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

NA /K ATP酶中阳离子结合位点的位置

基本信息

批准号：
2910479
负责人：
JOSE M ARGUELLO
金额：
$ 10.07万
依托单位：
WORCESTER POLYTECHNIC INSTITUTE
依托单位国家：
美国
项目类别：
财政年份：
1995
资助国家：
美国
起止时间：
1995-05-01 至 2001-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/2910479
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

项目摘要

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- NAK-ATPase的功能关系。NAK-ATPase存在于几乎所有动物细胞的质膜，在那里它负责维持跨膜Na和K梯度。催化剂和 NAK-ATPase的转运周期已被详细描述，但蛋白质结构和参与每种特殊情况的酶的区域机械事件在很大程度上是未知的。候选人计划测试是否保守的含氧残基，在细胞的跨膜节段催化亚基，构成假定的离子井的狭窄部分参与了Na和K的离子选择性和闭塞。将开始确定这些元素的三维排列为阳离子贡献氨基酸侧链的跨膜片段结合部位。选择性、保守性、含氧氨基的定点突变将执行酸化。功能参数，如ATPase活性，磷酸化、构象转变和阳离子结合测定分析各取代氨基酸的作用。与世隔绝亲和纯化异源表达蛋白的系统内源性异构体将被开发出来。半胱氨酸之间的交联空间接近处的残留物将使膜内标测成为可能蛋白质的区域。这些半胱氨酸将由SITE- 定向诱变。将用化学方法观察交联剂的作用。靶向跨膜片段的巯基修饰还原处理前后的特异性荧光试剂。