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

Abstract

Phospholipase C from the Dallas 1E strain of 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 -nitrophenylphosphorylcholine and confirmed by release of radioactivity from tritiated L-α-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 of 50000–54000. Phospholipase C activity was maximal at pH ≥ 8.4 and was enhanced in the presence of sorbitol and of several nonionic detergents but was eliminated by SDS. EDTA, Cu, Fe and Zn inhibited enzyme activity, whereas Ba, Ca, Co, Mg and Mn restored activity to EDTA-treated material. No haemolytic activity was demonstrated with the purified enzyme.

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1988-02-01
2024-03-29
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