Improved purification and biochemical properties of phosphatidylinositol-specific phospholipase C from Bacillus thuringiensis
- PMID: 2553399
- DOI: 10.1111/j.1432-1033.1989.tb15096.x
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.
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