LOCALIZATION OF LIGAND INTERACTIONS WITH THE SODIUM PUMP

这项工作的目的是确定 钠泵蛋白，其中生理配体相互作用， 对于主动钠转运机制来说是必不可少的。实验 过去三十年的研究已经确立了这种关系 在钠泵催化的生化反应和 运输反应是由它来调节的。一种针对 确定这类地区尤其及时，因为这是主要的 最近已经获得了α和β亚基的序列。这个 目前的工作将利用纯化的犬肾脏钠泵蛋白 并将采用光化学和化学亲和标记技术 蛋白质的特定区域。抑制作用的动力学和方式 各种新型试剂将被表征，它们的位置 使用无线标签进行本地化的操作。放射性标记的蛋白质将是 经过蛋白质降解和部分分离前分离的标记肽 一级结构中的测序和定位。用于治疗的试剂 包括一种新的羧基选择性荧光试剂的类似物 它最近被证明是K-Site标签，一种井的类似物 已知的利尿剂，阿米洛利，最近的研究表明，它也可能是一种 寻找K位的探针。感光试剂也将用于 定位ATP结合区中基本精氨酸附近的区域 阿尔法亚单位。此外，还将进一步使用光致亲和力 试剂，NAB-哇巴因，它与心脏糖苷特异结合 位于Na，K-ATPase外表面的位点，用于鉴定氨基 哇巴因结合中靠近糖苷部分的酸 域。总体目标是确定在初级序列中的什么位置 残基是与生理配体相互作用的残基 残基参与了重要的构象转变。 钠泵输送循环。钠泵在各种情况下都是至关重要的 维持体液和电解质平衡的器官。这些过程会受到干扰 在各种疾病状态下。在对这些内容进行充分描述之前 可以对病理情况做出更全面的了解 需要钠泵正常工作。

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.