title: Free protons do not substitute for sodium ions in buffer-mediated phosphorylation of (Na+ + K+)-ATPase.
abstract: In view of our recent finding of imidazole-activation of the phosphorylation of (Na+ + K+)-ATPase and the suggestion by others of an activating role of protons, in lieu of sodium ions, in the overall hydrolytic and phosphorylation processes of the enzyme, we have investigated the effect of pH on the phosphorylation process. No indication of proton activation is found. Rather, phosphorylation at low pH in the absence of Na+ is dependent on the buffer concentration. Imidazole-H+ stimulated phosphorylation at pH 5 reaches the same maximal steady-state level as Na+-stimulated phosphorylation. Low pH also elicits Tris-H+ stimulated phosphorylation, but due to a simultaneous inhibitory effect of this buffer the maximal steady-state level is no more than 50% of the Na+-stimulated phosphorylation level. Protons inhibit rather than activate phosphorylation. Upon decreasing the pH from 7 to 5, we observe for all ligands, whether activating or inhibiting phosphorylation (ATP, Na+, protonated imidazole, Mg2+ and K+), a decrease in affinity (largest for Mg2+) and a decrease in the maximal steady-state phosphorylation capacity. The effects of Na+ and imidazole-H+ on the phosphorylation step have been compared with those on the E2----E1 conformational change, which leads to the phosphorylation step. The different pH-dependence of the affinities for Na+ and protonated buffer in the E2----E1 transition suggests that there are separate activation sites with different pK values for Na+ and the buffer cation. The above findings rule out a role of free protons as a substitution for Na+ in the phosphorylation process.
mesh_terms: D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D002021:Buffers; D004789:Enzyme Activation / Q000187:drug effects; D006863:Hydrogen-Ion Concentration; D007093:Imidazoles / Q000494:pharmacology; D008274:Magnesium / Q000494:pharmacology; D010766:Phosphorylation; D011188:Potassium / Q000494:pharmacology; D011487:Protein Conformation; D011522:Protons; D011817:Rabbits; D012964:Sodium / Q000494:pharmacology*; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism*
chemical_list: D002021:Buffers; D007093:Imidazoles; D011522:Protons; C029899:imidazole; D000255:Adenosine Triphosphate; D012964:Sodium; D000254:Sodium-Potassium-Exchanging ATPase; D008274:Magnesium; D011188:Potassium