Characterization of zPIP5Kα Kinase Activity

The activity assay was modified from the approach reported previously [14 (link)]. The following components were included in reaction (50 μl): 100 mM Tris–HCl (pH 7.4), 50 mM EGTA, 100 mM MgCl₂, 100 ng of purified zPIP5Kα, 20 μM ATP with 1 μCi [³²P]-gamma ATP (PerkinElmer) and 10 μM PI(4)P (phosphatidylinositol 4-phosphate; Echelon Biosciences, Inc.). The reaction was initiated by adding 100 ng of purified enzyme. After processing at room temperature for 1 h, it was stopped by the addition of the lipid extraction solution containing chloroform, methanol and HCl with a volume ratio of 3.3 : 3.7 : 0.1, as well as 10 μg/ml Brain extract from bovine brain (Type I, Folch Fraction I, Sigma). After vortexing for 20 s, the sample was centrifuged at 2000×g for 1 min, and the lower organic phase was collected, spotted and separated by thin layer chromatography. The product of the reaction was quantified by a Storm 820 PhosphorImager (GE).
To determine the kinetic parameters, the measured initial reaction rates were plotted against different concentrations of the substrate (0.2–10 times of the corresponding K_m). The K_m and V_max were calculated by curve fitting to Michaelis–Menton equation in OriginPro® 8.0.