Figure 4.

Excitation wavelength scans indicate substantial lumenal de-esterification of Magfura-5 AM in canine cardiac SR vesicles. Vesicles were incubated with Magfura-5 AM (10 μM) for 120 min at 4°C. Each assay consists of 100 μg of protein in 1.5 ml of assay buffer (150 mM sucrose/50 mM KCl/5% PEG/10 mM Hepes, pH 7.0). Assays were performed at 30°C in a stirred cuvette. Traces describe excitation scans from 260–450 nm measuring emission at 510 nm; (a) Control; (b) addition of 20 μM Mn²⁺ (to quench external Magfura-5); (c) Addition of ionomycin (5 μM) plus Mn²⁺ (50 μM). The ion selectivity of ionomycin necessitates an increase in [Mn²⁺] to observe rapid quenching of lumenal Magfura-5.