The stoicheiometry of the sodium pump

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Abstract
When resealed ghosts containing ATP, Mg and Na were incubated in a medium containing K, ATP was hydrolysed vigorously by a ouabain-sensitive mechanism. If the ghosts contained K instead of or in addition to Na, and the external solution contained Na but no K, there was little ouabain-sensitive hydrolysis of ATP. As it is known that the ouabain-sensitive ATPase in fragmented ghosts requires both Na and K ions, these results show that the ATPase is activated by K externally and by Na internally, and suggest that the ions activating the ATPase are the ions that are transported. Resealed ghosts containing ATP, Mg and Na were incubated in Na-free media containing K, with and without ouabain, and the rate of loss of Na and rate of hydrolysis of ATP were measured. The hydrolysis of 1 molecule of ATP by the ouabain-sensitive mechanism was accompanied by the ouabain-sensitive loss of about 3 Na ions. 24Na and 42r were used to measure Na efflux and K influx in identical batches of fresh red cells under the same conditions and at the same time. Each flux was measured in the presence and absence of ouabain. The ratio (ouabain-sensitive Na efflux)/(ouabain-sensitive K influx) was significantly greater than 1 (1.20 [plus or minus]O.Ol and 1.35 [plus or minus]0.01 in 2 experiments). If a small fraction of the K influx represented a ouabain-sensitive K : K exchange, the ratio of the numbers of ions moved in the Na : K exchange catalysed by the pump must have been even further from unity. Resealed ghosts [[gamma]-32p]ATP, Mg, 24a and orthophosphate were incubated in balanced salt solutions with and without K and with and without ouabain. A comparison of Na efflux, estimated from 24Na loss, with ATP hydrolysis, estimated from the formation of [32p]Orthophosphate, showed that the Na : Na exchange in a K-free medium was accompanied by little or no ouabain-sensitive hydrolysis of ATP. Experiments on intact red cells loaded with 24Na showed that both Na : Na exchange in a K-free medium, and Na : K exchange in a medium conatining K, were partially inhibited by oligomycin (1-10 [mu]g/ml.). Inhibition of the Na : K exchange was not affected by raising the external K concentration.