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：（改编自研究者摘要）红细胞 解毒有毒亲电子异生物质和内源性产生的 脂质过氧化产物通过与谷胱甘肽（GSH）结合， 通过谷胱甘肽S-转移酶催化的反应。 在人类中 这些GSH结合物通过初级主动转运 由红细胞膜的不同ATP酶介导的机制， 被命名为S-二硝基苯基谷胱甘肽ATP酶（Dnp-SG ATP酶） 由于其在其存在下刺激ATP水解的能力， 底物，S-二硝基苯基谷胱甘肽（Dnp-SG）在无细胞系统中。 在 这一延续建议，进一步结构和功能 Dnp-SG ATP酶的特性。 纯化的Dnp-SG ATP酶 通过亲和层析将分析其氨基酸 组成、N-末端序列、亚基组成和分子组成 重量. Dnp-SG ATP酶的动力学特性，包括体外稳态 红细胞膜摄取底物（Dnp-SG）的状态动力学 由内而外囊泡（IOV），以及由重构的脂蛋白体 掺入纯化的Dnp-SG ATP酶进行研究。 为了 阐明DNP-SG ATP酶在保护心肌细胞中的生理作用。 红细胞膜，谷胱甘肽结合物的运输，亲电 红细胞的脂质过氧化产物将在原位进行研究， 在IOV中也是如此。 Dnp-SG ATP酶与细胞凋亡的相互关系 P-糖蛋白介导的药物外排泵，在多药物中过表达 耐药（MDR）癌细胞系，最近已经提出。 这是 初步研究表明，Dnp-SG的运输 阿霉素抑制红细胞的增殖， P-糖蛋白 之间的功能和结构相互关系 Dnp-SG ATP酶转运体和P-糖蛋白外排泵将 因此，可以使用红细胞和MDR细胞系进行研究。 动力学 阿霉素依赖性抑制甲状腺细胞摄取Dnp-SG 膜IOV和Dnp-SG和其他Dnp-SG ATP酶底物的作用 对P-糖蛋白介导的阿霉素从LZ III N细胞流出的影响将是 研究了 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 ATP酶的水解。 表达 其它组织以及肿瘤和MDR细胞系中的Dnp-SG ATP酶将 通过Western印迹分析，使用针对以下的多克隆抗体进行研究 Dnp-SG ATP酶。