FIGURE 8.

Stability of E1Ca₂·BeF_x in various Ca²⁺ concentrations. A, E1Ca₂·BeF_x was first produced in SR vesicles in 0.1 mm Ca²⁺ and 15 mm MgCl₂ with BeF_x. Subsequently, Ca²⁺ was changed to 0.1 (unchanged), 0.4, 0.7, 1.1, 3.1, and 10.1 mm, and the incubations continued at 25 °C for 72 h. At the indicated periods, the superfluorescence with 4 μm TNP-AMP was examined at 25 °C, and the maximum levels obtained as described in the legend to Fig. 6 are shown. E2·BeF₃⁻ formed without Ca²⁺, E1Ca₂ in 0.1–10 mm Ca²⁺, and E2 without Ca²⁺ were also incubated. B, in the presence of 0.7 mm Ca²⁺, E1Ca₂ in SR vesicles was first incubated with and without 1.2 μm A23187 (A23), then BeF_x was added (E1Ca₂·BeF_x). E1Ca₂·BeF_x (DOC-E1Ca₂·BeF_x) was also produced from E1Ca₂ in 0.7 mm Ca²⁺ of the Ca²⁺-ATPase purified and delipidated from SR vesicles by deoxycholate (DOC) treatment (30). The incubation was continued for 12 days, otherwise as indicated in A. E2·BeF₃⁻ (E2·BeF₃⁻, DOC-E2·BeF₃⁻) without Ca²⁺ and E1Ca₂ in 0.7 mm Ca²⁺ (E1Ca₂, DOC-E1Ca₂) and E2 without Ca²⁺ (E2, DOC-E2) were also incubated. C–G, the fluorescence traces upon TNP-AMP addition were shown for the representative samples with incubation periods and Ca²⁺ concentration (mm). Note that at 0.7 mm Ca²⁺, both with and without A23187, the development of the E1Ca₂·BeF_x characteristic transient superfluorescence remained perfectly the same for 12 days. By contrast, the transient superfluorescence was converted to the stable and higher superfluorescence characteristic of E2·BeF₃⁻ at 0.1 and 0.4 mm Ca²⁺, and it was markedly reduced by 3 and 10 mm Ca²⁺ due to decomposition to E1Ca₂.