INTERACTION OF DIGITALIS WITH ITS RECEPTOR NA+/K+ ATPASE

洋地黄与其受体 NA /K ATP 酶的相互作用

• 批准号: 2226862
• 负责人: EARL T WALLICK
• 金额: $ 25.79万
• 依托单位: UNIVERSITY OF CINCINNATI
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-08-01 至 1999-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2226862
• 关键词: 3T3 cells; HeLa cells; animal tissue; binding proteins; circular dichroism; digitalis; drug interactions; drug receptors; infrared spectrometry; isozymes; mutant; ouabain; protein sequence; site directed mutagenesis; sodium potassium exchanging ATPase; western blottings

The long term objective of this proposal is determination of the molecular details by which the drugs of the digitalis class (cardiac glycosides) interact with their pharmacological receptor, the (Na,K)-ATPase. To understand this process, the structure of the receptor and the drug, and the effect of changes in these structures on the stability (affinity) of the drug-receptor complex must be determined. Human heart contains three isoforms, alpha1 being predominant in muscle and alpha2 and alpha3 being enriched in the conducting system. If the alpha1 isoform is primarily responsible for the increase in contractility and alpha2 and/or alpha3 primarily responsible for the toxic effect, a cardiac glycoside having a relatively higher affinity for alpha1 (and therefore possessing an improved therapeutic index) might be discovered or synthesized. Specific Aim 1 is to characterize the three alpha isozymes of human (Na,K)-ATPase expressed in NIH 3T3 cells with respect to their differential interaction with a series of cardiac glycosides. Specific Aim 2 is to identify and locate within the primary sequence, amino acid residues in the (Na,K)- ATPase that are involved in interacting with cardiac glycosides. The sites of interest include ligand regulatory sites (Mg, ATP, Na and K) as well as the actual cardiac glycoside binding site. The structure of (Na,K)-ATPase will be altered using site-directed mutagenesis of sheep alpha1 isoform expressed in NIH 3T3 or in Hela cells. The structure of the drug will be varied by taking advantage of existing digitalis analogous. For Aims 1 and 2 the dissociation constant, KD, will be determined by saturation binding assays or by measurement of kinetic rate constants or alternatively, deduced from inhibition studies (150). The alteration in KD for binding of a series of cardiac glycosides to each mutant will define the role specific amino acids mutated play in the binding process. Specific Aim 3 is to obtain and characterize preparations of the alpha-beta complex, separated alpha and beta subunits and selected smaller protein fragments of sheep (Na,K)-ATPase of appropriate purity and in sufficient amounts for three dimensional structural studies. Purity will be assessed using native and denaturing gels and Western blots developed using antibodies against the sheep alpha and beta subunits. Degree of glycosylation, number and intactness of disulfide bonds and secondary structure (circular dichroism and infrared spectroscopy) will be determined. A working laboratory model of the receptor consistent with the experimental data will be constructed using techniques of energy minimization. The proteins, when pure, will be submitted for crystallization trials.

该提案的长期目标是确定分子 洋地黄类药物（强心苷）的详细信息 与其药理学受体（Na,K）-ATP酶相互作用。到 了解这个过程、受体和药物的结构，以及 这些结构的变化对稳定性（亲和力）的影响 必须确定药物-受体复合物。人的心脏包含三个 同工型，α1 在肌肉中占主导地位，α2 和 α3 在肌肉中占主导地位 丰富了传导系统。如果 alpha1 同工型主要是 负责收缩力和 alpha2 和/或 alpha3 的增加 主要负责毒性作用，强心苷具有 对 alpha1 的亲和力相对较高（因此具有 可能会发现或合成改善的治疗指数）。具体的 目标 1 是表征人类 (Na,K)-ATP 酶的三种 α 同工酶 在 NIH 3T3 细胞中表达的差异相互作用 具有一系列强心苷。具体目标 2 是确定并 位于一级序列内，氨基酸残基位于 (Na,K)- 参与与强心苷相互作用的 ATP 酶。网站 感兴趣的包括配体调节位点（Mg、ATP、Na 和 K）以及 实际的强心苷结合位点。 (Na,K)-ATP酶的结构 将使用绵羊 alpha1 同工型的定点诱变进行改变 在 NIH 3T3 或 Hela 细胞中表达。该药物的结构将是 通过利用现有的洋地黄类似物来改变。对于目标 1 和 2 解离常数 KD 将通过饱和结合来确定 测定或通过测量动力学速率常数或替代地， 从抑制研究中推导出来 (150)。 KD 的改变用于结合 每个突变体的一系列强心苷将定义其作用 特定氨基酸突变在结合过程中发挥作用。具体目标 3 是为了获得并表征α-β复合物的制剂， 分离α和β亚基并选择更小的蛋白质片段 适当纯度和足够量的羊 (Na,K)-ATP 酶 三维结构研究。 将使用以下方法评估纯度 使用抗体开发的天然和变性凝胶以及蛋白质印迹 对抗羊α和β亚基。 糖基化程度， 二硫键和二级结构（环状结构）的数量和完整性 二色性和红外光谱）将被测定。一个正在工作的 受体的实验室模型与实验数据一致 将采用能源最小化技术建造。蛋白质， 当纯净时，将提交进行结晶试验。

项目成果

Naturally occurring A pocket polymorphism in HLA-B*2703 increases the dependence on an accessory anchor residue at P1 for optimal binding of nonamer peptides.

HLA-B*2703 中天然存在的 A 口袋多态性增加了对 P1 处辅助锚定残基的依赖性，以实现九聚肽的最佳结合。

• 发表时间: 1997
• 期刊: Journal of immunology (Baltimore, Md. : 1950)
• 作者: Griffin,TA;Yuan,J;Friede,T;Stevanovic,S;Ariyoshi,K;Rowland-Jones,SL;Rammensee,HG;Colbert,RA
• 通讯作者: Colbert,RA