将是 DNP-SG亲和纯化人红细胞及其他组织 层析法和免疫亲和层析法测定其 结构和功能特性。其氨基酸序列 CNBr裂解产生的DNP-SG-ATPase的多肽片段 用高效液相色谱法和/或在转印后的十二烷基硫酸钠凝胶中分离 将对P-PVDF膜进行测定。这些序列将是 用于设计和合成核苷酸探针以克隆和 对DNP-SG ATPase基因进行测序，推测其一级结构。 重组DNP-SG-ATPase在E. Coli和/或其他合适的载体以获得足够的蛋白质 它的结构和功能表征。抗病毒抗体 DNP-SG ATPase将用于以下替代方法 克隆。DNP-SG ATPase基因座可能存在的基因组杂合性将 被调查以检查是否存在其他相关的 这个地点的传送者。三磷酸腺苷水解酶的动力学 谷胱甘肽结合物对DNP-SG-ATPase活性的影响 异生物质和脂质过氧化的有毒产物，如4- 羟基壬烯醛(4-HNE)和P-糖蛋白底物(例如 阿霉素、长春新碱)将被研究。此外，动力学和 这些化合物在体内依赖于ATP转运的机制 由红细胞膜和红细胞膜制备的内翻式囊泡 在含有天然重组DNP-SG的重组蛋白脂质体中 将对ATPase进行研究。我们将检验假设DNP-SG是否 ATPase是阿霉素转运的中介物，因此产生耐药性 P糖蛋白阴性、阿霉素耐药小细胞肺 我们从亲代NCI H-69细胞系中培育出癌细胞系。 本项目中提出的研究将确定DNP-SG的作用 ATPase在结构多样性保护机制中的作用 外源生物和有毒内生生物(如4-HNE)，并将测试 DNP-SG-ATPase可能参与发病机制的假说 癌细胞的抗药性，特别是那些不耐药的癌细胞 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.