DETOXICATION OF XENOBIOTICS IN ERYTHROCYTES

红细胞中异生物质的解毒

• 批准号: 2391935
• 负责人: YOGESH Chandra AWASTHI
• 金额: $ 18.91万
• 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/2391935
• 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

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.

在该项目的资助年度中，我们描述了 参与 ATP 依赖性初级主动转运的转运蛋白 谷胱甘肽 (GSH) 与人红细胞膜结合， 将其命名为二硝基苯基 S-谷胱甘肽 (Dnp-SG) ATP 酶 因为使用Dnp-SG作为模型底物。随后我们 表明 Dnp-SG ATPase 在人类细胞血浆中普遍存在 膜，除了 GSH 结合物之外，它还 参与胆红素结合物的 ATP 依赖性转运， 白三烯和结构不相关的化合物，例如 阿霉素和 P-糖蛋白的其他底物 多药中过度表达的 ATP 依赖性泵特征 抵抗癌细胞。这些研究首次 证明了一种极其通用的运输工具的存在 （与 P-糖蛋白不同）在人体细胞中可以主动 运输多种异生物质、药物及其 I 相 和 II 相代谢物。 为会议确立一个统一的主题 这些结构多样的化合物的运输机制 Dnp-SG ATPase，本申请中提出了对其的研究 结构和功能表征。 Dnp-SG ATP酶将是 通过 Dnp-SG 亲和力从人红细胞和其他组织中纯化 层析法和免疫亲和层析法测定其 结构和功能特性。 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 凝胶中分离，然后进行转印迹 对 P-PVDF 膜的影响将被确定。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 的 cDNA 进行测序，推断其一级结构。 重组 Dnp-SG ATPase 将通过在大肠杆菌中表达来制备。 大肠杆菌和/或其他合适的载体以获得足够的蛋白质 其结构和功能特征。 抗体针对 Dnp-SG ATPase 将成为替代方法中的 usssl 克隆。 Dnp-SG ATPase 位点可能存在基因组异质性 进行调查以检查是否存在其他相关的 该位点的转运蛋白。 ATP水解动力学 GSH 结合物刺激 Dnp-SG ATPase 的活性 异生物质和脂质过氧化的有毒产物，例如 4- 羟基壬烯醛 (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 这些化合物的 ATP 依赖性转运机制 由红细胞膜制备的内向外囊泡 (IOV) 在用天然重组 Dnp-SG 重建的蛋白脂质体中 将研究 ATP 酶。我们将检验 Dnp-SG 是否成立的假设 ATP酶是阿霉素转运的介质，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 我们从亲本 NCI H-69 细胞系中开发出癌细胞系。 本项目提出的研究将确定 Dnp-SG 的作用 ATP酶在结构多样性保护机制中的作用 异生素和有毒内生素（例如4-HNE），并将测试 Dnp-SG ATPase 可能参与该机制的假设 癌细胞的耐药性，特别是那些不耐药的细胞 表达P糖蛋白。