title: A novel hydrophobic amine, (Z)-5-methyl-2-[2-(1-naphthyl)ethenyl]-4-piperidinopyridine, as a probe of the K+ occlusion center of Na+/K(+)-ATPase.
abstract: A hydrophobic amine, (Z)-5-methyl-2-[2-(1-naphthyl)ethenyl]-4-piperidinopyridine (AU-1421), was examined as a probe of the K+ occlusion center of Na+/K(+)-ATPase. Treatment of the enzyme with AU-1421 at 37 degrees C and pH 7.0 produced irreversible inactivation of the enzyme. This inactivation was prevented, with simple competitive kinetics, by K+ or its congeners in the order of Tl+ greater than Rb+ greater than NH+4 greater than Cs+. The concentrations of these cations required for the protection, were consistent with the affinities for transport and ATPase activity. The apparent binding constant for K+ was calculated to be 0.03 mM, from the competition with AU-1421. This protection was cancelled by a high concentration of ATP or ADP. A high concentration of Na+ (Kd = 6.5-6.9 mM), as a substitute for K+, also prevented the inactivation by AU-1421. Thus, the enzyme was protected from AU-1421 when the occlusion center was occupied by a monovalent cation, irrespective of the enzyme conformation, E1 (Na(+)-bound form) or E2 (K(+)-bound form). On the other hand, the enzyme was most sensitive to AU-1421 in the presence of low concentration of Na+ (0.4-0.8 mM) or a high concentration of ATP. Tris, imidazole or choline, which favors the E1 state, also accelerated the inactivation by AU-1421. These suggest that AU-1421 reacts with the occlusion center through the E1 state.
authors.affiliation: Central Research Laboratories, Banyu Pharmaceutical Co., Tokyo, Japan.
authors.forename: J
authors.initials: J
authors.lastname: Takada
authors.forename: Y
authors.initials: Y
authors.lastname: Hioki
authors.forename: M
authors.initials: M
authors.lastname: Yano
authors.forename: Y
authors.initials: Y
authors.lastname: Fukushima
mesh_terms: D000244:Adenosine Diphosphate / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000494:pharmacology; D001665:Binding Sites; D001667:Binding, Competitive; D002414:Cations, Monovalent; D004789:Enzyme Activation / Q000187:drug effects; D007700:Kinetics; D009281:Naphthalenes / Q000494:pharmacology*; D011188:Potassium / Q000378:metabolism / Q000494:pharmacology*; D011725:Pyridines / Q000494:pharmacology*; D012964:Sodium / Q000494:pharmacology; D000254:Sodium-Potassium-Exchanging ATPase / Q000037:antagonists & inhibitors* / Q000378:metabolism
chemical_list: D002414:Cations, Monovalent; D009281:Naphthalenes; D011725:Pyridines; C063370:AU 1421; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D012964:Sodium; D000254:Sodium-Potassium-Exchanging ATPase; D011188:Potassium