title: Improved purification and biochemical properties of phosphatidylinositol-specific phospholipase C from Bacillus thuringiensis.
abstract: Monophosphatidylinositol inositol phosphohydrolase (phosphatidylinositol-specific phospholipase C. PtdIns-PLC. EC 3.1.4.10) has been purified from a Bacillus thuringiensis culture supernatant and from the cellular fraction of a recombinant Escherichia coli clone containing the PtdIns-PLC gene from B. thuringiensis. The two-step purification procedure involved ion-exchange chromatography on DEAE-Sepharose followed by separation on a Mono-Q/FPLC-column with yields of 32% and 50%, respectively. The molecular mass was determined to be 34 kDa by SDS/PAGE. The isoelectric point of the enzyme was 5.15. The amino-terminal sequences were shown to be identical for the enzymes purified from both organisms. PtdIns-PLC was inhibited by divalent cations using mixed micelles of Triton X-100 and pure phosphatidylinositol. PtdIns-PLC activity was detectable on polyacrylamide gels by activity staining on phosphatidylinostiol-containing agarose.
authors.affiliation: Institut für Mikrobielle Genetik, Universität Tübingen, Federal Republic of Germany.
authors.forename: T
authors.initials: T
authors.lastname: Kupke
authors.forename: M
authors.initials: M
authors.lastname: Lechner
authors.forename: G
authors.initials: G
authors.lastname: Kaim
authors.forename: F
authors.initials: F
authors.lastname: Götz
mesh_terms: D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D001413:Bacillus thuringiensis / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D054801:Phosphoinositide Phospholipase C; D010727:Phosphoric Diester Hydrolases / Q000032:analysis / Q000302:isolation & purification*
chemical_list: D000596:Amino Acids; D010727:Phosphoric Diester Hydrolases; D054801:Phosphoinositide Phospholipase C; D043265:Phosphatidylinositol Diacylglycerol-Lyase