Determination of rate constants for nucleotide dissociation from Na,K-ATPase.
A method for determining individual rate constants for nucleotide binding to and dissociation from membrane bound pig kidney Na,K-ATPase is presented. The method involves determination of the rate of relaxation when Na,K-ATPase in the presence of eosin is mixed with ADP or ATP in a stopped-flow fluorescence apparatus. It is shown that the nucleotide dependence of this rate of relaxation--taken together with measured equilibrium binding values for eosin and ADP--makes possible a reasonably reliable determination of the rate constant for dissociation of nucleotide, i.e., determination of the rate constant k-1 in the following model (where E denotes Na,K-ATPase): [formula: see text] All experiments are carried out at about 4 degrees C in a buffer containing 200 mM sucrose, 10 mM EDTA, 25 mM Tris and 73 mM NaCl (pH 7.4). Values obtained for the rate constants for dissociation are about 6 s-1 for ADP and 2-3 s-1 for ATP.
Esmann
M
M
Institute of Biophysics, University of Aarhus, Denmark.
D000244:Adenosine Diphosphate / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D002462:Cell Membrane / Q000201:enzymology; D004801:Eosine Yellowish-(YS) / Q000378:metabolism; D007679:Kidney Medulla / Q000201:enzymology*; D007700:Kinetics; D008027:Light; D008433:Mathematics; D008954:Models, Biological; D011489:Protein Denaturation / Q000528:radiation effects; D000254:Sodium-Potassium-Exchanging ATPase / Q000302:isolation & purification / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013552:Swine
D000244:Adenosine Diphosphate; D000254:Sodium-Potassium-Exchanging ATPase; D004801:Eosine Yellowish-(YS)