title: Rapid inhibition of the K(+)-sensitive phosphoenzyme of Na+/K(+)-ATPase by (Z)-5-methyl-2-[2-(1-naphthyl)ethenyl]-4-piperidinopyridine.
abstract: Membrane-bound Na+,K(+)-ATPase (0.1 mg/ml) was incubated with the K(+)-site-directed probe (Z)-5-methyl-2-[2-(1-naphthyl)ethenyl]-4-piperidinopyridine (AU-1421) (Takada, J. et al. (1990) Biochim. Biophys. Acta 1037, 373-379) at 37 degrees C for 30 min in the absence of ligands, then the Na(+)-dependent phosphorylation level was examined in the presence of 10 microM [32P]ATP at 0 degrees C. The level was decreased to 50% and 0% by about 50 microM and 100 microM AU-1421, respectively. Addition of 1 mM K+ during the treatment with AU-1421 resulted in complete maintenance of the phosphorylation level. When the preincubation was performed at 0 degrees C for 10 s, even 100 microM AU-1421 did not impair the phosphorylation. In contrast to the non-phospho form of the enzyme, the K(+)-sensitive phosphoenzyme formed from ATP was immediately inhibited by the addition of AU-1421 at 0 degrees C. The reactivity of the inhibited phosphoenzyme was restored by the addition of K+. About 1 mM K+ gave the same maximal reactivity in the presence of various fixed concentrations (8-41 microM) of AU-1421, but the apparent affinity for K+ decreased simply with the increase of AU-1421 concentration. From this simple competitive relationship, the apparent Ki value of AU-1421 for the phosphoenzyme was calculated to be 7.2 microM. Compared to the non-phospho form of the enzyme, the phospho form appears to be rather susceptible to AU-1421, probably because the K(+)-site of the phosphoenzyme is exposed to the extracellular aqueous phase.
authors.affiliation: Central Research Laboratories, Banyu Pharmaceutical Co., Ltd., Tokyo, Japan.
authors.forename: J
authors.initials: J
authors.lastname: Takada
authors.forename: Y
authors.initials: Y
authors.lastname: Fukushima
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D002118:Calcium / Q000494:pharmacology; D007679:Kidney Medulla / Q000201:enzymology; D008345:Manganese / Q000494:pharmacology; D009281:Naphthalenes / Q000494:pharmacology*; D010042:Ouabain / Q000378:metabolism / Q000494:pharmacology; D010766:Phosphorylation; D011188:Potassium / Q000494:pharmacology*; D011725:Pyridines / Q000494:pharmacology*; D012756:Sheep; D000254:Sodium-Potassium-Exchanging ATPase / Q000037:antagonists & inhibitors*
chemical_list: D009281:Naphthalenes; D011725:Pyridines; C063370:AU 1421; D008345:Manganese; D010042:Ouabain; D000255:Adenosine Triphosphate; D000254:Sodium-Potassium-Exchanging ATPase; D011188:Potassium; D002118:Calcium