LOCALIZATION OF LIGAND INTERACTIONS WITH THE SODIUM PUMP

这项工作的目的是确定区域和网站上的 钠泵蛋白，其中生理配体相互作用， 对主动钠转运机制至关重要。 实验 过去三十年的研究已经建立了 钠泵催化的生化反应和 它介导的运输反应。 一种针对 确定这些区域特别及时，因为 最近已经获得了α和β亚基的序列。 的 本工作将利用纯化的钠泵蛋白从犬肾 并将采用光化学和化学亲和标记， 蛋白质的特定区域。 抑制的动力学和模式 各种新的试剂将被表征和它们的位点， 使用放射性标记定位作用。 放射性标记的蛋白质将被 进行蛋白水解，并在部分水解之前分离标记的肽。 一级结构中的测序和定位。 的试剂 包括新的羧基选择性荧光试剂的类似物 它最近被证明是一个K位标记， 已知利尿剂阿米洛利，最近的研究表明，它也可能是一种 探测K位点 光敏试剂也将用于 定位ATP结合域中必需精氨酸附近的区域 α-亚基。 此外，将进一步使用光亲和性 特异性结合强心苷的NAB-哇巴因试剂 在Na，K-ATP酶的外表面上的位点，以识别氨基 在哇巴因结合中靠近糖苷部分的酸 域 总的目标是确定在主序列上 所述残基与生理配体相互作用， 残基参与的基本构象转变， 钠泵转运循环 钠泵在各种 体液和电解质平衡的器官。 这些过程受到干扰 in a variety品种of disease疾病state状态. 在充分描述这些 病理情况可以作出更全面的了解， 需要钠泵的功能。

The purpose of this work is to identify the regions and sites on the sodium pump protein where physiological ligands interact and which are essential for the mechanism of active sodium transport. Experimental studies over the past three decades have established the relationships between the biochemical reactions catalyzed by the sodium pump and the transport reactions it mediates. An approach directed at the identification of such regions is particularly timely as the primary sequence of the alpha and beta subunits has recently been obtained. The present work will utilize purified sodium pump protein from canine renal medulla and will employ photochemical and chemical affinity labeling of specific regions of the protein. The kinetics and mode of inhibition of a variety of novel reagents will be characterized and their sites of action localized using radiolabels. The radiolabeled protein will be subjected to proteolysis and labeled peptides isolated prior to partial sequencing and localization in the primary structure. The reagents to be used include analogs of a new carboxyl-selective fluorescent reagent which has recently been shown to be a K-site label, an analog of a well known diuretic, amiloride, which recent work has shown may also be a probe for the K-site. Photosensitive reagents will also be used to localize the region near an essential arginine in the ATP binding domain of the alpha-subunit. Also, further use will be made of a photoaffinity reagent, NAB-ouabain, which binds specifically to the cardiac glycoside site on the outside surface of the Na,K-ATPase, to identify the amino acids which are close to the glycoside moiety in the ouabain binding domain. The overall aim is to determine where on the primary sequence the residues are which interact with physiological ligands and which residues are involved in essential conformational transitions in the sodium pump transport cycle. The sodium pump is vital in a variety of organs for fluid and electrolyte balance. These processes are disturbed in a variety of disease states. Before an adequate description of these pathological situations can be made a more complete understanding of the functioning of the sodium pump is required.