LOCATION OF CATION BINDING SITES IN NA+/K+ ATPASE

NA /K ATP酶中阳离子结合位点的位置

• 批准号: 2211613
• 负责人: JOSE M ARGUELLO
• 金额: $ 9.59万
• 依托单位: UNIVERSITY OF CINCINNATI
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-05-01 至 2000-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2211613
• 关键词: 3T3 cells; HeLa cells; active sites; aminoacid; cations; chemical binding; chemical structure function; conformation; crosslink; cysteine; enzyme activity; fluorescent dye /probe; molecular biology; phosphorylation; protein isoforms; site directed mutagenesis; sodium potassium exchanging ATPase; transfection

The long term goal of the candidate's research is to define structure- function relationship of the NaK-ATPase. The NaK-ATPase is present in the plasma membrane of nearly all animal cells, where it is responsible for maintaining the transmembrane Na and K gradients. The catalytic and transport cycles of the NaK-ATPase have been described in detail but the protein structure and regions of the enzyme involved in each particular mechanistic event are largely unknown. The candidate plans to test if conserved oxygen containing residues, in the transmembrane segments of the catalytic subunit, constitute the narrow portion of a putative ion well involved in ion selectivity and occlusion of Na and K. In addition, he will start determining the three dimensional arrangement of those transmembrane segments that contribute amino acid side chains to the cation binding site. Site directed mutagenesis of selected, conserved, oxygen containing amino acids will be performed. Functional parameters such as ATPase activity, phosphorylation, conformation transitions, and cation binding, will be measured to analyze the role of each substituted amino acid. An isolation system to affinity purify the heterologous expressed protein from the endogenous isoforms will be developed. The crosslinking between cysteine residues in spatial proximity will allow mapping of the intramembrane region of the protein. These cysteines will be introduced by site- directed mutagenesis. The crosslinking will be observed by chemical modification of the targeted transmembrane segments with sulfhydryl specific fluorescent reagents before and after reductive treatment.

候选人研究的长期目标是定义结构