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S. J. D. Karlish^* and W. D. Stein

Biochemistry Department, Weizmann Institute of Science, Rehovot, Israel

Institute of Life Science, Hebrew University, Jersusalem, Israel

1. The passive Rb fluxes mediated by the Na—K pump in reconstituted vesicles, described by Karlish & Stein (1982), are affected by ATP or by phosphate acting separately.

2. Rb—Rb exchange through inside-out pumps is stimulated by ATP at low concentrations and is inhibited at high concentrations. There are mutual effects of Rb at cytoplasmic sites and ATP. The higher is the Rb concentration, the greater is the degree of stimulation and the less is the inhibition of exchange by ATP, and the higher are the concentrations of ATP required to produce effects. ATP stimulates Rb—Rb exchange maximally by about 5-fold.

3. There are similar effects of ATP on zero-trans net Rb uptake through inside-out pumps. However, much lower degrees of stimulation and greater inhibition of the net flux by ATP are observed, and much lower concentrations of ATP are required for these effects, by comparison with those on Rb—Rb exchange.

4. Rb uptake on inside-out pumps in Na-loaded vesicles shows only inhibition by ATP.

5. Phosphate effects require the presence of Mg₀ ions. At low Mg₀ concentrations (up to 100 µM) phosphate moderately stimulates Rb uptake into Rb-free or Rb-loaded vesicles (about 50%), but has no effect on Rb uptake into Na-loaded vesicles. At millimolar concentrations of Mg₀ ions, phosphate strongly inhibits the Rb uptake into Rb-free or Na-loaded vesicles but has no effect on Rb uptake into Rb-loaded vesicles.

6. The separate effects of ATP and of phosphate are explained in terms of the model proposed by Karlish & Stein (1982), modified to take into account stimulation of the conformational transition E₂(Rb)_occ E₁ Rb by ATP, and stimulation of the conformational transition E₂(Rb)_occ E₂ Rb by phosphate due to phosphorylation of the protein.