FIGURE 9.

Two Ca²⁺ are bound in E1Ca₂·BeF_x formed from E1Ca₂ and from E2·BeF₃⁻ at 0.7 mm Ca²⁺. A, SR vesicles in 0.7 mm ⁴⁵Ca²⁺ were incubated with BeF_x in the absence or presence of 5 μm A23187, otherwise as described in the legend to Fig. 4. The amount of bound Ca²⁺ was obtained with subtraction of the background level (10.1 ± 0.5 nmol/mg (n = 6)) determined by EGTA washing the vesicles incubated without BeF_x and A23187. The occluded Ca²⁺ was determined by EGTA washing in the absence of A23187 and by subtraction of the background level. (Here the determination of occluded Ca²⁺ in A23187 by EGTA washing was not feasible, because in the absence of (or even in 0.1 mm) Ca²⁺, A23187 converts E1Ca₂·BeF_x very rapidly to E2·BeF₃⁻ releasing Ca²⁺ (supplemental Figs. 4 and 5). B, E2·BeF₃⁻ was first produced in SR vesicles without A23187 and Ca²⁺. Subsequently, the samples were diluted 10 times with the buffer containing ⁴⁵CaCl₂ and BeF_x with and without 5 μm A23187, to give 0.7 mm ⁴⁵Ca²⁺ and the same buffer conditions as in A. At 15 s after dilution, the amount of bound ⁴⁵Ca²⁺ was determined without EGTA washing and by subtracting the nonspecific Ca²⁺ binding (1.0 ± 0.1 nmol/mg (n = 6)) determined by EGTA washing the sample incubated without BeF_x and A23187.