A Phospholipase C from the Dallas 1E Strain of Legionella pneumophila Serogroup 5: Purification and Characterization of Conditions for Optimal Activity with an Artificial Substrate

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Abstract
Phospholipase C from the Dallas 1E strain of Legionella pneumophila serogroup 5 was purified from buffered yeast extract culture supernate by ion-exchange chromatography followed by fractionation by manganous chloride and ammonium sulphate precipitation steps. Enzyme activity was assayed by hydrolysis of p-nitrophenylphosphorylcholine and confirmed by release of radioactivity from tritated L-.alpha.-dipalmitoylphosphatidylcholine labelled in the methyl groups of choline. After SDS-PAGE, the purified preparation yielded a single band upon Coomassie-blue staining. This protein migrated with an apparent Mr of 50000-54000. Phospholipase C activity was maximal at pH .gtoreq. 8.4 and was enhanced in the presence of sorbitol and of several nonionic detergents but was eliminated by SDS .cntdot. EDTA, Cu2+, Fe2+ and Zn2+ inhibited enzyme activity, whereas Ba2+, Ca2+, Co2+, Mg2+ and Mn2+ restored activity to EDTA-treated material. No haemolytic activity was demonstrated with the purified enzyme.