DETOXICATION OF XENOBIOTICS IN ERYTHROCYTES

红细胞中异生物质的解毒

• 批准号: 2176515
• 负责人: YOGESH Chandra AWASTHI
• 金额: $ 15.62万
• 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 1995-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2176515
• 关键词: P glycoprotein; adenosinetriphosphatase; affinity chromatography; biological transport; chemical conjugate; chemical substitution; detoxification; environmental toxicology; enzyme mechanism; erythrocyte membrane; erythrocytes; glutathione; glutathione transferase; human subject; human tissue; in situ hybridization; lipid peroxides; neoplastic cell culture for noncancer research; protein purification; protein structure function; toxin metabolism

DESCRIPTION: (Adapted from the investigator's abstract) Erythrocytes detoxify toxic electrophilic xenobiotics and endogenously generated products of lipid peroxidation by conjugating then to glutathione (GSH), through a reaction catalyzed by glutathione S-transferases. In humans these GSH-conjugates are effluxed through a primary active transport mechanism mediated by a different ATPase of erythrocyte membranes which have been designated as S-dinitrophenyl glutathione ATPase (Dnp-SG ATPase) because of its ability to stimulate ATP hydrolysis in the presence of its substrate, S-dinitrophenyl glutathione (Dnp-SG) in a cell free system. In this continuation proposal, further structural and functional characterization of Dnp-SG ATPase is proposed. Dnp-SG ATPase purified through affinity chromatography will be analyzed for its amino acid composition, N-terminal sequence(s), subunit composition, and molecular weight. Kinetic properties of Dnp-SG ATPase including in vitro steady state kinetics of uptake of the substrate (Dnp-SG) by erythrocyte membrane inside out vesicles (IOVs), as well as by reconstituted proteoliposomes incorporated with purified Dnp-SG ATPase will be studied. In order to elucidate the physiological role of DNP-SG ATPase in the protection of erthyrocyte membrane, the transport of GSH-conjugates of electrophilic products of lipid peroxidation by erythrocytes will be studied in situ as well as in the IOVs. A mechanistic interrelationship between Dnp-SG ATPase and the P-glycoprotein mediated drug efflux pump, overexpressed in multi-drug resistant (MDR) cancer cell lines, has been recently suggested. This is supported by preliminary studies, which show that the transport of Dnp-SG from erthryocytes is inhibited by adriamycin, a model substrate for P-glycoprotein. The functional and structural interrelationship(s) between Dnp-SG ATPase transporter and the P-glycoprotein efflux pump will therefore, be studied using erthyrocytes and MDR cell lines. The kinetics of adriamycin dependent inhibition of the uptake of Dnp-SG by erthyrocyte membrane IOVs and the effect of Dnp-SG, and other Dnp-SG ATPase substrates on P-glycoprotein mediated efflux of adriamycin from LZ III N cells will be studied. The effect of P-glycoprotein substrates on Dnp-SG stimulated ATP hydrolysis by purified Dnp-SG ATPase will also be studied. Expression of Dnp-SG ATPase in other tissues as well as in tumor and MDR cell lines will be studied by Western blot analyses using the polyclonal antibodies against Dnp-SG ATPase raised by the investigators.

描述：（改编自调查员的摘要）红细胞 排毒有毒的亲电异种生物和内源性产生 脂质过氧化的产物通过结合然后与谷胱甘肽（GSH）， 通过谷胱甘肽S-转移酶催化的反应。 在人类中 这些GSH结合物通过主要的主动运输被排出 由不同的红细胞膜ATPase介导的机制 已被指定为S-二苯基谷胱甘肽ATPase（DNP-SG ATPase） 由于它在存在下刺激ATP水解的能力 底物，无细胞系统中的S-二硝基苯基谷胱甘肽（DNP-SG）。 在 该延续建议，进一步的结构和功能 提出了DNP-SG ATPase的表征。 DNP-SG ATPase纯化 通过亲和色谱法将分析其氨基酸 组成，N末端序列，亚基组成和分子 重量。 DNP-SG ATPase的动力学特性，包括体外稳定 红细胞膜的基材摄取（DNP-SG）的状态动力学 内而外的囊泡（IOV）以及通过重构的蛋白质体 将研究与纯化的DNP-SG ATPase合并。 为了 阐明DNP-SG ATPase在保护中的生理作用 细胞肌细胞膜，亲电的GSH偶联物的运输 红细胞的脂质过氧化产物将在原位研究 以及在IOV中。 DNP-SG ATPase与 P-糖蛋白介导的药物外泵，在多药中过表达 最近已经提出了抗性（MDR）癌细胞系。 这是 在初步研究的支持下，这表明DNP-SG的运输 阿霉素抑制了从细胞的模型底物抑制。 P-糖蛋白。 功能和结构相互关系 DNP-SG ATPase Transporter和P-糖蛋白外排泵将 因此，可以使用细胞和MDR细胞系进行研究。 动力学 依赖性肌霉素抑制rthyrocyte对DNP-SG的抑制作用 膜IOV和DNP-SG和其他DNP-SG ATPase底物的效果 从LZ III N细胞中的Adrimycin介导的P-糖蛋白介导的外排将是 研究。 P-糖蛋白底物对DNP-SG刺激ATP的影响 还将研究通过纯化的DNP-SG ATPase水解。 表达 其他组织以及肿瘤和MDR细胞系中的DNP-SG ATPase将 使用蛋白质印迹分析使用多克隆抗体进行研究 研究人员培养的DNP-SG ATPase。