SPECIFIC DRUG-BINDING BY PURIFIED LIPID-RECONSTITUTED P-GLYCOPROTEIN - DEPENDENCE ON THE LIPID-COMPOSITION
SPECIFIC DRUG-BINDING BY PURIFIED LIPID-RECONSTITUTED P-GLYCOPROTEIN - DEPENDENCE ON THE LIPID-COMPOSITION
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DOI：
10.1016/0005-2736(92)90334-i
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发表时间：
1992-06-11
期刊：
影响因子：
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通讯作者：
KOMANO, T
中科院分区：
文献类型：
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作者：
SAEKI, T;SHIMABUKU, AM;KOMANO, T
我们将p -糖蛋白与β -半乳糖苷酶在c端融合，目的是在重组实验中研究p -糖蛋白的药物结合机制。融合蛋白在NIH 3T3细胞中的表达赋予了多药耐药表型，这表明β -半乳糖苷酶在c端融合不影响p糖蛋白的功能。用抗-半乳糖苷酶多克隆抗体对融合蛋白进行部分免疫沉淀纯化，并在不同组成的脂质体存在下研究其[H-3]叠氮嘧啶结合特性。纯化后的p -糖蛋白重组为脂质体，能够与[H-3]叠氮多啶结合。当脂质体中胆固醇含量增加到重量比为20%时，刺激部分纯化融合蛋白的特异性结合活性，胆固醇含量增加越高，结合活性越低。与长春花碱竞争可明显降低这种结合。在刺激特异性结合方面，豆甾醇效果较差，麦角甾醇效果最差。
We fused P-glycoprotein with beta-galactosidase at the C-terminus aiming to study the mechanism of drug binding of P-glycoprotein in reconstitution experiments. Expression of the fusion protein in NIH 3T3 cells conferred a multidrug-resistant phenotype, suggesting that beta-galactosidase fusion at the C-terminus does not affect the functions of P-glycoprotein. The fusion protein was partially purified by simple immunoprecipitation with anti-beta-galactosidase polyclonal antibody, and its [H-3]azidopine binding property was investigated in the presence of various compositions of liposomes. The purified P-glycoprotein, after reconstitution into liposomes, was capable of binding [H-3]azidopine. When the cholesterol content of liposomes was increased to a weight ratio of 20%, the specific binding activity of the partially purified fusion protein was stimulated, and when the cholesterol content was increased higher, the binding activity decreased. The binding was specifically decreased by competition with vinblastine. Stigmasterol was less effective, and ergosterol was the least effective in stimulating the specific binding.