Figure 4. Dephosphorylation reactions of PPIP5K2^KD and substrate saturation plots for ATP and ADP.

(A) Either no enzyme (broken line) or 0.56 μg/ml PPIP5K2^KD (solid line) was incubated with 10 μM [³H]InsP₈ and 5 mM ADP for 10 min as indicated in the Materials and methods section. The reactions were quenched and neutralized and analysed by Partisphere SAX HPLC as described in the Materials and methods section. Representative HPLC data are shown. (B) The initial velocity of InsP₈ dephosphorylation was determined for reverse reactions under pseudo first-order rate conditions [38] in which the concentration of the designated inositol phosphate was fixed at saturating levels (10 μM; Table 1), and the concentration of nucleotide was varied as indicated. Each individual data point was analysed by HPLC as described under the Materials and methods section. The Michaelis–Menten equation was fitted to the data using non-linear regression (GraphPad Prism v5.03). Results are presented as the means±S.E.M. (C) Either no enzyme (broken line) or 27 μg/ml PPIP5K2^KD (solid line) was incubated with 10 μM 1-[³H]InsP₇ and 28 μM ADP for 45 min as indicated in the Materials and methods section. The reactions were quenched and neutralized and analysed by Partisphere SAX HPLC as described in the Materials and methods section. Representative HPLC data are shown. (D) The initial velocity of 1-InsP₇ dephosphorylation was determined as described in the legend to panel (B) for InsP₈.