BIOCHEMICAL INVESTIGATION OF P-GLYCOPROTEIN

多药耐药性是癌症患者在治疗过程中遇到的一种情况。 使肿瘤对多种细胞毒性抗癌药物产生耐药性 化疗剂。 它通常涉及P- 糖蛋白是一种位于质膜的蛋白质，约1280个氨基酸 酸，由两个重复的一半组成，每个一半含有六个 预测的跨膜螺旋和一个核苷酸结合位点。 那里 是P-糖蛋白以ATP依赖性方式起作用的有力证据， 排除药物和各种其他疏水化合物， 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ATP酶活性，和最广泛的- 目前认为的假设是P-糖蛋白作为ATP- 驱动的药物外排泵。 我们最近建立了一个中国仓鼠卵巢细胞系， 强烈和组成型过表达P-糖蛋白，高达32% （w/w）在分离的质膜中。 以这个系统为源 材料，我们建议进行彻底的生物化学调查 P-糖蛋白，包括纯化至均一，复溶 在脂质体中，表征转运性质，详细研究 核苷酸结合位点和ATP水解的催化作用，以及 催化抑制剂的表征。 这类基本知识在设计方法方面将是无价的， 使细胞中的P-糖蛋白失活，克服细胞的多药耐药性， 患者

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.