DETOXICATION OF XENOBIOTICS IN ERYTHROCYTES

描述：(改编自研究人员摘要)红细胞 解毒有毒的亲电性异源物质和内源性生成 通过结合形成谷胱甘肽(GSH)的脂质过氧化产物， 通过谷胱甘肽S转移酶催化的反应。在人类身上 这些GSH结合物通过主要的主动转运而外流。 红细胞膜不同ATPase介导的机制 已被命名为S-二硝基苯基谷胱甘肽-ATP酶(DNP-SG-ATPase) 因为它能够在其存在的情况下刺激ATP水解酶 底物S-二硝基苯基谷胱甘肽(DNP-SG)。在……里面 这项延续建议，进一步在结构和功能上 对DNP-SG ATPase的性质进行了研究。纯化的DNP-SG ATPase 通过亲和层析对其氨基酸进行分析 组成、N-末端序列(S)、亚基组成和分子 重量。DNP-SG-ATPase的动力学性质包括体外稳定性 红细胞膜摄取底物(DNP-SG)的状态动力学 内翻囊泡(IOV)以及重组蛋白脂质体 与纯化的DNP-SG ATPase结合将进行研究。为了 阐明DNP-SG-ATPase在心肌保护中的生理作用 甲状腺细胞膜上GSH结合物的亲电转运 红细胞的脂质过氧化产物将被现场研究为 IOV也是如此。 DNP-SG-ATPase与血管紧张素转换酶的相互作用 P-糖蛋白介导的药物外排泵，在多药中过表达 耐药(MDR)癌细胞系，是最近提出的。这是 得到初步研究的支持，这些研究表明DNP-SG的运输 阿霉素，一种模型底物，抑制从麦角细胞中释放 P-糖蛋白。功能与结构的相互关系(S) DNP-SG ATPase转运体和P-糖蛋白外排泵将 因此，可利用麦角细胞和多药耐药细胞株进行研究。动力学 阿霉素依赖抑制甲状腺细胞摄取DNP-SG的研究 膜IOV及DNP-SG和其他DNP-SG ATPase底物的作用 对P-糖蛋白介导的阿霉素从LZ III N细胞外排的影响 学习。P-糖蛋白底物对DNP-SG刺激ATP的影响 还将研究纯化的DNP-SG ATPase的水解性。的表达 DNP-SG ATPase在其他组织以及肿瘤和MDR细胞系中将 用多克隆抗体进行免疫印迹分析 DNP-SG-ATPase由调查人员提出。

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.