H,K-ATPase and Na,K-ATPase show the highest degree of sequence similarity among all other members of the P-type ATPases family. To explore their common features in terms of ligand binding, we evaluated conformational transitions due to the binding of Na⁺, K⁺ and Pi in the H,K-ATPase, and compared the results with those obtained for the Na,K-ATPase. This work shows that eosin fluorescence time courses provide a reasonably precise method to study the kinetics of the E1−E2 conformational changes in the H,K-ATPase. We found that, although Na⁺ shifts the equilibrium toward the E1 conformation and seems to compete with H⁺ in ATPase activity assays, it was neither possible to isolate a Na⁺-occluded state, nor to reveal an influx of Na⁺ related to H,K-ATPase activity. The high rate of the E2K → E1 transition found for the H,K-ATPase, which is not compatible with the presence of a K⁺-occluded form, agrees with the negligible level of occluded Rb⁺ (used as a K⁺ congener) found in the absence of added ligands. The use of vanadate and fluorinated metals to induce E2P-like states increased the level of occluded Rb⁺ and suggests that—during dephosphorylation—the probability of K⁺ to remain occluded increases from the E2P-ground to the E2P-product state. From kinetic experiments we found an unexpected increase in the values of k_obs for E2P formation with [Pi]; consequently, to obey the Albers-Post model, the binding of Pi to the E2 state cannot be a rapid-equilibrium reaction.

H，K-ATPase和Na，K-ATPase在P型ATPase家族的所有其他成员中显示出最高程度的序列相似性。为了探索它们在配体结合方面的共同特征，我们评估了由于H，K-ATPase中Na ⁺，K ⁺和Pi的结合而导致的构象转变，并将结果与​​Na，K-ATPase的结果进行了比较。这项工作表明曙红荧光时间进程提供了一种合理精确的方法来研究H，K-ATPase中E 1- E 2构象变化的动力学。我们发现，尽管Na ⁺使平衡朝E 1构象移动，并且似乎与H ⁺竞争在ATP酶活性测定中，既不可能分离出Na ⁺闭塞的状态，也不能揭示与H，K-ATPase活性有关的Na ⁺流入。H，K-ATPase的高E 2K→  E 1跃迁速率与存在K ^+-闭环形式不相容，与可忽略的Rb ⁺（用作K ⁺在没有添加配体的情况下发现）。使用钒酸盐和氟化金属诱导类似E 2P的状态会增加Rb ⁺的吸留水平，并表明-在去磷酸化过程中-K ⁺的可能性保持被遮挡的状态从E 2P接地增加到E 2P乘积状态。从动力学实验，我们发现在的值意外增加ķ _OBS用于é 2P形成与[PI]; 因此，为了服从Albers-Post模型，Pi与E 2状态的结合不能成为快速平衡反应。