Esmann M
Institute of Biophysics, University of Aarhus, Denmark.
Biochim Biophys Acta. 1992 Sep 21;1110(1):20-8. doi: 10.1016/0005-2736(92)90289-x.
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.
本文介绍了一种测定核苷酸与膜结合猪肾钠钾 - ATP酶结合和解离的个体速率常数的方法。该方法包括在停流荧光装置中，将存在曙红的钠钾 - ATP酶与ADP或ATP混合时，测定弛豫速率。结果表明，这种弛豫速率对核苷酸的依赖性，结合测得的曙红和ADP的平衡结合值，使得合理可靠地测定核苷酸解离速率常数成为可能，即在以下模型中测定速率常数k - 1（其中E表示钠钾 - ATP酶）：[公式：见原文] 所有实验均在约4℃下，于含有200 mM蔗糖、10 mM EDTA、25 mM Tris和73 mM NaCl（pH 7.4）的缓冲液中进行。测得的ADP解离速率常数约为6 s - 1，ATP解离速率常数约为2 - 3 s - 1。
