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 cultur e cells (C.M. Okpodu, W. Cross, W.F. Boss [1990] Plant Physiol 93: S-6 3). We have purified the enzyme over 1000-fold using Q-Sepharose ion e xchange, hydroxylapatite, and G-100 gel filtration column chromatograp hy. The M(r) of the enzyme was estimated to be 83,000 by gel filtratio n. 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 west ern blots. The isolated lipid kinase phosphorylated PI but not lysopho sphatidylinositol or phosphatidylinositol monophosphate. Maximal PI ki nase 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 increasi ng 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 activ ity. 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 ch aracteristics of the carrot soluble PI 4-kinase are compared with the previously reported PI il-kinases from animals and yeast.