Stimulation of p-nitrophenylphosphatase activity of Na+/K+-ATPase by NaCl with oligomycin or ATP.
It is known that the addition of NaCl with oligomycin or ATP stimulates ouabain-sensitive and K+-dependent p-nitrophenylphosphatase (pNPPase) activity of Na+/K+-ATPase. We investigated the mechanism of the stimulation. The combination of oligomycin and NaCl increased the affinity of pNPPase activity for K+. When the ratio of Na+ to Rb+ was 10 in the presence of oligomycin, Rb+-binding and pNPPase activity reached a maximal level and Na+ was occluded. Phosphorylation of Na+/K+-ATPase by p-nitrophenylphosphate (pNPP) was not affected by oligomycin. Because oligomycin stabilizes the Na+-occluded E1 state of Na+/K+-ATPase, it seemed that the Na+-occluded E1 state increased the affinity of the phosphoenzyme formed from pNPP for K+. On the other hand, the combination of ATP and NaCl also increased the affinity of pNPPase for K+ and activated ATPase activity. Both activities were affected by the ligand conditions. Oligomycin noncompetitively affected the activation of pNPPase by NaCl and ATP. Nonhydrolyzable ATP analogues could not substitute for ATP. As NaE1P, which is the high-energy phosphoenzyme formed from ATP with Na+, is also the Na+-occluded E1 state, it is suggested that the Na+-occluded E1 state increases the affinity of the phosphoenzyme from pNPP for K+ through the interaction between alpha subunits. Therefore, membrane-bound Na+/K+-ATPase would function as at least an (alphabeta)2-diprotomer with interacting alpha subunits at the phosphorylation step.
Homareda
Haruo
H
Department of Biochemistry, Kyorin University School of Medicine, Mitaka, Tokyo, Japan. homareda@kyorin-u.ac.jp
Ushimaru
Makoto
M
D009597:4-Nitrophenylphosphatase / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000494:pharmacology*; D000818:Animals; D004285:Dogs; D004789:Enzyme Activation; D006863:Hydrogen-Ion Concentration; D009840:Oligomycins / Q000494:pharmacology*; D010042:Ouabain / Q000494:pharmacology; D012965:Sodium Chloride / Q000494:pharmacology; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism*
D009840:Oligomycins; D012965:Sodium Chloride; D010042:Ouabain; D000255:Adenosine Triphosphate; D009597:4-Nitrophenylphosphatase; D000254:Sodium-Potassium-Exchanging ATPase