FIGURE 9.

Kinetics of lumenal Ca²⁺-induced change of E2P to E1PCa₂ in the wild type in the presence of K⁺ without ADP. A, the microsomes expressing the wild type were phosphorylated with ³²P_i in the presence of A23187 and absence of Ca²⁺ and chilled on ice, as described in Fig. 7. Subsequently, the phosphorylated sample was mixed at 0 °C with a 20-fold volume of chase solution containing 105 mm KCl and various concentrations of CaCl₂ without ADP, otherwise as described in Fig. 7. The final free Ca²⁺ concentrations were indicated in the figure. At the indicated time periods after this addition, the chase reaction was terminated by trichloroacetic acid, and the amount of EP was determined. Solid lines show the least squares fit to a double exponential decay. The EP remaining in the second and slow phase was all in the ADP-sensitive form (thus E1PCa₂, data not shown), and the first and rapid phase is the forward hydrolysis of E2P without bound Ca²⁺. B, the fraction of EP in the second phase in the total amount of EP was obtained by extrapolating to the zero time in the double exponential decay fitting, and plotted versus the Ca²⁺ concentration. The data were fitted well with the Hill equation (solid line), and the Ca²⁺ concentration giving the 50% saturation and the Hill coefficient were found to be 750 μm and 1.5. Here note that the EP amount in the second phase is dependent on the ratio between the rate of the forward E2P hydrolysis and that of the reverse E2P to E1PCa₂ conversion upon the lumenal Ca²⁺ binding to E2P: the plot reflects the relative values between these forward and reverse rates of E2P rather than the lumenal Ca²⁺ affinity of E2P. C, by using the data obtained in A and B with the wild type in the presence of K⁺, the rate of the lumenal Ca²⁺-induced E1PCa₂ formation from E2P (k_rev (○)) was calculated at each Ca²⁺ concentration by the equation, k_rev = k_hF_s/(1 - F_s). Here, F_s is the fraction of EP in the second phase, and k_h is the forward hydrolysis rate of E2P without Ca²⁺. For comparison with the wild type in the absence of K⁺ (•) and Y122A in the presence of K⁺ (▵), their lumenal Ca²⁺ access rates (the rates of the lumenal Ca²⁺-induced reverse E2P decay via E1PCa₂ in the presence of ADP) obtained in Fig. 8A are plotted.