Analysis of MDR1 P-glycoprotein conformational changes in permeabilized cells using differential immunoreactivity
Analysis of MDR1 P-glycoprotein conformational changes in permeabilized cells using differential immunoreactivity
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DOI：
10.1021/bi001371v
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发表时间：
2001-04-10
期刊：
影响因子：
2.9
通讯作者：
Roninson, IB
中科院分区：
文献类型：
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作者：
Druley, TE;Stein, WD;Roninson, IB
atp依赖的多药转运蛋白p糖蛋白（Pgp）与构象敏感的单克隆抗体UIC2的反应性在Pgp转运底物、atp消耗剂或降低Pgp核苷酸结合水平的突变存在时增加。我们研究了核苷酸和长春碱（一种Pgp转运底物）对Pgp在被金黄色葡萄球菌α -毒素渗透的细胞中UIC2反应性的影响。ATP、ADP和非水解ATP类似物降低了UIC2的反应活性；这种作用被钒酸盐（一种核苷酸捕获剂）增强了。核苷酸诱导的构象转变的希尔数对于ATP和ADP为2，而对于不可水解的ATP类似物为1。Pgp的两个核苷酸结合位点中的一个发生突变，使ATP和ADP的Hill数降至1，而这两个位点的突变大大降低了核苷酸的总体作用。长春花碱逆转了所有核苷酸（包括不可水解的类似物）导致的UIC2反应性下降；长春花碱的这种作用被钒酸盐阻断。这些数据表明，uic2检测到的Pgp构象变化是由核苷酸的结合和脱粘驱动的，核苷酸水解影响其Pgp相互作用的希尔数，Pgp转运底物促进核苷酸与Pgp的解离。这些发现与传统的E1/E2模型一致，该模型通过一系列连锁平衡解释了转运蛋白的构象转变。
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 vinblastine, a Pgp transport substrate, on the UIC2 reactivity of Pgp in cells permeabilized by Staphylococcus aureus alpha -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 E1/E2 model that explains conformational transitions of a transporter protein through a series of linked equilibria.