title: Na+-like effect of imidazole on the phosphorylation of (Na+ + K+)-ATPase.
abstract: A high basal level of phosphorylation (approx. 70% of the optimal Na+-dependent phosphorylation level) is observed in 50 mM imidazole-HCl (pH 7.0), in the absence of added Na+ and K+ and the presence of 10-100 microM Mg2+. In 50 mM Tris-HCl (pH 7.0) the basal level is only 5%, irrespective of the Mg2+ concentration. Nevertheless, imidazole is a less effective activator of phosphorylation than Na+ (Km imidazole-H+ 5.9 mM, Km Na+ 2 mM under comparable conditions). Imidazole-activated phosphorylation is strongly pH dependent, being optimal at pH less than or equal to 7 and minimal at pH greater than or equal to 8, while Na+-activated phosphorylation is optimal at pH 7.4. This suggests that imidazole-H+ is the activating species. Imidazole facilitates Na+-stimulated phosphorylation. The Km for Na+ decreases from 0.63 mM at 5 mM imidazole-HCl to 0.21 mM at 50 mM imidazole-HCl (pH 7; 0.1 mM Mg2+ in all cases). Imidazole-activated phosphorylation is more sensitive to inhibition by K+ (I50 = 12.5 microM) than Na+-activated phosphorylation (I50 = 180 microM). Mg2+ antagonizes activation by imidazole-H+ and also inhibition by K+. The Ki value for Mg2+ (approx. 0.3 mM) is the same for the two antagonistic effects. Tris buffer (pH 7.0) inhibits imidazole-activated phosphorylation with an I50 value of 30 mM in 50 mM imidazole-HCl (pH 7.0) plus 0.1 mM Mg2+. We conclude that imidazole-H+, but not Tris-H+, can replace Na+ as an activator of ATP-dependent phosphorylation, primarily by shifting the E2----E1 transition to the right, leading to a phosphorylating E1 conformation which is different from that in Tris buffer.
authors.forename: F M
authors.initials: FM
authors.lastname: Schuurmans Stekhoven
authors.forename: H G
authors.initials: HG
authors.lastname: Swarts
authors.forename: J J
authors.initials: JJ
authors.lastname: de Pont
authors.forename: S L
authors.initials: SL
authors.lastname: Bonting
mesh_terms: D000818:Animals; D006863:Hydrogen-Ion Concentration; D007093:Imidazoles / Q000494:pharmacology*; D007679:Kidney Medulla / Q000201:enzymology; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D010766:Phosphorylation; D011188:Potassium / Q000494:pharmacology; D011817:Rabbits; D012964:Sodium / Q000494:pharmacology*; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism*; D014325:Tromethamine / Q000494:pharmacology
chemical_list: D007093:Imidazoles; D014325:Tromethamine; C029899:imidazole; D012964:Sodium; D000254:Sodium-Potassium-Exchanging ATPase; D008274:Magnesium; D011188:Potassium