Combined effects of ATP and phosphate on rubidium exchange mediated by Na-K-ATPase reconstituted into phospholipid vesicles

1. Phospholipid vesicles reconstituted with Na—K-ATPase show an (ATP+phosphate)-stimulated Rb—Rb exchange, with properties similar to the K—K exchange of human red cells. This includes a rate 15-20% of the rate of active ATP-dependent Na—K exchange. 2. We have studied activation of this Rb—Rb exchange by ATP at fixed phosphate concentrations and by phosphate at fixed ATP concentrations. It is found for both ATP and phosphate that with low concentrations of the fixed ligand an increase in concentration of the complementary ligand produces first stimulation and then inhibition of Rb—Rb exchange. At high concentrations of the fixed ligand the complementary ligand shows only saturation behaviour. 3. The pattern of activation and of inhibition by ATP and by phosphate is affected by the Rb 0 concentration in the exterior medium, in that higher concentrations of Rb 0 counteract inhibitory effects of high concentrations of ATP and phosphate. 4. (ATP+phosphate)-stimulated Rb—Rb exchange is activated by Rb 0 in the exterior medium along a sigmoid curve. An increase of Rb i within the vesicles, which raises the maximal velocity of Rb—Rb exchange, is accompanied by a smaller increase in the Rb 0 concentration required for half-maximal stimulation of the Rb—Rb exchange. 5. The data are interpreted in terms of a model similar to those proposed by Karlish & Stein (1982 a , b ), but extended to include simultaneous effects of ATP and phosphate. Inhibitions by high concentration of ATP or phosphate arise as a result of stabilization of E 1 ATP or E 2 -P forms respectively, in the presence of low concentrations of the complementary ligand. With high concentrations of the fixed ligand, saturation behaviour of the varying ligand is observed because the occluded Rb forms become the dominant transport intermediates. The occluded Rb forms bind both ATP and phosphate weakly and independently. The effects of ATP together with phosphate are accounted for by a simple combination of their separate effects on the Rb—Rb exchange. 6. We suggest that the functional role of the occluded Rb form E 2 (Rb) occ in active transport is to minimize passive cation leaks through the system and allow control of the direction of cation movements by binding of physiological ligands such as ATP or phosphate.