A study of some aspects of the kinetics, mechanism and lipid dependence of the NA(^+)-K(^+)-ATPase

Abstract

The kinetics of the NA(^+)-K(^+)-ATPase with respect to ouabain nhibition, potassium activation and the effect of temperature on its catalytic activity has been studied, and attempts were made to define these kinetic parameters using computer assisted procedures. The pattern of ouabain inhibition of the enzyme was described within the context of the enzyme being a cooperative kinetic dimer. The theoretical basis for the non-ideal Arrhenius temperature kinetics of this enzyme was also studied and its behaviour was described in terms of a thermodynamic equilibrium between a high temperature active state and a low temperature inactive state. The sigmoidal response of the enzyme to increasing potassium ion concentrations was quantified in terms of its stoichiometric requirement for two moles of potassium ion per mole of ATP hydrolysed. The effects of lipid targeted and protein targeted modulators on the kinetic properties of the NA(^+)-K(^+)-ATPase were studied. The kinetic properties of the enzyme was insensitive to the perturbations in the membrane brought about by peroxidisation of membrane lipids, and the detergent extraction procedures used for partial purification of the enzyme provided that the final preparation was not labile. Those kinetic properties were also insensitive to the membrane lipid changes coincident with the adaptation of a given species to different temperatures. The kinetic properties of the NA(^+)-K(^+)-ATPase (or its K(^+)-phosphatase activity) were found to be sensitive to a glycoprotein aimed modulator (Concanavalin A), and a sulphydryl blocking reagent (thimerosal). A partial thermal inactivation of the NA(^+)-K(^+)-ATPase was also found to result in significant changes in its kinetic properties. Studies on the thermal inactivation of the NA(^+)-K(^+)-ATPase revealed a biphasic decay pattern which was interpreted in terms of a sequential decay by a dimeric species. A comparative study of the thermal inactivation of the enzyme showed that NA(^+)-K(^+)-ATPases prepared from animals that were naturally adapted to function at 'high' temperatures were of greater thermal stability than those prepared from animals that normally function at low temperatures. However, the thermal stability of the enzyme was not affected by acclimation of a species to different temperature conditions. The kinetic stability of the enzyme was not affected by a partial purification by sodium dodecylsulpliate extraction, but was significantly reduced on incubation with octanol. It is proposed that membrane lipids play little (if any) role in the fine control of the NA(^+)-K(^+)-ATPase, and that their primary role is that of restricting the NA(^+)-K(^+)-ATPase to its biologically active conformational states. It is also proposed that fine control of the NA(^+)-K(^+)-ATPase can be brought about by a variety of mechanisms aimed at the protein-protein interactions as well as allosteric modulations by regulatory ligand binding sites.