BIOCHEMICAL INVESTIGATION OF P GLYCOPROTEIN

P 糖蛋白的生化研究

• 批准号: 2857186
• 负责人: ALAN E. SENIOR
• 金额: $ 23.49万
• 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 2000-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2857186
• 关键词: CHO cells; P glycoprotein; active sites; adenosinetriphosphatase; affinity labeling; atomic force microscopy; binding sites; catalyst; fluorescent dye /probe; glycoprotein structure; membrane transport proteins; molecular site; multidrug resistance; nucleotide analog; protein purification; protein reconstitution; protein structure function; structural biology; tryptophan

DESCRIPTION: Multi-drug resistance is a situation encountered in cancer patients in which the tumor becomes resistant to a variety of cytotoxic anti-cancer chemotheraputic agents. It often involves overexpression of P-glycoprotein (Pgp), a plasma membrane protein of 1280 amino acids, composed of two related halves, each of which contains six predicted transmembrane helices and one nucleotide binding site. There is firm evidence that Pgp acts in an ATP-dependent manner to exclude drugs and a wide range of other hydrophobic compounds from cells. The investigator's lab and others have established that Pgp displays substantial drug-stimulated ATPase activity, and the most widely considered current model is that Pgp is an ATP-driven drug efflux pump. The investigator has generated a Chinese hamster ovary cell line that over-expresses Pgp. Two defined systems for biochemical investigation of Pgp have been developed from these cells: 1) isolated plasma membanes which are considerably enriched in Pgp (up to 32 percent w/w) and 2) purified, reconstituted Pgp. A further system will be developed to allow combined mutagenic and biochemical analyses. The aim of this proposal is to characterize the structure and function of the Pgp catalytic sites. Approaches include covalent labeling, specific insertion of tryptophans as fluorescent probes, mutational analyses, tests of the proposed catalytic cycle and direct structural analysis by scanning force microscopy. Basic knowledge of this kind will be invaluable in devising ways to disable P-glycoprotein in cells and overcome multidrig resistanmce in patients

描述：多重耐药性是癌症中遇到的一种情况 肿瘤对多种细胞毒药物产生抗药性的患者 抗癌化疗剂。 它通常涉及过度表达 P-糖蛋白（Pgp），一种由 1280 个氨基酸组成的质膜蛋白， 由两个相关的半部分组成，每半部分包含六个预测 跨膜螺旋和一个核苷酸结合位点。 有坚定的 有证据表明 Pgp 以 ATP 依赖性方式发挥作用以排除药物和 来自细胞的多种其他疏水性化合物。 调查员的 实验室和其他人已经确定 Pgp 显示出显着的 药物刺激的 ATP 酶活性，以及​​目前最广泛考虑的 模型是 Pgp 是 ATP 驱动的药物外排泵。 调查员有 产生了过度表达 Pgp 的中国仓鼠卵巢细胞系。二 已开发出用于 Pgp 生化研究的明确系统 从这些细胞中：1）分离的质膜，其相当大 富含 Pgp（高达 32% w/w）和 2) 纯化、重构的 Pgp。一个 将开发进一步的系统以允许结合诱变和生化 分析。 该提案的目的是描述结构和 Pgp 催化位点的功能。 方法包括共价标记、 色氨酸的特异性插入作为荧光探针、突变 所提出的催化循环和直接结构的分析、测试 通过扫描力显微镜进行分析。 此类基础知识将 对于设计使细胞中的 P-糖蛋白失效并克服 患者多药耐药