FIGURE 4.

Accumulation of ADP-insensitive EP in the presence (A) and absence (B) of K⁺. Microsomes expressing the wild type or mutant were phosphorylated with 10 μm [γ-³²P]ATP at 0 °C for 15 s in 50 μl of a mixture containing 2.5 μg of microsomal protein, 1 μm A23187, 7 mm MgCl₂, 0.05 mm CaCl₂, 50 mm MOPS/Tris (pH 7.0), and 0.1 m KCl (A) or 0.1 m LiCl without KCl (B). The total amount of EP formed was determined by the acid-quenching. For determination of ADP-insensitive EP (E2P), an equal volume of a mixture containing 10 mm ADP, 7 mm MgCl₂, 10 mm EGTA, 50 mm MOPS/Tris (pH 7.0), and 0.1 m KCl (A) or LiCl without KCl (B) was added to the above phosphorylation mixture, and the reaction was quenched at 1 s after the ADP addition. ADP-sensitive EP (E1PCa₂) disappeared entirely within 1 s after the ADP addition. The amount of E2P is shown as percentage of the total amount of EP.