DETOXICATION OF XENOBIOTICS IN ERYTHROCYTES

红细胞中异生物质的解毒

• 批准号: 2176516
• 负责人: YOGESH Chandra AWASTHI
• 金额: $ 18.34万
• 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 1999-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2176516
• 关键词: P glycoprotein; adenosinetriphosphatase; affinity chromatography; biological transport; chemical conjugate; detoxification; doxorubicin; enzyme activity; enzyme mechanism; erythrocyte membrane; glutathione; human subject; immunoaffinity chromatography; laboratory rabbit; lipid peroxides; liposomes; membrane transport proteins; molecular cloning; nucleic acid probes; protein purification; protein sequence; protein structure function; P glycoprotein; adenosinetriphosphatase; affinity chromatography; biological transport; chemical conjugate; detoxification; doxorubicin; enzyme activity; enzyme mechanism; erythrocyte membrane; glutathione; human subject; immunoaffinity chromatography; laboratory rabbit; lipid peroxides; liposomes; membrane transport proteins; molecular cloning; nucleic acid probes; protein purification; protein sequence; protein structure function

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.

在本项目的资助年中，我们表征了 参与ATP依赖性的一级主动转运的转运蛋白 谷胱甘肽（GSH） - 人类红细胞膜中的缀合物 将其指定为二硝基苯基S-glutathione（DNP-SG）ATPase 因为将DNP-SG用作模型基板。随后我们 表明DNP-SG ATPase在人类细胞血浆中无处不在 膜，除了gsh偶联之外，它也是 参与胆红素偶联物的ATP依赖性运输， 白细胞和结构无关的化合物，例如 阿霉素和其他P-糖蛋白的底物，孔 特征在多饮中表征了ATP依赖性泵过多的泵 抗性癌细胞。这些研究，第一次 证明了极限运输蛋白的存在 （与p-糖蛋白不同）在人类细胞中，可以主动 运输多样化的异种生物，药物及其第一阶段 和II期代谢产物。 为建立一个统一的主题 通过 DNP-SG ATPase，在本申请中提出了研究 结构和功能表征。 DNP-SG ATPase将是 通过DNP-SG亲和力从人的红细胞和其他组织中纯化 色谱和免疫亲和力色谱法确定其 结构和功能特性。 的氨基酸序列 CNBR裂解产生的DNP-SG ATPase的肽片段 并通过HPLC和/或在SDS凝胶中孤立 将确定在P-PVDF膜上。这些序列将是 用于设计和合成核苷酸探针以克隆和 序列DNP-SG ATPase的cDNA推断其主要结构。 重组DNP-SG ATPase将通过在E中表达。 大肠杆菌和/或其他合适的向量，以获取足够的蛋白质 它的结构和功能表征。 反对的抗体 DNP-SG ATPase将是USSSL的替代方法 克隆。 DNP-SG ATPase基因座的可能的基因组杂种将 进行调查以检查其他相关的存在 该基因座的运输商。 ATP水解的动力学 DNP-SG ATPase的活性由GSH偶联物刺激 脂质过氧化的异种生物和有毒产物，例如4- 羟基烯烯（4-HNE）和P-糖蛋白的底物（例如 将研究阿霉素，长春新碱）。 还有动力学和 这些化合物在ATP依赖性转运的机制 由红细胞膜制备的内部囊泡（IOV）和 在具有天然重组DNP-SG的重构蛋白脂质体中 将研究ATPase。我们将测试是否dnp-sg的假设 ATPase是阿霉素转运的介体，因此抗性 p糖蛋白阴性，阿霉素抗性小细胞肺 我们从父母NCI H-69细胞系开发的癌细胞系。 该项目提出的研究将定义DNP-SG的作用 ATPase在防止结构上多样的保护机制中 异种生物和有毒的内生物学（例如4-HNE），将测试 DNP-SG ATPase可能涉及机理的假设 癌细胞的耐药性，尤其是那些 表达P糖蛋白。