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

(NA K ) - ATP酶亚基的生物发生和功能

• 批准号: 3303519
• 负责人: KUNIO TAKEYASU
• 金额: $ 15.34万
• 依托单位: OHIO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-07-01 至 1995-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3303519
• 关键词: Escherichia coli; adenosine triphosphate; catalyst; cell membrane; chemical structure function; complementary DNA; cytoplasm; endoplasmic reticulum; extracellular; fluorescence microscopy; glycoproteins; glycosylation; immunofluorescence technique; ion transport; mannose; membrane proteins; membrane transport proteins; monoclonal antibody; oligosaccharides; ouabain; potassium; radiotracer; sarcoplasmic reticulum; site directed mutagenesis; sodium; sodium potassium exchanging ATPase; 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， 在等离子体中建立离子梯度时， 膜的 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，和 较小的糖蛋白（β-）亚基，其作用被怀疑是 将α-亚单位从内质网转运至质膜。 然而，关于结构、功能和组装知之甚少 子单元之间的关系。 该提案的目的是定义 α-亚基结构-功能所需的关键区域 以及与β亚基的相互作用。 具体目标是：（1） 确定α-亚基中的细胞外和细胞内结构域，（2） 为了鉴定抑制剂结合结构域，例如哇巴因结合位点， 已知定位于细胞外结构域，和（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 将寡糖转移到人工引入的N-连接糖基化上 信号（Asn-X-Thr（或-Ser））的α-亚基通过定点 诱变，和（ii）检查定点单克隆抗体的结合， 在透化细胞之前和之后针对α-亚基的抗体。 的 第二个和第三个目标将通过以下方式实现：（i）测试哇巴因， 结合和组装能力的部分缺失突变体的 α-亚基，和（ii）检查哇巴因敏感功能， 钙泵/钠泵α亚单位嵌合体的组装能力 分子。 大多数问题将在基因转移中得到解决 表达野生型和/或突变的禽类cDNA的E. 大肠杆菌或哺乳动物细胞中，并且基因产物通过禽流感病毒检测。 特异性单克隆抗体。 代谢标记实验和 将采用免疫荧光显微镜检查稳定性， 在哺乳动物中表达的禽类分子的细胞内定位 细胞 ATP水解、离子转运和配体（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。 结果从这些 研究将为理解生物起源和 钠泵子单元的功能。