FIGURE 9.

MgATP dependence of the decay rate of E1PCa₂ formed from ATP. E1PCa₂ was first formed in 50 μl of a microsomes suspension in 10 μm [γ-³²P]ATP, 10 μm Ca²⁺ (0.98 mm CaCl₂ with 1 mm EGTA), and 0.1 m KCl, otherwise as in Fig. 4A. Phosphorylation was terminated by 100 μl of a buffer containing 8 mm EGTA, 1 μm A23187, 0.1 m KCl, 50 mm MOPS/Tris (pH 7.0), and various concentrations of ATP and MgCl₂ (producing MgATP (more than 97% of the total ATP) with 6.2 mm free Mg²⁺). At different times after this MgATP addition, the decay reaction of E1PCa₂ at 0 °C was quenched by acid. The rate of the single exponential decay of E1PCa₂ obtained was plotted versus the MgATP concentration. Solid lines show the least squares fit to the Hill equation, and the parameters V₀ (the rate at the lowest 10 μm MgATP), V_max (the maximum rate), and K_0.5 (MgATP concentration giving the half-maximal change) are given in Table 2.