This project aims to discover basic features of the mechanism and structure of the Na/K pump of animal cell membranes using (Na, K)ATPase purified from mammalian kidney. Phospholipid vesicles reconstituted with the purified (Na, K)ATPase are to be used for a study of Na and K fluxes, protein conformational changes monitored with fluorescence probes, and of functional site labelling and selective proteolytic digestion. Following our recent discovery of slow passive Na/K-pump mediated fluxes of Rb(K) in the vesicles, we intend now to investigate conditions which may affect these """"""""leak"""""""" fluxes and alter their rates relative to normal active K pumping. These conditions include transmembrane potentials and temperature, and the concentrations of protons, and K congeners.
The aim i s to enquire into the possibility of a varying efficiency of active K transport and to define in detail the roles of K occlusion and K/ligand binding interactions on E1 and E2 forms of the protein, in regulating optimal rates and efficiency of active K transport. Na fluxes in the vesicles will be characterized kinetically with the purpose of both testing predictions of the conventional transport models, and especially the possibility of variable Na/ATP coupling ratios and efficiency of active Na transport. In another application of the vesicles we shall attempt to identify and to label covalently carboxyl residues involved in cation binding and transport, and locate those residues along the a-chain utilizing selective proteolytic digestion. This should permit a distinction to be made between different transport models. The Na/K pump generates the normal trans-membrane Na and K gradients and is responsible for volume control, electrical and secretory activities etc. The significance of an understanding of the working and structure of the pump at the molecular level derives ultimately from its essential physiological roles, and a growing recognition that abnormalities in active transport may be involved in the aetiology of diseases such as essential hypertension.
Showing the most recent 10 out of 16 publications
