Fig. 1.

Accumulation of⁴⁵Ca²⁺ into resealed vesicles derived from SPM. A, Freshly prepared SPM (○, •) or previously resealed SPM vesicles (▵, ▴) was incubated with⁴⁵Ca²⁺, and aliquot (100 μl) at each incubation time was used for determination of⁴⁵Ca²⁺ incorporation, as described in Experimental Procedures. Results in the figure are representative profiles of the time course of⁴⁵Ca²⁺ accumulation. Vehicle (○, ▵) or 5 μm A-23187 (•, ▴) was added to the assay tube at 10 min. B, An electron microscopic (negative-staining) image of the resealed SPM vesicles.C, Time course of ATP- and calmodulin-dependent⁴⁵Ca²⁺ accumulation into previously resealed SPM vesicles, which was prepared in Experimental Procedures. Vehicle (▴), 5 μg/ml of calmodulin (▵), 1 mm ATP (•), or 5 μg/ml calmodulin plus 1 mm ATP (○) was added simultaneously with⁴⁵Ca²⁺ to the tube containing previously resealed SPM vesicles. Experiments were performed under the condition of free [Ca²⁺] at 10 μm using 0.109 mmCaCl₂ and 0.1 mm EGTA. Each point of data represents the mean ± SEM from three separate experiments. D, Blockade of⁴⁵Ca²⁺ accumulation into resealed SPM vesicles and unlysed microsomes by pretreatment with various concentrations of saponin. In both experiments using resealed SPM vesicles and unlysed microsomes, incubation was carried out at 37°C for 30 min under the condition of free [Ca²⁺] at 100 nm in the presence or absence of 1 mm ATP. In resealed SPM vesicles, the ATP-dependent⁴⁵Ca²⁺ incorporation in control (without saponin) resealed SPM vesicles (0.25 mg of protein/fraction) was 699 ± 20 cpm/fraction. Experiments using intact microsomes were performed as described in Experimental Procedures. The ATP-dependent ⁴⁵Ca²⁺incorporation in control microsomes (0.25 mg of protein/fraction) was 209 ± 9.3 cpm/fraction. Data represent the mean ± SEM from three separate experiments.