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: 3303520
• 负责人: KUNIO TAKEYASU
• 金额: $ 10.69万
• 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/3303520
• 关键词: Escherichia coli; adenosine triphosphate; adenosinetriphosphatase; 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; 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