BIOCHEMICAL INVESTIGATION OF P GLYCOPROTEIN

描述：多药耐药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 抗癌化疗药物。它经常涉及到过度表达 P糖蛋白(Pgp)是一种由1280个氨基酸组成的质膜蛋白， 由两个相关的部分组成，每个部分包含六个预测 跨膜螺旋和一个核苷酸结合位点。有坚定的信念 PGP以一种依赖于ATP的方式作用于排除药物和 细胞中广泛存在的其他疏水化合物。调查员的 Lab和其他人已经确定PGP显示出大量的 药物刺激的ATPase活性，也是目前被最广泛认为的 模型认为PGP是一种由ATP驱动的药物外排泵。调查员已经 产生了一个过表达Pgp的中国仓鼠卵巢细胞系。二 已开发出用于PGP生化研究的定义系统 从这些细胞中：1)分离的血浆膜脂，相当于 富含PGP(高达32%w/w)和2)纯化、重组的PGP。一个 将开发进一步的系统，以允许将诱变和生化相结合 分析。这项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， 色氨酸作为荧光探针的特异性插入，突变 对建议的催化循环和直接结构进行分析、测试 用扫描力显微镜进行分析。这方面的基本知识将是 在设计方法禁用细胞中的P-糖蛋白和克服 患者的多药耐药

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