(A, top) This graphic depicts how Ins(1,3,4)P3 stimulates Ins(1,3,4,5,6)P5 dephosphorylation by ITPK1 [47]. The 1-phosphate group (shown in red) that is removed from Ins(1,3,4,5,6)P5 is not released into the bulk phase but is retained by the nucleotide and donated to Ins(1,3,4)P3; this returns the enzyme to its ADP state, ready for another cycle of Ins(1,3,4,5,6)P5 dephosphorylation. (A, bottom) The analogous potential reaction sequence for PPIP5K. (B) Either no enzyme (broken line) or 0.63 μg/ml PPIP5K2KD (solid line; dotted line) was incubated with 10 μM [3H]InsP8 and 10 mM ADP for 10 min as indicated in the Materials and methods section. Where indicated (dotted line) incubations also contained 10 μM InsP6. 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.