BIOCHEMICAL INVESTIGATION OF P-GLYCOPROTEIN

P-糖蛋白的生化研究

• 批准号: 2187790
• 负责人: ALAN E. SENIOR
• 金额: $ 19.79万
• 依托单位: UNIVERSITY OF ROCHESTER
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-01-01 至 1996-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2187790
• 关键词: 6 thioguanine; 6 thiopurine; CHO cells; P glycoprotein; adenosinetriphosphatase; affinity labeling; binding proteins; biochemistry; catalyst; fluorescent dye /probe; glycoprotein structure; liposomes; maleimides; membrane transport proteins; molecular site; multidrug resistance; nucleotide analog; nucleotides; phosphatase inhibitor; protein purification; protein reconstitution; structural biology

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.

多重耐药是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耐药性 化疗剂。 它通常涉及 P- 的过度表达 糖蛋白，一种位于质膜上的蛋白质，约有 1280 个氨基酸 酸，由两个重复的半部分组成，每个半部分包含六个 预测的跨膜螺旋和一个核苷酸结合位点。 那里 有力的证据表明 P-糖蛋白以 ATP 依赖性方式发挥作用 将药物和多种其他疏水性化合物排除在外 细胞。 我们和其他人已经确定 P-糖蛋白显示 大量药物刺激的 ATP 酶活性，以及​​最广泛的 当前的假设是 P-糖蛋白充当 ATP- 驱动药物流出泵。 我们最近培育了一种中国仓鼠卵巢细胞系，可以生长 剧烈且组成型地过表达 P-糖蛋白，高达 32% (w/w) 在分离的质膜中。 使用该系统作为源 材料，我们建议进行彻底的生化调查 P-糖蛋白，包括纯化至同质、重构 在脂质体中，运输特性的表征，详细研究 核苷酸结合位点和 ATP 水解的催化，以及 催化抑制剂的表征。 此类基础知识对于设计方法非常宝贵 使细胞中的P-糖蛋白失效并克服多药耐药性 患者。