(Z)-5-methyl-2-[2-(1-naphthyl)ethenyl]-4-piperidinopyridine (AU-1421), calcium ions and ethylenediamine as the K(+)-site directed probe for Na+/K(+)-ATPase.
Recently, we have shown that a hydrophobic amine (AU-1421) produces an irreversible inactivation of Na+/K(+)-ATPase activity. This inactivation was prevented by K+ and its congeners. In this study, we examined the possibility of Ca2+ or ethylenediamine as a probe of the K+ occlusion center of Na+/K(+)-ATPase. The inactivation by AU-1421 was prevented by Ca2+ with an apparent high affinity (approximately 0.1 mM). Ca2+ protection was cancelled by high concentrations of ATP, ADP or Mg2+. Ca2+ and K+ were similar in these respects. Kinetic analyses of the above data indicated the presence of two AU-1421 occlusion sites on the enzyme, either one of which is susceptible to Ca2+ occlusion. Ethylenediamine also prevented the inactivation by AU-1421 or by C12E8 solubilization of the enzyme, suggesting that ethylenediamine, like K+, stabilized the enzyme. However, an apparent affinity of ethylenediamine (approximately 1.4 mM) was one order of magnitude lower than that of K+ (approximately 0.2 mM), and the protective manner did not show a simple competition. In addition, ethylenediamine binding was unaffected by ATP or ADP at a low affinity site, and antagonized K+ binding. From these results we concluded that ethylenediamine does not act like K+ or Ca2+ in protecting AU-1421 inactivation, since it can't stabilize the enzyme conformation as an E2 (K(+)-bound form).
Takada
J
J
Central Research Laboratories, Banyu Pharmaceutical Company, Tokyo, Japan.
Hioki
Y
Y
Yano
M
M
D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000494:pharmacology*; D005029:Ethylenediamines / Q000494:pharmacology*; D007425:Intracellular Membranes / Q000201:enzymology; D007679:Kidney Medulla / Q000201:enzymology; D007700:Kinetics; D008861:Microsomes / Q000201:enzymology; D009281:Naphthalenes / Q000494:pharmacology*; D011188:Potassium / Q000378:metabolism*; D011725:Pyridines / Q000494:pharmacology*; D000254:Sodium-Potassium-Exchanging ATPase / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D013552:Swine
D005029:Ethylenediamines; D009281:Naphthalenes; D011725:Pyridines; C063370:AU 1421; C031234:ethylenediamine; D000254:Sodium-Potassium-Exchanging ATPase; D011188:Potassium; D002118:Calcium