LOCALIZATION OF LIGAND INTERACTIONS WITH THE SODIUM

这项工作的目的是确定区域和地点上

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.