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：多药耐药性是癌症中遇到的一种情况 肿瘤对多种细胞毒性药物产生耐药性的患者 抗癌化学治疗剂。 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， 由两个相关的半部组成，每个半部包含六个预测的 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依赖性方式起作用，以排除药物， 广泛的其他疏水化合物从细胞。 研究者 实验室和其他机构已经证实，Pgp显示出大量的 药物刺激的ATP酶活性，以及最广泛认为的电流 Pgp是ATP驱动的药物外排泵。 研究者已 产生过表达Pgp的中国仓鼠卵巢细胞系。两 已经开发了用于Pgp的生物化学研究的限定系统 从这些细胞中：1）分离的质膜， 富含Pgp（高达32%w/w）和2）纯化的重构Pgp。一 将开发进一步的系统，以允许结合诱变和生物化学 分析。 本建议的目的是确定结构的特点， Pgp催化位点的功能。 方法包括共价标记， 作为荧光探针的特异性插入的突变的 分析、测试拟议的催化循环和直接结构 通过扫描力显微镜分析。 这类基本知识将是 在设计使细胞中的P-糖蛋白失活并克服 患者多药耐药