Comparison of rates of cation release and of conformational change in dog kidney Na, K-ATPase.

1. It is now widely believed that the main rate-limiting step in the sodium-potassium pump (Na, K-ATPase) cycle is a conformational change between two forms of the dephosphoenzyme (E2 and E1) and that this change releases to the cell interior potassium ions occluded within the E2 form. 2. If this hypothesis is correct, and if occluded ions cannot be released directly from dephosphoenzyme in the E2 conformation, we should expect that, under any given conditions, the rate of release of the occluded ions would be identical with the rate of the conformational change. 3. Using the potassium congeners 86Rb, 137Cs and 204Tl, the rates of release of the occluded ions can be measured by a rapid ion-exchange technique. Using the fluorescence probes fluorescein isothiocyanate (FITC), eosin or 5-iodoacetamido-fluorescein (5-IAF), the rates of the conformational change can be measured by stopped-flow fluorimetry. 4. A comparison of the two rates in the absence of ATP showed that the rate of release of the occluded ions was usually somewhat faster than the rate of the fluorescence change. The discrepancy was probably caused by a very slow direct release of occluded ions from enzyme in the E2 form, but we cannot exclude the possibility that it is the result of systematic errors. In the presence of 5 microM-ATP, both rates were increased and there was no significant difference between them. 5. The results are compatible with the hypothesis that the same conformational change alters the fluorescence of the fluorescent probes and releases the occluded potassium congener ions.