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

(NA K )-ATP酶亚基的生物发生和功能

• 批准号: 3303516
• 负责人: KUNIO TAKEYASU
• 金额: $ 17.06万
• 依托单位: UNIVERSITY OF VIRGINIA
• 依托单位国家: 美国
• 财政年份: 1990
• 资助国家: 美国
• 起止时间: 1990-04-01 至 1995-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3303516
• 关键词: Escherichia coli; adenosine triphosphate; adenosinetriphosphatase; calcium transporting ATPase; catalyst; cell membrane; chemical structure function; complementary DNA; cytoplasm; endoplasmic reticulum; extracellular; fluorescence microscopy; glycoproteins; glycosylation; immunofluorescence technique; ion transport; mannose; membrane proteins; monoclonal antibody; oligosaccharides; ouabain; radiotracer; sarcoplasmic reticulum; site directed mutagenesis; transfection; tritium

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.

(Na+ + K+)-ATP酶（钠泵）是一种完整的膜蛋白，其 函数对于建立等离子体中的离子梯度至关重要 膜。 钠泵由两个亚基组成：较大的 α- 携带 ATP 催化活性和哇巴因结合位点的亚基，以及 较小的糖蛋白（β-）亚基，其作用可疑 将α亚基从内质网转运至质膜。 然而，人们对其结构、功能和组装知之甚少。 子单元之间的关系。 该提案的目标是定义 α亚基中其结构功能所需的关键区域 以及它与β亚基的相互作用。 具体目标是（1） 定义 α 亚基中的细胞外和细胞内结构域，(2) 识别抑制剂结合域，例如哇巴因结合位点， 已知定位于细胞外结构域，并且（3）确定 与 β 亚基组装所需的 α ​​亚基中的结构域。 第一个目标将通过以下方式实现：(i) 监测添加 人工引入的 N-连接糖基化寡糖 α-亚基中的信号（Asn-X-Thr（或-Ser））通过位点定向 诱变，以及（ii）检查定点单克隆抗体的结合 透化细胞之前和之后针对α亚基的抗体。 这 第二个和第三个目标将通过（i）测试哇巴因来实现 部分缺失突变体的结合和组装能力 α-亚基，以及（ii）检查哇巴因敏感功能和 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 表达野生型和/或突变型禽类 cDNA 的实验 E. 大肠杆菌或哺乳动物细胞中的基因产物可通过禽类检测到 特异性单克隆抗体。 代谢标记实验和 免疫荧光显微镜将用于检查稳定性和 在哺乳动物中表达的鸟类分子的细胞内定位 细胞。 ATP 水解、离子传输和配体（抗体和 抑制剂）结合测定将用于监测结构和 基因操纵分子的功能。 这些结果 研究将为理解生物发生和 钠泵亚基的功能。