BIOCHEMICAL INVESTIGATION OF P-GLYCOPROTEIN

多药耐药是中国癌症患者遇到的一种情况 肿瘤对多种细胞毒性抗癌药物产生抗药性 化疗药物。它通常涉及P-蛋白的过度表达 糖蛋白，一种位于质膜上的蛋白质，约1280个氨基酸 酸，由两个重复的一半组成，每个一半包含六个 预测的跨膜螺旋和一个核苷酸结合位点。那里 是否有确凿证据表明P-糖蛋白以ATP依赖的方式作用于 排除药物和各种各样的其他疏水化合物 细胞。我们和其他人已经确定P-糖蛋白显示 大量的药物刺激的ATPase活性，以及最广泛的- 目前的假设是，P-糖蛋白作为一种ATP- 驱动的药物外排泵。 我们最近培育了一个中国仓鼠卵巢细胞系，它可以生长 强势和结构性过表达P-糖蛋白，高达32% (W/W)在分离的质膜中。以此系统为源 材料，我们建议进行彻底的生化调查 P-糖蛋白，涉及纯化到均一，重组 在脂质体中，传输特性的表征，详细研究 核苷酸结合部位和ATP水解酶的催化作用，以及 催化抑制剂的表征。 这方面的基本知识将对设计出 去细胞P-糖蛋白和克服多药耐药 病人。

Multidrug-resistance is a situation encountered in cancer patients in which the tumor becomes resistant to a variety of cytotoxic anti-cancer chemotherapeutic agents. It often involves overexpression of P- glycoprotein, a plasma-membrane located protein of around 1280 amino acids, composed of two duplicated halves, each of which contains six predicted transmembrane helices and one nucleotide-binding site. There is firm evidence that P-glycoprotein acts in an ATP-dependent manner to exclude drugs and a wide variety of other hydrophobic compounds from cells. We and others have established that P-glycoprotein displays substantial drug-stimulated ATPase activity, and the most widely- considered current hypothesis is that P-glycoprotein acts as an ATP- driven drug-efflux pump. We have recently generated a Chinese hamster ovary cell line that grows vigorously and constitutively overexpress P-glycoprotein, up to 32% (w/w) in isolated plasma membranes. Using this system as source material, we propose to carry out a thorough biochemical investigation of P-glycoprotein, involving purification to homogeneity, reconstitution in liposomes, characterization of transport properties, detailed study of the nucleotide binding sites and the catalysis of ATP hydrolysis, and characterization of inhibitors of catalysis. Basic knowledge of this kind will be invaluable in devising ways to disable P-glycoprotein in cells and overcome multidrug-resistance in patients.