Kinetic characterization of Na,K-ATPase from rabbit outer renal medulla: properties of the (alpha beta)(2) dimer.
We describe and compare the main kinetic characteristics of the (alpha beta)(2) form of rabbit kidney Na,K-ATPase. The dependence of ATPase activity on ATP concentration revealed high (K(0.5)=4 microM) and low (K(0.5)=1.4 mM) affinity sites for ATP, exhibiting negative cooperativity and a specific activity of approximately 700 U/mg. For p-nitrophenylphosphate (PNPP) as substrate, a single saturation curve was found, with a smaller apparent affinity of the enzyme for this substrate (K(0.5)=0.5 mM) and a lower hydrolysis rate (V(M)=42 U/mg). Stimulation of ATPase activity by K(+) (K(0.5)=0.63 mM), Na(+) (K(0.5)=11 mM) and Mg(2+) (K(0.5)=0.60 mM) all showed V(M)'s of approximately 600 U/mg and negative cooperativity. K(+) (K(0.5)=0.69 mM) and Mg(2+) (K(0.5)=0.57 mM) also stimulated PNPPase activity of the (alpha beta)(2) form. Ouabain (K(0.5)=0.01 microM and K(0.5)=0.1 mM) and orthovanadate (K(0.5)=0.06 microM) completely inhibited the ATPase activity of the (alpha beta)(2) form. The kinetic characteristics obtained constitute reference values for diprotomeric (alpha beta)(2)-units of Na,K-ATPase, thus contributing to a better understanding of the biochemical mechanisms of the enzyme.
de Lima Santos
Hérica
H
Departamento de Química, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto-FFCLRP, Universidade de São Paulo-USP, Av. Bandeirantes 3900, SP, 14040-901, Ribeirão Preto, Brazil.
Ciancaglini
Pietro
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D000255:Adenosine Triphosphate / Q000378:metabolism; D000814:Aniline Compounds / Q000378:metabolism; D000818:Animals; D019281:Dimerization; D006868:Hydrolysis / Q000187:drug effects; D007679:Kidney Medulla / Q000201:enzymology*; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism; D009943:Organophosphorus Compounds / Q000378:metabolism; D010042:Ouabain / Q000494:pharmacology; D011188:Potassium / Q000378:metabolism; D011817:Rabbits; D012964:Sodium / Q000378:metabolism; D000254:Sodium-Potassium-Exchanging ATPase / Q000037:antagonists & inhibitors / Q000378:metabolism*
D000814:Aniline Compounds; D009943:Organophosphorus Compounds; D010042:Ouabain; C068798:4-aminophenylphosphate; D000255:Adenosine Triphosphate; D012964:Sodium; D000254:Sodium-Potassium-Exchanging ATPase; D008274:Magnesium; D011188:Potassium