The Effect of Holding Potential on Charge Translocation by the Na+/K+-ATPase in the Absence of Potassium
1. The binding of [(3)H]ouabain to fresh Girardi and Hela cells and to those cultured in low concentrations of ouabain for 24 hr has been measured.2. Fresh cells bind 1.6-2.2 x 10(6) molecules of ouabain(*) per cell from K-free Krebs, but less than 0.3 x 10(6) molecules from 15 mM-[K](o) Krebs. The ouabain(*) binds with a t((1/2)) of about 8 min from K-free 2 x 10(-7)M ouabain(*) and is released with a t((1/2)) of about 20 hr. Cells in a poor condition probably exchange ouabain more quickly.3. Cells incubated in ouabain(*) for 24 hr bind ouabain in amounts dependent on the [ouabain] and the external [K]. At the highest [ouabain] used the total amount bound exceeds that bound by fresh cells. Lowering [K](o) in the medium increases the maximum ouabain which is bound.4. Cells incubated in ouabain(*) for 24 hr bind an additional amount of ouabain when exposed to 2 x 10(-7)M ouabain(*) in K-free Krebs.5. There is a close relationship between the% of the total ouabain bound in 24 hr and the% inhibition of the Na efflux suggesting that this ouabain is bound to the Na pumps.6. Radiochromatography of the counts recovered from the cells showed that it migrated to the same place as the applied [(3)H]ouabain. The wash-off rate of ouabain bound to cells during incubation is similar to that from fresh cells, both tests suggesting that the ouabain exists in the same state in the cells.7. The number of Na ions extruded per pump is constant at about 60 sec(-1) in fresh cells and those pre-incubated in ouabain.8. The total ouabain bound by the cells is closely related to the [Na](i) in cells pre-incubated in ouabain but is unaffected by it in fresh cells where [Na](i) is raised acutely.9. These results are compatible with the hypothesis that partial blocking of Na pumps leads to the production of more pumping sites by the cell.