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

候选人研究的长期目标是定义结构- NaK-ATPase的功能关系。 NaK-ATP酶存在于 几乎所有动物细胞的质膜，它负责 维持跨膜Na和K梯度。 催化和 已经详细描述了NaK-ATPase的转运循环， 蛋白质结构和酶的区域参与每个特定的 机械事件在很大程度上是未知。 候选人计划测试，如果 保守的含氧残基，在跨膜段的 催化亚单位，构成一个假定的离子阱的狭窄部分 参与Na和K的离子选择性和吸留。 此外他 将开始确定那些 为阳离子提供氨基酸侧链的跨膜区段 结合位点 选择的、保守的、含氧氨基酸的定点诱变 酸将被执行。 功能参数如ATP酶活性， 磷酸化，构象转换和阳离子结合，将是 测量以分析每个取代的氨基酸的作用。 隔离 系统，以从所述细胞亲和纯化异源表达的蛋白质。 将产生内源性同种型。 半胱氨酸之间的交联 在空间上接近的残基将允许膜内的映射 蛋白质的区域。 这些半胱氨酸将通过位点引入- 定向诱变 将通过化学分析观察交联。 用巯基修饰靶向跨膜区段 还原处理前后的特异性荧光试剂。

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.