Bacterial phosphatidylinositol-specific phospholipases C (PI-PLC) display similar substrate specificity as their eukaryotic counterparts involved in signal transduction of insulin and Ca2+-mobilizing hormones, and are used in the study of the novel glycosylphosphatidylinositol-protein anchors (GPI-anchors). For the investigation of structure-function aspects of the PI-PLC secreted from Bacillus cereus cells, a panel of murine monoclonal antibodies was generated and shown to be specific for the PI-PLC polypeptide in enzyme-linked immunosorbent assays and Western blots. Two of the monoclonals inhibited reactions catalyzed by the bacterial enzyme in vitro: hydrolysis of phosphatidylinositol and the release of bovine erythrocyte acerylcholinesterase from its GPI-anchor. At saturating concentrations of inhibitory antibody only a few percent of the enzyme activity remained. The epitope recognized by one of the inhibitory antibodies, A72−24, was mapped by proteolytic digestion, protein sequencing, and Western blotting of the generated fragments. The data indicate that at least part of the epitope resides within an 8 kDa-stretch of the bacterial PI-PLC (Gln-45 - Lys-122). Essentially the same segment of the bacterial polypeptide has previously been shown to display limited amino acid sequence similarity with several eukaryotic PI-specific phospholipases C (Kuppe, A., Evans, L.M., McMillen, D.A. and Griffith, O.H. (1989) J. Bacteriol. 171, 6077–6083). The results reported here suggest that the conserved peptide of these enzymes may contain functionally important residues.

The inositol phosphate products formed during the cleavage of phosphatidylinositol by phosphatidylinositol-specific phospholipase C from Bacillus cereus were analyzed by 31P NMR. 31P NMR spectroscopy can distinguish between the inositol phosphate species and phosphatidylinositol. Chemical shift values (with reference to phosphoric acid) observed are 0.41, 3.62, 4.45, and 16.30 ppm for phosphatidylinositol, myo-inositol 1-monophosphate, myo-inositol 2-monophosphate, and myo-inositol 1,2-cyclic monophosphate, respectively. It is shown that under a variety of experimental conditions this phospholipase C cleaves phosphatidylinositol via an intramolecular phosphotransfer reaction producing diacylglycerol and D-myo-inositol 1,2-cyclic monophosphate. We also report the new and unexpected observation that the phosphatidylinositol-specific phospholipase C from B. cereus is able to hydrolyze the inositol cyclic phosphate to form D-myo-inositol 1-monophosphate. The enzyme, therefore, possesses phosphotransferase and cyclic phosphodiesterase activities. The second reaction requires thousandfold higher enzyme concentrations to be observed by 31P NMR. This reaction was shown to be regiospecific in that only the 1-phosphate was produced and stereospecific in that only D-myo-inositol 1,2-cyclic monophosphate was hydrolyzed. Inhibition with a monoclonal antibody specific for the B. cereus phospholipase C showed that the cyclic phosphodiesterase activity is intrinsic to the bacterial enzyme. We propose a two-step mechanism for the phosphatidyl-inositol-specific phospholipase C from B. cereus involving sequential phosphotransferase and cyclic phosphodiesterase activities. This mechanism bears a resemblance to the well-known two-step mechanism of pancreatic ribonuclease, RNase A.

The phosphatidylinositol (PI)-specific phospholipase C (PLC) of Bacillus cereus was cloned into Escherichia coli by using monoclonal antibody probes raised against the purified protein. The enzyme is specific for hydrolysis of the membrane lipid PI and PI-glycan-containing membrane anchors, which are important structural components of one class of membrane proteins. The protein expressed in E. coli comigrated with B. cereus PI-PLC in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, as detected by immunoblotting, and conferred PI-PLC activity on the host. This enzyme activity was inhibited by PI-PLC-specific monoclonal antibodies. The nucleotide sequence of the PI-PLC gene suggests that this secreted bacterial protein is synthesized as a larger precursor with a 31-amino-acid N-terminal extension to the mature enzyme of 298 amino acids. From analysis of coding and flanking sequences of the gene, we conclude that the PI-PLC gene does not reside next to the gene cluster of the other two secreted phospholipases C on the bacterial chromosome. The deduced amino acid sequence of the B. cereus PI-PLC contains a stretch of significant similarity to the glycosylphosphatidylinositol-specific PLC of Trypanosoma brucei. The conserved peptide is proposed to play a role in the function of these enzymes.

Phosphatidylinositol‐specific phospholipase C was purified in a 27% yield from the culture medium of Bacillus cereus by a combination of ammonium sulfate precipitation and ion‐exchange and hydrophobic interaction chromatography. The purified enzyme was free of other phospholipase C‐type activities and exhibited a high specific activity of approximately 1,300 units/mg. Amino acid composition analysis and sodium dodecyl sulfate‐polyacrylamide gel electrophoresis indicated a molecular weight of about 35 kDa. The sequence of the first 29 N‐terminal amino acids was also determined.

The syntheses of two new radioactive probes derived from cardiolipin and phosphatidylcholine are reported. These probes are derivatives of natural lipids and contain an amine-specific benzaldehyde in the head-group region. This functional group allows a choice of timing of the reaction (e.g., after equilibration and detergent removal) because an irreversible covalent bond is formed only upon the addition of reducing agent. These probes, as well as a benzaldehyde analogue of phosphatidic acid, and a water-soluble benzaldehyde reagent were covalently attached to bovine heart cytochrome c oxidase. After reconstitution into vesicles, the lipid-benzaldehyde probes selectively incorporated into the smaller polypeptides of the enzyme, while the remaining subunits (I-IV) exhibited little incorporation of label. The accessibility of amine groups labeled under the conditions used here was independent of the structural and charge differences between the benzaldehyde probes. This suggests that all three lipid probes react with polypeptides of the cytochrome c oxidase complex at general contact sites for membrane phospholipids. A water-soluble benzaldehyde reagent predominantly labeled subunits IV, Va, and Vb and polypeptides of VII-VIII. A comparison of these results facilitates a more refined view of the disposition of polypeptides of cytochrome c oxidase in respect to the lipid and aqueous phases.