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: 2406664
• 负责人: Kathleen J Sweadner
• 金额: $ 27.01万
• 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/2406664
• 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， 申请者正在寻求支持，以继续研究 动物细胞质膜的Na+/K+-ATPase转位。这个 离子转运P型ATPase家族成员NAK-ATPase ATPase，催化Na+和K+的主动运输，是受体 心脏中的正性心脏糖苷类药物。关于它的许多信息都是众所周知的 酶的作用机制，但对其认识存在较大差距。 结构，包括其心脏糖苷结合部位。抗体 和其他与天然的、未经修饰的蛋白质相互作用的蛋白质 被用来测试这种传输器的跨膜拓扑模型。 斯威德纳博士现在提议获得新的针对该病毒的单抗 结构最不确定的部位，细胞外表面和 A亚基的第三个C-末端。抗体的特征将是 通过确定它们是结合到细胞内还是细胞外 通过映射它们的表位，并通过测量对 酶的功能。每种抗体的性质将决定 它将用于四个主要科学目标中的哪一个。第一，绘制地图 抗体将被用来区分不同的假设 折叠模型以确定酶的拓扑结构。第二，假设 将检测到的C末端的第三个a亚基是一个折叠的 结构域具有独特的、依赖于构象的结构不稳定性。热能 变性，加上使用抗体作为天然和 变性结构，将用于定义结构域并构建 一种主动运输的模式。第三，针对胞外部位的抗体 将用于探测心脏糖苷结合部位的结构， 以确定它是表面的还是埋在口袋里的。最后， 优先与天然酶结合的细胞外抗体将 被评估与洗涤剂中的酶结合。可变区 (Fv)将从杂交瘤中克隆并在大肠杆菌中表达 亲和力标签。将对修改后的FV片段进行酶检测 提纯和稳定，长期目标是共结晶 与NAK-ATPase结合的抗体片段。总而言之，这是一种 杂交瘤技术、蛋白质化学和分子方法将是 用于研究这种重要的质膜蛋白的结构。