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首页> 外文期刊>Biochemistry >Analysis of MDR1 P-Glycoprotein Conformational Changes in Permeabilized Cells Using Differential Immunoreactivity
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Analysis of MDR1 P-Glycoprotein Conformational Changes in Permeabilized Cells Using Differential Immunoreactivity

机译：使用差异免疫反应性分析通透性细胞中MDR1 P-糖蛋白的构象变化

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The reactivity of the ATP-dependent multidrug transporter P—glycoprotein (Pgp) with the conformation-sensitive monoclonal antibody UIC2 is increased in the presence of Pgp transport substrates, ATP-depleting agents, or mutations that reduce the level of nucleotide binding by Pgp. We have investigated the effects of nucleotides and vinbiastine, a Pgp transport substrate, on the UIC2 reactivity of Pgp in cells permeabilized by Staphylococcus aureus a-toxin. ATP, ADP, and nonhydrolyzable ATP analogues decreased the UIC2 reactivity; this effect was potentiated by vanadate, a nucleotide-trapping agent. The Hill number for the nucleotide-induced conformational transition was 2 for ATP and ADP but 1 for nonhydrolyzable ATP analogues. The Hill numbers for ATP and ADP were decreased to 1 by mutations in one of the two nucleotide binding sites of Pgp, whereas mutation of both sites greatly diminished the overall effect of nucleotides. Vinblastine reversed the decrease in the UIC2 reactivity brought about by all the nucleotides, including nonhydrolyzable analogues; this effect of vinblastine was blocked by vanadate. These data indicate that UIC2-detectable conformational changes of Pgp are driven by binding and debinding of nucleotides, that nucleotide hydrolysis affects the Hill number for its Pgp interactions, and that Pgp transport substrates promote nucleotide dissociation from Pgp. These findings are consistent with a conventional E1IE2 model that explains conformational transitions of a transporter protein through a series of linked equilibria.
机译：在存在Pgp传输底物，ATP消耗剂或降低Pgp核苷酸结合水平的突变的情况下，ATP依赖性多药转运蛋白P-糖蛋白（Pgp）与构象敏感的单克隆抗体UIC2的反应性增加。我们已经研究了核苷酸和长春新碱（一种Pgp转运底物）对金黄色葡萄球菌α-毒素透化的细胞中Pgp的UIC2反应性的影响。 ATP，ADP和不可水解的ATP类似物会降低UIC2反应性;核苷酸捕获剂钒酸盐可增强这种作用。对于ATP和ADP，核苷酸诱导的构象转变的希尔数为2，对于不可水解的ATP类似物为1。通过Pgp的两个核苷酸结合位点之一的突变，ATP和ADP的Hill值降低到1，而两个位点的突变都大大降低了核苷酸的总体作用。长春碱逆转了所有核苷酸（包括不可水解的类似物）引起的UIC2反应性降低;长春碱的这种作用被钒酸盐阻断。这些数据表明，UIG2可检测到的Pgp构象变化是由核苷酸的结合和脱脂驱动的，核苷酸水解会影响其Pgp相互作用的Hill数，并且Pgp转运底物会促进核苷酸从Pgp上解离。这些发现与常规的E1IE2模型一致，该模型解释了转运蛋白通过一系列连锁平衡的构象转变。

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