Choi Y R, Akera T
Biochim Biophys Acta. 1977 Apr 12;481(2):648-59. doi: 10.1016/0005-2744(77)90298-4.
The association and dissociation rate constants for the interaction of [3H]-ouabain with partially purified rat brain (Na+,K+)-ATPase (ATP phosphohydrolase, EC 3.6.1.3) in vitro were estimated from the time course of the [3H]-ouabain binding observed in the presence of Na+, Mg2+ and ATP by a polynomial approximation-curve-fitting technique. The reduction of the association rate constant by K+ was greater than its reduction of the dissociation rate constant. Thus, the affinity of Na+,K+)-ATPase for ouabain was reduced by K+. The binding-site concentration was unaffected by K+. Consistent with these findings, the addition of KCl to an incubation mixture at the time when [3H]-ouabain binding to (Na+,K+)ATPase is close to equilibrium, caused an immediate decrease in bound ouabain concentration, apparently shifting towards a new, lower equilibrium concentration. Dissociation rate constants which were estimated following the termination of the ouabain-binding reaction were different from those estimated with above methods and may not be useful in predicting the ligand effects on equilibrium of the ouabain-enzyme interaction.
通过多项式近似曲线拟合技术，根据在Na⁺、Mg²⁺和ATP存在的情况下观察到的[³H]哇巴因结合的时间进程，估算了[³H]哇巴因与部分纯化的大鼠脑（Na⁺，K⁺）-ATP酶（ATP磷酸水解酶，EC 3.6.1.3）体外相互作用的缔合和解离速率常数。K⁺对缔合速率常数的降低作用大于对解离速率常数的降低作用。因此，K⁺降低了（Na⁺，K⁺）-ATP酶对哇巴因的亲和力。结合位点浓度不受K⁺的影响。与这些发现一致的是，当[³H]哇巴因与（Na⁺，K⁺）-ATP酶的结合接近平衡时，向孵育混合物中加入KCl会导致结合的哇巴因浓度立即降低，显然是向一个新的、更低的平衡浓度转变。在哇巴因结合反应终止后估算的解离速率常数与用上述方法估算的不同，可能无助于预测配体对哇巴因-酶相互作用平衡的影响。
