FIGURE 10.

Formation of E1Ca₂·BeF₃⁻ from E2·BeF₃⁻ by lumenal Ca²⁺ binding. E2·BeF₃⁻ was first produced in SR vesicles with BeF_x in the presence and absence of 1.2 μm A23187 at 25 °C in 0.5 mm EGTA, 15 mm MgCl₂, 0.1 m KCl, and 30 mm MOPS/Tris (pH 7.0). Then a small volume of CaCl₂ was added to give various Ca²⁺ concentrations as indicated. At 10 s after this Ca²⁺ addition, TNP-AMP fluorescence was monitored with 4 μm TNP-AMP. In A and B, the maximum intensity in the presence of A23187 was plotted in semi-log scale at 0–0.9 mm Ca²⁺ (A) and 0.7–15 mm Ca²⁺ (B). Note also the different scales in the ordinates. The inset in A is the linear plot at 0–0.9 mm Ca²⁺ to clearly show the saturation of the first phase of the Ca²⁺-dependent change, i.e. the formation of E1Ca₂·BeF₃⁻ from E2·BeF₃⁻. In C, the maximum superfluorescence intensity in the absence of A23187 was plotted at 0–15 mm Ca²⁺. In D and E, the traces of superfluorescence development upon TNP-AMP addition in the presence of A23187 (D) or its absence (E) were shown at the representative Ca²⁺ concentrations (0, 0.7 or 1.0, and 10 mm). Solid lines in A and B show the least squares fit to the Hill equation. Apparent Ca²⁺ affinity (K_0.5) and Hill coefficients at 0–0.9 mm Ca²⁺ are 0.43 mm and 3.8 with the intensity decrease from 170 to 142, and those at Ca²⁺ over 0.7 mm are 1.7 mm and 1.0 with a further intensity decrease to 36.