Partial Reactions of the Na,K-ATPase: Determination of Activation Energies and an Approach to Mechanism

Abstract

Kinetic experiments were performed with preparations of kidney Na,K-ATPase in isolated membrane fragments or reconstituted in vesicles to obtain information of the activation energies under turnover conditions and for selected partial reactions of the Post-Albers pump cycle. The ion transport activities were detected with potential or conformation sensitive fluorescent dyes in steady-state or time-resolved experiments. The activation energies were derived from Arrhenius plots of measurements in the temperature range between 5 °C and 37 °C. The results were used to elaborate indications of the respective underlying rate-limiting reaction steps and allowed conclusions to be drawn about possible molecular reaction mechanisms. The observed consequent alteration between ligand-induced reaction and conformational relaxation steps when the Na,K-ATPase performs the pump cycle, together with constraints set by thermodynamic principles, provided restrictions which have to be met when mechanistic models are proposed. A model meeting such requirements is presented for discussion. Graphic Abstract

Molecular determinants for cold adaptation in an Antarctic Na+/K+-ATPase

“…For example, it has been suggested that the E 1 -P(Na 3 ) → E 2 -P transition, associated with the release of Na + to the extracellular environment, could become rate limiting at cold temperatures because its rate decreases steeply below 15 °C in the shark rectal gland Na + /K + -ATPase ( 37 ). In a more recent, detailed study, it has been shown that as temperature becomes colder, the opening of the extracellular gate that allows Na + to be released becomes the rate-limiting step in the transport cycle ( 38 ). Indeed, the occlusion/deocclusion transition that exposes the first Na + to the extracellular environment carries large entropic and enthalpic changes ( 39 ).…”