Fig. 6

Effect of 1,2-DCB on functional states of SERCA as determined by ATP phosphorylation. SR vesicles at 0.1 mg of protein/ml were incubated at 25 °C in a medium containing: a 20 mM Mops, pH 7.0, 80 mM KCl, 5 mM MgCl₂, 15 μM A23187, 0.1 mM EGTA, 10 μM free Ca²⁺, and a certain 1,2-DCB concentration starting with 50 μM [γ-³²P]ATP (●). Inset: The E₂ + 1,2-DCB prior phosphorylation was starting with 10 μM free Ca²⁺ and 50 μM [γ-³²P]ATP (○). The phosphorylation in each case was maintained for 2 s at 0 °C. b For phosphorylation initiating from E₂ATP Ca²⁺ free, the enzyme (0.1 mg of protein/ml) was first treated with 1,2-DCB in a media containing 20 mM Mops, pH 7.0, 80 mM KCl, 5 mM MgCl₂, 15 μM A23187, 0.1 mM EGTA, followed by phosphorylation at 0 °C or 25 °C by adding [γ-³²P]ATP and 10 μM free Ca²⁺. For more details see Methods