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。是 对跨上皮转运（如肾脏）和离子转运至关重要 支持肌肉和神经电兴奋性的梯度。是 心脏中的强心苷受体，许多研究指出， 在高血压中的作用。它的催化亚基属于一个离子家族 运输ATP酶。其中第一种的晶体结构，即Ca 2 + ATP， 肌浆网，最近被确定，提供了新的见解 离子传输的可能机制我们的理论分析 Na，K-ATPase与Ca 2 +-ATPase的比对表明，某些过去的假设 对Na，K-ATPase结构的认识是错误的，对Ca ~（2+）-ATPase结构的认识是错误的 提供了一个可行的框架，以测试有关钠，钾-ATP酶的具体假设 结构这一提议将检验Na，K-ATP酶α 亚基采用相同的折叠作为Ca 2 +-ATP酶，使用单克隆抗体， 用定位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纯化。 Na，K ATP酶的结晶。我们也会把注意力集中在这两个 Na，K-ATP酶亚基，在Ca 2 + -ATP酶中没有对应物：β 和γ亚基。我们将测试具体的假设，在哪里以及如何这些 亚基与α亚基缔合，使用天然和 诱变酶我们发现γ亚基调节了 Na，K-ATP酶的生理学显著性质，其对Na+的亲和力 和K+，我们将利用这一点来绘制 γ-饱和诱变。我们将检验这样一个假设， γ，就像类似但不同的受磷蛋白，调节 Ca ~（2+）-ATP酶在细胞膜上发生可逆的低聚反应。总之，a 结合蛋白质化学、杂交瘤技术和分子生物学， 方法将被用来研究基本血浆的结构 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.