BIOGENESIS AND FUNCTION OF THE (NA++K+) - ATPASE SUBUNIT

(Na++K+)-ATPase(钠泵)是一种完整的膜蛋白，其 函数是建立跨等离子体的离子梯度的关键 薄膜。钠泵由两个亚单位组成：较大的α- 携带ATP催化活性和哇巴因结合位点的亚基，以及 具有可疑作用的较小的糖蛋白(β-)亚单位 将α-亚基从内质网运输到质膜。 然而，人们对其结构、功能和组装知之甚少 亚基之间的关系。这项提案的目标是界定 α亚基结构功能所需的临界区 以及它与β-亚基的相互作用。具体目标是(1) 定义阿尔法亚单位的胞外区和胞内区，(2) 识别抑制物结合域，如哇巴因结合位点， 已知定位于细胞外域，以及(3)确定 结构域(S)在与β-亚基组装所需的α-亚基中。 第一个目标将通过以下方式实现：(1)监测增加 低聚糖与人工引入的N-连接糖基化反应 定位于α亚基的信号(Asn-X-Thr(或-Ser)) 诱变，以及(Ii)检测定点单抗的结合 使细胞通透性前后的阿尔法亚单位抗体。这个 第二和第三个目标将通过(I)测试哇巴因- 部分缺失突变体的结合和组装能力 和(Ii)检测哇巴因敏感功能和 钙泵/钠泵α-亚基嵌合体的组装能力 分子。大多数问题将在基因转移中得到解决。 野生型和/或突变的禽类基因在E. 大肠杆菌或哺乳动物细胞中，基因产物由禽类检测到- 特异性的单抗。代谢标记实验和 将使用免疫荧光显微镜检查稳定性和 哺乳动物表达的禽类分子的细胞内定位 细胞。ATP-水解性、离子转运和配基(抗体和 抑制剂)结合试验将用于监测结构和 基因操纵分子的功能。这些研究的结果 研究将为理解生物发生和 钠泵亚单位的功能。

The (Na+ + K+)-ATPase (sodium-pump) is an integral membrane protein whose function is critical in establishing ion gradients across the plasma membrane. The sodium-pump consists of two subunits: the larger alpha- subunit carrying ATP-catalytic activity and an ouabain-binding site, and the smaller glycoprotein (Beta-) subunit with a suspected role of transporting the alpha-subunit from the ER to the plasma membrane. However, little is known regarding the structure, function and assembly relationships between the subunits. The goal of this proposal is to define critical regions in the alpha-subunit required for its structure-function and for its interaction with the beta-subunit. The specific aims are (1) to define extracellular and intracellular domains in the alpha-subunit, (2) to identify inhibitor binding domains, such as ouabain-binding sites, which are known to localize on the extracellular domain, and (3) to determine the domain(s) in the alpha-subunit required for assembly with the beta-subunit. The first aim will be accomplished by (i) monitoring the addition of oligosaccharides onto artificially-introduced N-linked glycosylation signals (Asn-X-Thr(or-Ser)) in the alpha-subunit by site-directed mutagenesis, and (ii) examining the binding of site-directed monoclonal antibodies to the alpha-subunit before and after permeabilizing cells. The second and third aims will be accomplished by (i) testing the ouabain- binding and the assembling ability of the partial-deletion mutants of the alpha-subunit, and (ii) examining the ouabain-sensitive function and assembling ability of the calcium-pump/sodium-pump alpha-subunit chimeric molecules. Most of the questions will be addressed in gene transfer experiments in which wild-type and/or mutated avian cDNAs are expressed E. coli or in mammalian cells and the gene products are detected by avian- specific monoclonal antibodies. Metabolic labeling experiments and immunofluorescent microscopy will be employed to examine the stability and intracellular localization of the avian molecules expressed in mammalian cells. ATP-hydrolysis, ion-transport, and ligand (antibodies and inhibitors) binding assays will be used to monitor the structure and function of genetically manipulated molecules. The results from these studies will provide a structural bases for understanding biogenesis and function of the sodium-pump subunits.