Structure of Na,K-ATPase: monoclonal antibody probes

描述(申请人提供)：Na，K-ATPase(P型离子泵) 在几乎所有动物细胞中催化Na+和K+的主动转运。它是 对跨上皮运输是必不可少的，如在肾脏中，以及对离子 支持肌肉和神经的电兴奋性的梯度。它是 心脏中的正性心脏糖苷受体，许多研究指出 在高血压中扮演了一个角色。它的催化亚基属于一个离子家族 运输ATPase。其中第一个的晶体结构是钙离子--三磷酸腺苷 肌浆网，最近被确定，提供了新的见解 研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的结构是错误的，而钙-ATPase的结构是错误的 为测试有关Na，K-ATPase的特定假说提供了一个可行的框架 结构。这一提议将检验Na，K-ATPaseα 亚基采用与钙-ATPase相同的折叠，使用单抗 以映射的表位作为结构探针。抗Na，K-ATPase抗体 胞外表面将被产生，以便于纯化和最终 Na，K-ATPase的结晶。我们还将重点关注这两个方面。 Ca~(2+)-ATPase中没有对应的Na，K-ATPase亚基：β 和伽马亚基。我们将测试关于这些在哪里以及如何实现的特定假设 亚基与阿尔法亚单位相关联，使用原生和 诱变酶。我们发现伽马亚基的调节作用最强 Na，K-ATPase的生理特性及其与Na+的亲和力 和K+，我们将利用这一点来绘制基本的结构特征 通过饱和诱变产生伽马。我们将检验这样一种假设，即通过 伽马，就像类似但不同的磷蛋白蛋白，它调节 Ca~(2+)-ATPase在膜上发生可逆的齐聚反应。总而言之，一个 蛋白质化学、杂交瘤技术和分子的结合 这些方法将被用来研究基本等离子体的结构 膜蛋白。

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.