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. Copyright © 1989, Wiley Blackwell. All rights reserved