Characterization of a nuclear phosphatidylinositol 4-kinase in carrot suspension culture cells

We have shown previously that a nuclear phosphatidylinositol (PI) 4-kinase activity was present in intact nuclei isolated from carrot suspension culture cells (Daucus carota L.). Here, we further characterized the enzyme activity of the nuclear enzyme. We found that the pH optimum of the nuclear-associated PI kinase varied with assay conditions. The enzyme had a broad pH optimum between 6.5–7.5 in the presence of endogenous substrate. When the substrate was added in the form of phosphatidylinositol/phosphatidylserine (PI/PS) mixed micelles (1 mM PI and 400 μM PS), the enzyme had an optimum of pH 6.5. In comparison, the pH optimum was 7.0 when PI/Triton X-100 mixed micelles (1 mM PI in 0.025 %, v/v final concentration of Triton X-100) were used. The nuclear-associated PI kinase activity increased 5-fold in the presence of low concentrations of Triton X-100 (0.05 to 0.3 %, v/v); however, the activity decreased by 30 % at Triton X-100 concentrations greater than 0.3 % (v/v). Calcium at 10 μM inhibited 100 % of the nuclear-associated enzyme activity. The Km for ATP was estimated to be between 36 and 40 μM. The nuclear-associated PI kinase activity was inhibited by both 50 μM ADP and 10 μM adenosine. Treatment of intact nuclei with DNase, RNase, phospholipase A2 and Triton X-100 did not solubilize the enzyme activity. Based on sensitivity to calcium, ADP, detergent, pH optimum and the product analysis, the nuclear-associated PI 4-kinase was compared with previously reported PI kinases from plants, animals and yeast.