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Mechanism of the ATP-dependent membrane transporter

ATP依赖性膜转运蛋白的机制

基本信息

批准号：
02660091
负责人：
UEDA Kazumitsu
金额：
$ 1.34万
依托单位：
Kyoto University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1990
资助国家：
日本
起止时间：
1990 至 1991
项目状态：
已结题

项目摘要

The ATP-dependent membrane transport proteins of eukaryotes includes P-glycoprotein and cystic fibrosis transmembrane conductance regulator(CFIR). P-glycoprotein functions as an ATP-dependent efflux pump of anticancer drugs and is involved in multidrug resistance of cancer cells which is a major obstacle of cancer chemotherapy. CFTR is involved in cystic fibrosis, the most common lethal, recessively inherited disease among Caucasians. This family of membrane transport proteins has a high degree of symmetry between its two halves, each composed of six membrane-spanning alpha-helices and a hydrophilic region containing an ATP-binding sites. To investigate the involvement of the two ATP-binding domains in the ATPase activity of P-glycoprotein, fulllength and the 5'-half of human MDR1 cDNA, which encodes P-glycoprotein, were fused with the E. coli lacZ gene and expressed in NIH3T3 cells. Immunoprecipitated full-length P-glycoprrotein-beta-galactosidase showed ATPase activity in the presenc … More e of phospholipids suggesting that stabilization of the transmembrane domains is necessary for ATP hydrolysis. N-terminal half P-glycoprotein-beta-galactosidase also showed ability to hydrolyze ATP but with slightly lower specific activity. Both ATPase activities showed similar characteristics when the effect of several inhibitors was analyzed, indicating that the N-terminal ATP-binding domain contains all residues necessary to hydrolyze ATP without interacting with the C-terminal ATP-binding domain. Then, MDRI cDNA fragment encoding the NH_2- and COOH-cytoplasmic domains of P-glycoprotein(N-half and C-half, respectively)were isolated and fused with E. coli lacZ gene. The fusion genes were expressed in E. coli strain BL21 using the T7 RNA polymerase expression system. The fusion proteins were purified by affinity chromatography against the beta-galactosidase moiety to near homogeneity. Both ATP-binding doipains of P-glycoprotein showed ATPase activity with similar Km values but the Vmax for the activity of C-half was about half that of N-half. This difference in turnover rates suggests different roles in the transport process. Less

真核生物的ATP依赖性膜转运蛋白包括P-糖蛋白和囊性纤维化跨膜传导调节因子（CFIR）。P-糖蛋白作为ATP依赖的抗癌药物外排泵发挥作用，并参与肿瘤细胞的多药耐药，是肿瘤化疗的主要障碍。CFTR与囊性纤维化有关，囊性纤维化是白种人中最常见的致死性复发性遗传性疾病。这个膜转运蛋白家族在其两个半部分之间具有高度对称性，每个半部分由六个跨膜α-螺旋和含有ATP结合位点的亲水区域组成。为了研究P-糖蛋白ATP酶活性与两个ATP结合区的关系，将人MDR 1全长cDNA和5 ′端cDNA分别与E. colilacZ基因在NIH 3 T3细胞中表达。免疫沉淀的全长P-糖蛋白-β-半乳糖苷酶显示ATP酶活性， ...更多信息表明跨膜结构域的稳定是ATP水解所必需的。N-末端半P-糖蛋白-β-半乳糖苷酶也显示出水解ATP的能力，但比活性略低。两种ATP酶活性显示出相似的特性时，几种抑制剂的影响进行了分析，表明N-末端ATP结合结构域包含所有必要的残基水解ATP，而不与C-末端ATP结合结构域相互作用。然后，分离编码P-糖蛋白NH_2-和COOH-胞浆区（分别为N-半和C-半）的MDRI cDNA片段，并与E.大肠杆菌lacZ基因。融合基因在E. coli菌株BL 21中进行表达。通过针对β-半乳糖苷酶部分的亲和层析将融合蛋白纯化至接近同质。P-糖蛋白的两种ATP结合蛋白酶都具有相似的Km值，但C-半的Vmax约为N-半的一半。周转率的这种差异表明在运输过程中的不同作用。少