Mechanism of the ATP-dependent membrane transporter

真核生物依赖于ATP的膜转运蛋白包括P-糖蛋白和囊性纤维化跨膜电导调节因子(CFIR)。P-糖蛋白是一种依赖于ATP的抗癌药物外排泵，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，囊性纤维化是高加索人中最常见的致死性隐性遗传病。这个膜转运蛋白家族在其两半之间具有高度的对称性，每一半由六个跨膜的α-螺旋和一个包含一个ATP结合位点的亲水区组成。为探讨这两个三磷酸腺苷结合区在P-糖蛋白ATPase活性中的作用，将编码P-糖蛋白的人mdr1基因的全长和5‘端与大肠杆菌LacZ基因融合，并在NIH3T3细胞中表达。免疫沉淀全长β-糖蛋白-β-半乳糖苷酶在存在的…中显示ATPase活性更多的磷脂表明，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-半乳糖苷酶也表现出对ATP的水解性，但比活力略低。当分析几种抑制剂的作用时，这两种ATPase活性表现出相似的特征，表明N-末端的ATP结合结构域包含了水解ATP所需的所有残基，而不与C-末端的ATP结合结构域相互作用。然后，分离出编码P-糖蛋白NH2-和COOH-胞质结构域的MDRI基因片段(N-Half和C-Half)，并将其与E.coliLacZ基因融合。利用T7RNA聚合酶表达系统在大肠杆菌BL21中表达融合基因。融合蛋白通过亲和层析纯化，达到接近均一的目的蛋白。P-糖蛋白的两个ATP结合区都显示出相似的Km值的ATPase活性，但C-Half的Vmax约为N-Half的一半。这种周转率的差异表明，在运输过程中扮演着不同的角色。较少

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