FIGURE 4.

Ca²⁺ binding and occlusion at transport sites. The E1Ca₂ state ATPase of SR vesicles in various concentrations of ⁴⁵Ca²⁺ and 15 mm MgCl₂ was incubated with BeF_x, AlF_x, and AlF_x·ADP or without these compounds (E1Ca₂) for 30 min at 25 °C. The amounts of bound (upper panel) and occluded (lower panel) ⁴⁵Ca²⁺ were determined without and with the perfusion of the membrane filter with a 5 mm EGTA-containing washing solution (without CaCl₂ and fluoride compounds otherwise as the above incubation solution). The nonspecific Ca²⁺ binding was determined by including 10 μm thapsigargin before the addition of fluoride compounds, and subtracted. When ADP was used for E1Ca₂·AlF₄⁻·ADP, 5 μm A23187 was included to avoid Ca²⁺ accumulation in the vesicles by ATP produced from ADP due to adenylate kinase in the vesicles. In the inset, the stoichiometries of bound Ca²⁺ (open bars) and occluded Ca²⁺ (closed bars) to the phosphorylation site (P-site) were determined at saturating 50 μm ⁴⁵Ca²⁺. Solid lines in the upper panel show the least squares fit to the Hill equation. K_0.5 of Ca²⁺ and Hill coefficients obtained were 1.3 μm and 1.7 (E1Ca₂), 4.3 μm and 1.7 (E1Ca₂·BeF_x), 2.3 μm and 1.7 (E1Ca₂·AlF_x), and 0.4 μm and 1.4 (E1Ca₂·AlF₄⁻·ADP). In the lower panel, the values for E1Ca₂·BeF_x and E1Ca₂·AlF₄⁻·ADP are essentially not altered by EGTA washing (4.7 μm and 1.9, and 0.4 μm and 1.9, respectively).