FIGURE 3.

IP production, identity, and kinetics. A, production of IP₄ and IP₅ over time. Time courses of generation of IP₄ and IP₅ by wild-type and K121W A. thaliana IPMK from [³H]Ins(1,4,5)P₃. Note the simultaneous appearance of IP₄ and IP₅ in the wild-type reactions, but no IP₅ signal for the K121W reactions, even after 60 min. B, identification of the K121W IP₄ product as Ins(1,4,5,6)P₄. Samples containing [³²P]IP₄ produced by K121W and [³H]Ins(1,3,4,5)P₄ as an internal standard treated or not treated with 5-ptas and separated by HPLC, and radioactivity was counted on ³H and ³²P channels simultaneously. 5-Ptase product is observed in only for [³H]IP₄. C, representative TLC plate of [³²P]IP₄ assay. D, representative HPLC trace of [³H]Ins(1,4,5)P₃ assay. E, Michaelis-Menten steady-state kinetic curves for Ins(1,4,5)P₃ and Ins(1,4,5,6)P₄ with wild-type and K121W A. thaliana IPMK.