PURIFICATION AND CHARACTERIZATION OF A SOLUBLE PHOSPHATIDYLINOSITOL 4-KINASE FROM CARROT SUSPENSION-CULTURE CELLS

Previously we reported the presence of a soluble phosphatidylinositol 4-kinase (PI il-kinase) in carrot (Daucus carota L.) suspension culture cells (C.M. Okpodu, W. Cross, W.F. Boss [1990] Plant Physiol 93: S-63). We have purified the enzyme over 1000-fold using Q-Sepharose ion exchange, hydroxylapatite, and G-100 gel filtration column chromatography. The M(r) of the enzyme was estimated to be 83,000 by gel filtration. PI 4-kinase activity was recovered after renaturation of the 80-kD region of polyacrylamide gels, and an 80-kD peptide cross-reacted with antibodies to the yeast 55-kD membrane-associated PI 4-kinase on western blots. The isolated lipid kinase phosphorylated PI but not lysophosphatidylinositol or phosphatidylinositol monophosphate. Maximal PI kinase activity occurred when the substrate was added as Triton X-100/PI mixed micelles at pH 8. The enzyme required divalent cations. At low concentrations (1-5 mM), Mn2+ was more effective than Mg2+ in increasing enzyme activity; however, maximal activity occurred at 25 to 40 mM Mg2+. Calcium from 0.01 mu M to 1 mM had no effect on the enzyme activity. The K-m of the enzyme for ATP was estimated to be between 400 and 463 mu M. The enzyme was inhibited by adenosine (100 mu M); however, ADP (up to 100 mu M) had no effect on the activity. The biochemical characteristics of the carrot soluble PI 4-kinase are compared with the previously reported PI il-kinases from animals and yeast.