LOCALIZATION OF LIGAND INTERACTIONS WITH THE SODIUM

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， 其对于主动钠转运机制是必需的。 过去三十年的实验研究已经建立了 催化的生化反应之间的关系 钠泵及其介导的运输反应。 然而，关于哪些区域或 残基参与生理配体的结合，或者 参与运输周期的重要步骤。 方法 特别是针对这些地区的识别， 及时的，因为α和β亚基的主要序列 最近获得的。 本工作将利用纯化的 钠泵蛋白，并将采用 特定区域的光化学和化学亲和标记 蛋白质。 对各种不同的抑制剂的动力学和抑制模式进行了研究。 新的试剂将被表征，并且它们的作用位点 用放射性标记定位。 放射性标记的蛋白质将被 进行蛋白水解，并在 一级结构中的部分测序和定位。 的 使用的试剂包括用于阳离子位点的新的光亲和标记， 该蛋白质，一种用于核苷酸结合精氨酸残基的光标记物， 酰基磷酸酯反应性羟胺的光标记。 光敏试剂将用于表征独特的 构象敏感的赖氨酸残基先前显示为 α-亚基中的DIDS反应性，并研究 NH 2和CO2 H基团足够接近以交联， 阿托咪胺诱导的抑制作用。 这些试剂在研究 锁定在特定构象中的酶和侧酶 系统将产生新的信息的折叠安排， 蛋白质。 抑制和定位研究也将在 用有效的疏水-SH基团试剂进行。 的 总体目标是确定在初级序列上， 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 钠泵在一个 各种器官的液体和电解质平衡。 这些 这些过程在各种疾病状态中受到干扰。 之前 可以对这些病理情况进行充分的描述 更全面地了解钠的功能 泵是必需的。

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.