title: Purification and characterization of phosphatidylinositol synthase from human placenta.
abstract: Phosphatidylinositol synthase (CDP-1,2-diacyl-sn-glycerol:myoinositol 3-phosphatidyltransferase, EC 2.7.8.11) was purified from the microsomal fraction of human placenta. The Triton X-100-extracted enzyme was purified 8300-fold over the microsomal fraction by affinity chromatography on CDP-diacylglycerol-Sepharose followed by ion-exchange chromatography on Mono Q. The purified enzyme had a molecular mass of 24,000 Da on SDS/PAGE. The enzyme had a pH optimum at 9.0, required Mn2+ or Mg2+, and was inhibited by Ca2+ and Zn2+. The Km for myo-inositol was determined to be 0.28 mM. Optimal activity was obtained at 0.2-0.4 mM CDP-diacylglycerol; higher concentrations of the lipid substrate inhibited the enzyme reaction. The enzyme was inhibited by nucleoside di- and tri-phosphates, Pi and PPi. CDP competitively inhibited the enzyme reaction with a Kis of 4 mM. The optimal temperature for the PtdIns synthase reaction was 50 degrees C.
authors.affiliation: Glaxo Institute for Molecular Biology, Plan-les-Ouates/Geneva, Switzerland.
authors.forename: B E
authors.initials: BE
authors.lastname: Antonsson
mesh_terms: D051607:CDP-Diacylglycerol-Inositol 3-Phosphatidyltransferase; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D005260:Female; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008565:Membrane Proteins; D008670:Metals; D008861:Microsomes / Q000201:enzymology; D010920:Placenta / Q000201:enzymology*; D011257:Pregnancy Proteins / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D013379:Substrate Specificity; D013696:Temperature; D017855:Transferases (Other Substituted Phosphate Groups) / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism
chemical_list: D008565:Membrane Proteins; D008670:Metals; D011257:Pregnancy Proteins; D017855:Transferases (Other Substituted Phosphate Groups); C496766:CDIPT protein, human; D051607:CDP-Diacylglycerol-Inositol 3-Phosphatidyltransferase