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

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.