LOCALIZATION OF LIGAND INTERACTIONS WITH THE SODIUM

这项工作的目的是确定 钠泵蛋白与生理配体相互作用和 它们对主动钠转运的机制是必不可少的。 过去三十年的实验研究已经建立了 黄曲霉毒素催化的生化反应之间的关系 钠泵和它所介导的运输反应。 然而，几乎没有关于哪些地区或 残基参与生理配体的结合或 参与运输周期的基本步骤。一种方法 特别是针对这些区域的识别 正如α和β亚基的初级序列一样及时 是最近获得的。本工作将利用提纯的 从犬肾髓质中提取钠泵蛋白 特定区域的光化学和化学亲和标记 蛋白质的含量。几种酶的抑制动力学和抑制方式 将表征新型试剂及其作用部位 使用无线电标签定位的。放射性标记的蛋白质将是 经过蛋白水解物和在此之前分离的标记肽 一级结构中的部分测序和定位。这个 使用的试剂包括一种新的阳离子位点的光亲和标记 这种蛋白质是核苷酸结合精氨酸残基的光标签， 酰基磷酸-反应性羟胺的光标记。 光敏试剂将被用来表征独特的 构象敏感的赖氨酸残基之前被证明是 DDS-在阿尔法亚基中的反应性，并调查在哪里 NH2和CO2H基团足够紧密，可以在 碳二亚胺诱导的抑制作用。这些试剂在研究中的作用 锁定在特定构象中的酶和侧链酶 系统将产生有关折叠排列的新信息 蛋白质。抑制和本地化研究也将 使用强大的疏水-SH基试剂进行。这个 总体目标是确定主序列上的位置 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。钠泵在一个 维持体液和电解质平衡的各种器官。这些 在各种疾病状态下，这些过程都会受到干扰。在此之前 可以对这些病理情况进行充分的描述 对钠的功能有更全面的了解 需要泵。

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. However little information is available as to which regions or residues are involved in binding of physiological ligands or are involved in essential steps of the transport cycle. 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 labelling 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 radiolabelled proteins will be subjected to proteolysis and labelled peptides isolated prior to partial sequencing and localization in the primary structure. The reagents used include a new photoaffinity label for cation sites on the protein, a photolabel for nucleotide-binding arginine residues, a photolabel for acylphosphate-reactive hydroxylamines. Photosensitive reagents will be used to characterize the unique conformationally-sensitive lysine residue previously shown to be DIDS-reactive in the alpha-subunit and also to investigate where NH2 and CO2H groups are sufficiently close to be cross-linked in the carbodiimide-induced inhibition. These reagents in studies of enzyme locked in specific conformations and of a sided enzyme system will yield new information on the folded arrangement of the protein. Inhibition and localization studies will also be performed with a potent hydrophobic-SH group reagent. 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.