Figure 7. Effect of ATP/ADP and AMP upon the equilibrium condition for PPIP5K2^KD.

(A) and (B) show a time course of InsP₆ and 5-InsP₇ phosphorylation, respectively. In (A), 20 μg/ml enzyme was incubated with reaction buffer (see the Materials and methods section) containing 15 μM InsP₆, 5 mM ATP and 0.5 mM ADP. In (B), 0.33 μg/ml enzyme was incubated with reaction buffer (see the Materials and methods section) containing 1 μM 5-InsP₇ and 5 mM ATP. Assays were quenched and neutralized and analysed by Partisphere SAX HPLC as described in the Materials and methods section. (C) and (D) show the equilibrium point for the phosphorylation of 5-InsP₇ to InsP₈ in which 0.33 μg/ml enzyme was incubated as described in the Materials and methods section under initial conditions of 1 μM 5-InsP₇ and either 1 (squares) or 5 (diamonds) mM ATP; either the ATP/ADP ratio (C) or the ATP/AMP ratio (D) was varied as indicated. Assays were conducted for 60 min. The reactions were then quenched and neutralized and analysed by Partisphere SAX HPLC as described in the Materials and methods section.