title: Purification and characterization of a phosphatidylinositol 4-kinase from bovine uteri.
abstract: Phosphatidylinositol 4-kinase has been purified 10,148-fold to a specific activity of 2.7 mumol/mg/min from bovine uteri. This purification was accomplished by detergent extraction of an acetone powder, ammonium sulfate precipitation, and chromatography on MonoQ, S-Sepharose, MonoP, and hydroxylapatite columns. The purified enzyme has a molecular mass of 55 kDa and appears to be monomeric. Kinetic analyses of the enzymatic activity demonstrated apparent Km values of 18 microM and 22 micrograms/ml (approximately 26 microM) for ATP and phosphatidylinositol, respectively, optimal activity in the pH range of 6.0-7.0, and a sigmoidal dependence of enzymatic activity on [Mg2+]. Ca2+ inhibited the enzyme at nonphysiological concentrations with 50% inhibition observed at a free [Ca2+] of approximately 300 microM. The purified enzyme efficiently utilized both ATP and 2'-deoxy-ATP as phosphoryl donors and specifically phosphorylated phosphatidylinositol on the fourth position. No phosphatidylinositol-4-phosphate 5-kinase activity was observed in the purified enzyme preparations. To our knowledge, this is the first reported purification of a phosphatidylinositol-specific phosphatidylinositol 4-kinase.
authors.affiliation: Molecular Biology and Biochemistry Program, Washington University Medical School, St. Louis, Missouri 63110.
authors.forename: F D
authors.initials: FD
authors.lastname: Porter
authors.forename: Y S
authors.initials: YS
authors.lastname: Li
authors.forename: T F
authors.initials: TF
authors.lastname: Deuel
mesh_terms: D019870:1-Phosphatidylinositol 4-Kinase; D000818:Animals; D002417:Cattle; D002845:Chromatography; D002850:Chromatography, Gel; D017886:Durapatite; D005260:Female; D006882:Hydroxyapatites; D007700:Kinetics; D050356:Lipid Metabolism; D010770:Phosphotransferases / Q000302:isolation & purification* / Q000378:metabolism; D012265:Ribonucleotides / Q000378:metabolism; D013379:Substrate Specificity; D014599:Uterus / Q000201:enzymology*
chemical_list: D006882:Hydroxyapatites; D012265:Ribonucleotides; D017886:Durapatite; D010770:Phosphotransferases; D019870:1-Phosphatidylinositol 4-Kinase