STRUCTURE OF NA, K-ATPASE--MONOCLONAL ANTIBODY PROBES

NA、K-ATP酶的结构--单克隆抗体探针

• 批准号: 2735105
• 负责人: Kathleen J Sweadner
• 金额: $ 26.45万
• 依托单位: MASSACHUSETTS GENERAL HOSPITAL
• 依托单位国家: 美国
• 财政年份: 1987
• 资助国家: 美国
• 起止时间: 1987-07-01 至 2001-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2735105
• 关键词: animal genetic material tag; antibody specificity; blocking antibody; chemical binding; hybridomas; isozymes; laboratory rat; molecular cloning; molecular site; monoclonal antibody; protein structure; sodium potassium exchanging ATPase

DESCRIPTION (Adapted from applicant's abstract): In this application, the applicant is seeking support to continue studies of the structure of the Na+/K+-translocating ATPase of animal cell plasma membranes. The NaK-ATPase, a member of the P-type ATPase family of ion-translocating ATPases, catalyzes the active transport of Na+ and K+ and is the receptor for inotropic cardiac glycosides in the heart. Much is known about its enzymatic mechanism, but there are major gaps in understanding its structure, including that of its cardiac glycoside binding site. Antibodies and other proteins that interact with the native, unmodified protein have been used to test models of the transmembrane topology of this transporter. Dr. Sweadner now proposes to obtain new monoclonal antibodies against the sites where the structure is most uncertain, the extracellular surface and the C-terminal third of the a subunit. The antibodies will be characterized by determining whether they bind to the intracellular or extracellular surfaces, by mapping their epitopes, and by measuring any effects on the function of the enzyme. The properties of each antibody will determine which of four principal scientific aims it will be used for. First, mapped antibodies will be used to discriminate between different hypothetical folding models to determine the enzyme's topology. Second, the hypothesis will be tested that the C-terminal third of the a subunit is a folding domain with unique, conformation-dependent structural lability. Thermal denaturation, coupled with the use of antibodies as probes of native and denatured structure, will be used to define the structural domains and build a model of active transport. Third, antibodies against extracellular sites will be used to probe the structure of the cardiac glycoside binding site, to determine whether it is superficial or buried in a pocket. Finally, extracellular antibodies that bind preferentially to the native enzyme will be evaluated for binding to the enzyme in detergent. The variable domains (Fv) will be cloned from the hybridomas and expressed in E. coli with an affinity tag. The modified Fv fragments will be tested for enzyme purification and stabilization, with the long-term goal of co-crystallizing the antibody fragment with the NaK-ATPase. In sum, a combination of hybridoma technology, protein chemistry, and molecular approaches will be used to investigate the structure of this important plasma membrane protein.

描述（改编自申请人的摘要）：在本申请中， 申请人正在寻求支持，以继续研究 动物细胞质膜Na +/K+转运ATP酶。 的 NaK-ATP酶是P型ATP酶家族中的一员， ATP酶催化Na+和K+的主动转运，是 心脏中的强心苷 很多人都知道， 酶的机制，但在理解其 结构，包括其强心苷结合位点。 抗体 和其他与天然未修饰蛋白质相互作用的蛋白质， 已用于测试该转运蛋白的跨膜拓扑结构模型。 博士斯韦德纳现在建议获得新的单克隆抗体， 结构最不确定的部位，细胞外表面和 a亚基的C端第三个。 抗体将被表征 通过确定它们是否与细胞内或细胞外 表面，通过绘制它们的表位，并通过测量对它们的任何影响， 酶的功能。 每种抗体的特性将决定 它将被用于四个主要科学目的中的哪一个。 首先，映射 抗体将用于区分不同的假设 折叠模型来确定酶的拓扑结构。 第二，假设 将测试a亚基的C末端第三个是折叠的 结构域具有独特的构象依赖性结构不稳定性。 热 变性，再加上使用抗体作为天然和 变性结构，将用于定义结构域和构建 一种主动交通模式。 第三，针对细胞外位点的抗体 将用于探测强心苷结合位点的结构， 以确定它是表面的还是埋在口袋里。 最后， 优先结合天然酶的细胞外抗体 评估与洗涤剂中酶的结合。 可变结构域 (Fv)将从杂交瘤中克隆并在E.大肠杆菌， 亲和标记。 将测试修饰的Fv片段的酶活性。 纯化和稳定，长期目标是共结晶 将抗体片段与NaK-ATPase结合。 总之， 杂交瘤技术、蛋白质化学和分子方法将成为 用于研究这种重要的质膜蛋白的结构。