Orthophosphate-promoted ouabain binding to Na/K pumps of resealed red cell ghosts. Evidence for E*P preferentially binding ouabain.
Modulation of phosphoenzyme forms of the Na/K pump by Na+ and K+ was studied by measuring the rate of Pi-promoted ouabain binding to resealed ghosts made from human red cells. This system permits distinguishing the effects of the ions at intracellular and external binding sites. Internal K+, Ki, inhibited the rate of Pi-promoted ouabain binding, contrary to a prediction based on a current model of the pump. External K+, Ko, failed to inhibit ouabain binding in the absence of Ki. However, Ko enhanced the inhibition by Ki. Nai also inhibited ouabain binding; this inhibition was much less affected by Ko than was inhibition by Ki, suggesting that Ki and Nai affect ouabain binding at different internal sites. Nao inhibited ouabain binding in the absence of Ki or Ko, so Nao and Ko also act at different sites. With Nao present, Ki stimulated ouabain binding. Thus a condition was found in which the predicted stimulation of binding by Ki was observed. The results of this study are interpreted in terms of three phosphoenzyme forms of the pump: E1P, E*P, and E2P. E*P is the form binding ouabain with highest affinity. Ki promotes E*P----E2P, thereby inhibiting ouabain binding. Ko binds only to E2P, therefore Ki is required for inhibition by Ko, and there is little E2P present with no Ki. Nao inhibits binding by stabilizing E1P whereas Nai inhibits by stabilizing E1. The stimulation by Ki with Nao present means that Ki and Nao together favor formation of E*P. Furthermore, Ki and Nao may bind to the pump simultaneously. Ki may play a role in the normal pump cycle, binding at allosteric sites to promote E*P----E2P.
Guerra
M
M
Department of Pharmacology, State University of New York Health Science Center, Syracuse 13210.
Steinberg
M
M
Dunham
P B
PB
D000255:Adenosine Triphosphate / Q000494:pharmacology; D004910:Erythrocyte Membrane / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008954:Models, Biological; D010042:Ouabain / Q000097:blood*; D010710:Phosphates / Q000494:pharmacology*; D011188:Potassium / Q000097:blood*; D011485:Protein Binding; D012964:Sodium / Q000494:pharmacology; D000254:Sodium-Potassium-Exchanging ATPase / Q000187:drug effects / Q000502:physiology*
D010710:Phosphates; D010042:Ouabain; D000255:Adenosine Triphosphate; D012964:Sodium; D000254:Sodium-Potassium-Exchanging ATPase; D008274:Magnesium; D011188:Potassium