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: 6638259
• 负责人: Kathleen J Sweadner
• 金额: $ 34.6万
• 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 2005-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6638259
• 关键词: Escherichia coli; antibody specificity; binding sites; cardiac glycosides; cell line; cell membrane; crosslink; crystallization; gene mutation; hybridomas; immunologic substance development /preparation; immunologic techniques; intermolecular interaction; laboratory mouse; laboratory rat; model design /development; monoclonal antibody; physical model; polymerization; protein folding; protein purification; protein sequence; protein structure function; sodium potassium exchanging ATPase; structural biology; surface property

DESCRIPTION ( provided by applicant): The Na,K-ATPase (a P-type ion pump) catalyzes active transport of Na+ and K+ in almost all animal cells. It is essential for transepithelial transport, such as in the kidney, and for the ion gradients that support electrical excitability in muscle and nerve. It is the receptor for inotropic cardiac glycosides in the heart, and many studies point to a role in hypertension. Its catalytic subunit belongs to a family of ion transport ATPases. The crystal structure of the first of these, the Ca2+ ATP of sarcoplasmic reticulum, has recently been determined, providing new insight into the possible mechanism of ion transport. Our theoretical analysis of Na,K-ATPase aligned with Ca2+-ATPase suggests that certain past assumptions about Na,K-ATPase structure were wrong, and that the Ca2+-ATPase structure provides a workable framework for testing specific hypotheses about Na,K-ATPase structure. This proposal will test the hypothesis that the Na,K-ATPase alpha subunit adopts the same fold as the Ca2+-ATPase, using monoclonal antibodies with mapped epitopes as structural probes. Antibodies against the Na,K-ATPase extracellular surface will be produced to facilitate purification and eventual crystallization of the Na,K ATPase. We will also focus our attention on the two Na,K-ATPase subunits that have no counterpart in the Ca2+ - ATPase: the beta and gamma subunits. We will test specific hypotheses about where and how these subunits associate with the alpha subunit, using cross-linking of native and mutagenized enzyme. We have found that the gamma subunit modulates the most physiologically significant properties of the Na,K-ATPase, its affinity for Na+ and K+, and we will exploit that to map the essential structural features of gamma by saturation mutagenesis. We will test the hypothesis that regulation by gamma, like the similar but distinct phospholamban protein that modulates the Ca2+-ATPase, entails reversible oligomerization in the membrane. In sum, a combination of protein chemistry, hybridoma technology, and molecular approaches will be used to investigate the structure of an essential plasma membrane protein.

描述（由申请人提供）：Na,K-ATP酶（P型离子泵） 催化几乎所有动物细胞中 Na+ 和 K+ 的主动转运。这是 对于跨上皮运输（例如在肾脏中）和离子运输至关重要 支持肌肉和神经电兴奋性的梯度。它是 心脏中正性肌力强心苷的受体，许多研究表明 对高血压有一定的作用。其催化亚基属于离子家族 转运ATP酶。第一个的晶体结构，Ca2+ ATP 最近确定了肌浆网，提供了新的见解 探究离子传输的可能机制。我们的理论分析 Na,K-ATPase 与 Ca2+-ATPase 一致表明过去的某些假设 关于Na,K-ATPase结构错误，Ca2+-ATPase结构 提供了一个可行的框架来测试有关 Na,K-ATP 酶的特定假设 结构。该提案将检验 Na,K-ATPase α 的假设 亚基采用与 Ca2+-ATPase 相同的折叠，使用单克隆抗体 以映射的表位作为结构探针。 Na,K-ATP 酶抗体 将产生细胞外表面以促进纯化和最终 Na,K ATP酶的结晶。我们也将重点关注这两个方面 Na,K-ATP 酶亚基在 Ca2+ 中没有对应物 - ATP 酶：β 和伽马亚基。我们将测试有关这些内容在何处以及如何进行的具体假设 亚基与α亚基结合，利用天然和亚基的交联 诱变酶。我们发现伽玛亚基的调节作用最强 Na,K-ATP 酶的生理学重要特性及其对 Na+ 的亲和力 和 K+，我们将利用它来绘制基本结构特征 γ饱和诱变。我们将检验以下假设：监管 γ，类似于类似但不同的受磷蛋白，调节 Ca2+-ATP酶需要在膜中进行可逆的寡聚化。总而言之，一个 蛋白质化学、杂交瘤技术和分子生物学的结合 方法将用于研究基本等离子体的结构 膜蛋白。