Kinetic evaluation of the Na-K pump reaction mechanism

1. The ouabain-sensitive K influx was measured at varying external K concentrations ([K]o) and at several fixed internal Na concentrations ([Na]c). The cells were nominally K-free and the solutions Na-free. Both the apparent maximal velocity (VM) and the apparent Michaelis constant for K (KK) increased as Nac increased. The ratio app. VM/app. KK increased with increasing Nac. 2. The ouabain-sensitive Cs influx was measured at varying external Cs concentrations and at several fixed Nac in K-free cells and Na-free solutions. Both app. VM and app. Kcs increased as Nac increased and the ratio app. VM/app. Kcs increased with increasing Nac. 3. The data were evaluated in terms of ping-pong model and a simultaneous model for the pump reaction mechanism. The simultaneous model described the data adequately and the ping-pong models did not. 4. The K influx was measured at varying external K concentrations in solutions containing Na and at a low and high Nac; the cells contained K. The relation between the pump rate and the external K concentration was sigmoid. A Hill equation was fitted to the data. KK was higher in the high Nac cells, but the Hill coefficient (n) was not altered as Nac increased. 5. The K influx was measured at varying internal Na concentrations and two fixed external K concentrations; the cells contained K. The relation between the pump rate and Nac was sigmoid. When a Hill equation was fitted to the data, it was found that KNac was higher at the high external K concentration, but n was the same at both K concentrations.