SUMMARY1. Measurements were made of the binding of 73H]ouabain to a variety of cell types.2. Two components of binding could usually be distinguished: a component that saturated at low glycoside concentrations and a component that increased up to the highest ouabain concentrations examined.3. Detailed studies with HeLa cells and kidney slices from guinea-pigs showed that the saturable component is probably associated with inhibition of the Na pump. The main evidence for this is (a) at low concentrations of ouabain there is a close correspondence between the concentration of ouabain giving half-maximum binding and the concentration giving halfmaximum inhibition of the Na pump; (b) at low glycoside concentrations, binding precedes inhibition of the Na pump; (c) the rate of binding is very sensitive to external K ions, being highest in the absence of K; (d) binding is reversible and the release of ouabain is associated with reactivation of the Na pump, (e) binding is reduced in the absence of Na ions and in the presence of metabolic inhibitors; (f) binding has a Q10 of about 4; and (g) in the presence of Na and ATP, lysed HeLa cells bind a similar amount of ouabain and the binding is sensitive to K ions.4. The linear component of binding does not seem to involve the Na pump and it may reflect uptake of ouabain into the cell interior. It has a Q10 of 2-5 and is unaffected by K concentrations which have a large effect on the saturable component.5. Bound ouabain could be removed from HeLa cells by low pH, trichloroacetic acid, urea, high temperatures and 100 % ethanol. These agents did not distinguish between the two components of binding. 6. Criteria are developed for estimating the number of Na pumping sites in cells and the data for ouabain-binding to a number of cells is compared * Present address:

It is generally recognized that the oxygen consumption of cells is related to the amount of free energy being utilized by such processes as secretion, contraction, and the synthesis of complex molecules. Two questions arise concerning the nature of the coupling between energy release from metabolism and its utilization. First, what fraction of the cell's total metabolism is devoted to a particular energy-requiring process; and secondly, how is an increase in oxygen consumption elicited in order to ensure a supply of the requisite energy? This paper deals with these problems in connexion with the active movements of ions in kidney cortex slices.In slices of kidney cortex from adult rabbits and guinea-pigs the establishment and maintenance of concentration gradients of sodium and potassium ions between intracellular and extracellular fluid depends on energy from respiration (Mudge, 1951 a, b;Whittam & Davies, 1953). The evidence for this is chiefly the fact that active transport is stopped when the energy supply is decreased by the inhibition of respiration. Such studies have not revealed the fraction of the total respiratory energy expended on these ion movements or whether the latter in turn influence the rate of respiration. Kidney cortex is a particularly suitable tissue for studying the interdependence of ion transport and metabolism, because both its rate of respiration and the turnover rate of its potassium are high, so that a reduction in oxygen uptake which might result from stopping transport could easily be measured. The coupling between respiration and potassium transport in slices of kidney cortex has been investigated in the present study by comparing the effects of ouabain, sodium ion, oc-oxoglutarate, and temperature on tissue potassium concentration and respiration during incubation at 25 and 380 C. A preliminary account of this work has been reported previously (Whittam & Willis, 1962).

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SUMMARY1. The rate of inhibition of the Na pump by ouabain was examined both by direct measurement of the rate of decline of the Na efflux and by the binding of [3H]ouabain.2. The onset of inhibition of the Na efflux was concentration-dependent; but did not follow simple first order kinetics. The time course of inhibition was roughly exponential although in about 30 % of the axons inhibition was preceded by a transient stimulation of the Na efflux.3. Inhibition of the Na efflux by both ouabain and strophanthidin was apparently irreversible.4. The onset of inhibition was slowed markedly at low temperatures. 5. Replacement of external Na by choline, dextrose or potassium slowed the rate of inhibition. Li behaved like Na and inhibition was faster in K-ASW than in choline-ASW.6. The rate of inhibition of Na-Na exchange was similar to that of Na-K exchange, but ouabain failed to bind securely to fully poisoned axons.7. Two components of [3H]ouabain-binding could be distinguished. A linear component which probably reflects uptake into the cells and a saturable component which seems to reflect binding to Na-pumping sites.8. The saturable component of binding followed a similar time course to the inhibition of the Na efflux and the rate of binding was reduced in choline-ASW and in fully poisoned axons.9. Measurements of [3H]ouabain-binding indicate that the number ofNa pumping sites in the axon membrane is probably between 103 and 104/,u2.

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Ouabain-sensitive K influx into ground squirrel and guinea pig red cells was measured at 5 and 37°C as a function of external K and internal Na. In both species the external K affinity increases on cooling, being three-and fivefold higher in guinea pig and ground squirrel, respectively, at 5 than at 37°C. Internal Na affinity also increased on cooling, by about the same extent . The effect of internal Na on ouabain-sensitive K influx in guinea pig cells fits a cubic Michaelis-Menten-type equation, but in ground squirrel cells this was true only at high [Na]i . There was still significant ouabain-sensitive K influx at low [Na]i . Ouabain-binding experiments indicated around 800 sites/cell for guinea pig and Columbian ground squirrel erythrocytes, and 280 sites/cell for thirteenlined ground squirrel cells. There was no significant difference in ouabain bound per cell at 37 and 5°C. Calculated turnover numbers for Columbian and thirteen-lined ground squirrel and guinea pig red cell sodium pumps at 37°C were about equal, being 77-100 and 100-129 s-1 , respectively . At 5°C red cells from ground squirrels performed significantly better, the turnover numbers being 1.0-2 .3 s-1 compared with 0.42-0.47 s-1 for erythrocytes of guinea pig. The results do not accord with a hypothesis that cold-sensitive Na pumps are blocked in one predominant form.

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