E1P(3Na ⁺ )–E2P distribution evaluated by ADP sensitivity and E2(2K ⁺ ) → E1 conformational transition evaluated by ATP dependence of Na ⁺ ,K ⁺ -ATPase activity. (A) ADP-dependent dephosphorylation. Phosphorylation was carried out for 5 s at 0°C with 2 µM [γ-³²P]ATP in 20 mM Tris (pH 7.5), 100 mM NaCl, 50 mM choline chloride, 3 mM MgCl₂, 1 mM EGTA, and 10 µM ouabain. Dephosphorylation was initiated by the addition of 2.5 mM ADP and 1 mM unlabeled ATP. The dephosphorylation reaction was terminated by acid quenching after the indicated time intervals. Line plots represent the best fit of a bi-exponential decay function (Eq. 3 in Materials and methods) with the rate constant corresponding to the rapid phase, reflecting the ADP reaction with E1P, set to 2 s⁻¹. The relative fraction of E2P together with the rate constant corresponding to the slow phase, reflecting E2P dephosphorylation, are indicated in Table 1 as mean ± SD and number of independent experiments. (B) ATP dependence of Na⁺,K⁺-ATPase activity. ATPase activity was determined at 37°C in 130 mM NaCl, 20 mM KCl, 3 mM MgCl₂, 30 mM histidine (pH 7.4), 1 mM EGTA, 10 µM ouabain, and the indicated ATP concentrations. Line plots represent the best fit of a Hill function (Eq. 1 in Materials and methods). K_0.5 values (in µM ± SD) and number of experiments (n) in parentheses are: WT, 402 ± 74 (12); Y780A, 63 ± 20 (6); Y780L, 362 ± 122 (4); Y780F, 368 ± 19 (4); Y780Q, 121 ± 61 (7). In all panels, symbols are mean ± SEM (seen only when larger than the symbols). For each assay, the WT data from the left panel is reproduced as broken lines in the other panels.