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Physiological Laboratory, University of Cambridge, Cambridge CB2 3EG

1. A method has been developed for regenerating [³²P]ATP of constant specific activity within resealed red cell ghosts, and for measuring its hydrolysis. The method may be used to follow the hydrolysis of ATP at concentrations down to 1 µM, and for periods long enough for the ATP at these very low concentrations to turn over several hundred times.

2. Using this method we have been able to show that the `uncoupled' efflux of Na caused by the Na pump when resealed red cell ghosts are incubated in (Na + K)-free media is associated with a hydrolysis of ATP. The stoicheiometry is roughly 2-3 Na ions expelled per molecule of ATP hydrolysed.

3. Measurements of ATP hydrolysis and Na efflux as functions of intracellular ATP concentration have shown that uncoupled Na efflux, and its associated ATP hydrolysis, are saturated at intracellular ATP concentrations in the region of 1 µM.

4. Measurement of ATP hydrolysis as a function of ATP concentration in resealed ghosts incubated in a K-containing medium gave a complicated activation curve suggesting the involvement of high-affinity (K_m ca. 1 µM) and low-affinity (K_m ca. 100 µM) sites.

5. When resealed ghosts containing about 1 µM-ATP were incubated in a Na-free or in a high-Na medium, the addition of K to the medium reduced the rate of ouabain-sensitive ATP hydrolysis.

6. Ouabain-sensitive ATP hydrolysis in resealed ghosts incubated in K-free choline media was inhibited by external Na at low concentrations (K_i < 1 mM), but this inhibition was reversed as the external Na concentration was further increased.

7. The results show that uncoupled Na efflux may be thought of as the transport mode associated with Na-ATPase activity, just as Na-K exchange is the transport mode associated with (Na + K)-ATPase activity. The significance of the differences between uncoupled Na efflux and Na-ATPase activity, on the one hand, and Na—K exchange and (Na + K)-ATPase activity, on the other, is discussed.

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