A chromogenic substrate for phosphatidylinositol-specific phospholipase C: 4-nitrophenyl myo-inositol-1-phosphate.

A chromogenic water-soluble substrate for phosphatidylinositol-specific phospholipase C was synthesized starting from myo-inositol employing isopropylidene and 4-methoxytetrahydropyranyl protecting groups. In this analogue of phosphatidylinositol, 4-nitrophenol replaces the diacylglycerol moiety, resulting in synthetic, racemic 4-nitrophenyl myo-inositol-1-phosphate. Using this synthetic substrate a rapid, convenient and sensitive spectrophotometric assay for the phosphatidylinositol-specific phospholipase C from Bacillus cereus was developed. Initial rates of the cleavage of the nitrophenol substrate were linear with time and the amount of enzyme used. At pH 7.0, specific activities for the B. cereus enzyme were 77 and 150 mumol substrate cleaved min-1 (mg protein)-1 at substrate concentrations of 1 and 2 mM, respectively. Under these conditions, less than 50 ng quantities of enzyme were easily detected. The chromogenic substrate was stable during long term storage (6 months) as a solid at -20 degrees C.     

Phosphatidylinositol-specific phospholipase C from Bacillus cereus combines intrinsic phosphotransferase and cyclic phosphodiesterase activities: a 31P NMR study

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.     

A fluorescent substrate for the continuous assay of phosphatidylinositol-specific phospholipase C: synthesis and application of 2-naphthyl myo-inositol-1-phosphate.

A fluorescent water-soluble substrate for phosphatidylinositol-specific phospholipase C was synthesized. The diacylglycerol moiety of the natural substrate, phosphatidylinositol, was replaced by the fluorescent moiety, 2-naphthol, resulting in the synthetic substrate, racemic 2-naphthyl myo-inositol-1-phosphate. The synthetic substrate provided a continuous fluorometric assay for the phosphatidylinositol-specific phospholipase C from Bacillus cereus. Initial rates of the cleavage of the 2-naphthyl substrate by the phospholipase measured by fluorometry were linear with time and the amount of enzyme added. The specific enzyme activity at pH 8.5 and 25 degrees C was about 0.04 mumol/min mg protein at an initial substrate concentration of 0.8 mM. 31P NMR experiments suggest that, as with phosphatidylinositol itself, cleavage of the fluorescent substrate proceeds in two steps via a myo-inositol-1,2-cyclic phosphate intermediate, and that only the D-isomer is a substrate for the B. cereus phospholipase. The synthetic substrate was stable during long-term storage as a solid in the dark at -20 degrees C. It was also stable for several weeks when stored in the dark frozen in aqueous solution near neutral pH.     

Inhibition of phosphatidylinositol-specific phospholipase C by phosphonate substrate analogues

Non-hydrolysable analogues of phosphatidylinositol were synthesized and tested as inhibitors of phosphatidylinositol-specific phospholipase C from Bacillus cereus. In these molecules, the phosphodiester bond of phosphatidylinositol hydrolyzed by the phospholipase was replaced by a phosphonate linkage and a simpler hydrophobic group replaced the diacylglycerol moiety. One of the phosphonates also contained a carboxylate functional group suitable for matrix attachment. All three synthetic phosphonates inhibited the phospholipase C activity in a concentration-dependent manner, with the analogue most closely resembling the structure of the natural substrate, phosphatidylinositol, being the most potent inhibitor. The data indicate that phosphonate analogues of phosphatidylinositol may be useful for study of phospholipase C and other proteins interacting with myo-inositol phospholipids.     

Allosteric interactions within subsites of a monomeric enzyme: kinetics of fluorogenic substrates of PI-specific phospholipase C

Two novel water-soluble fluorescein myo-inositol phosphate (FLIP) substrates, butyl-FLIP and methyl-FLIP, were used to examine the kinetics and subsite interactions of Bacillus cereus phosphatidylinositol-specific phospholipase C. Butyl-FLIP exhibited sigmoidal kinetics when initial rates are plotted versus substrate concentration. The data fit a Hill coefficient of 1.2-1.5, suggesting an allosteric interaction between two sites. Two substrate molecules bind to this enzyme, one at the active site and one at a subsite, causing an increase in activity. The kinetic behavior is mathematically similar to that of well-known cooperative multimeric enzymes even though this phosphatidylinositol-specific phospholipase C is a small, monomeric enzyme. The less hydrophobic substrate, methyl-FLIP, binds only to the active site and not the activator site, and thus exhibits standard hyperbolic kinetics. An analytical expression is presented that accounts for the kinetics of both substrates in the absence and presence of a nonsubstrate short-chain phospholipid, dihexanoylphosphatidylcholine. The fluorogenic substrates detect activation at much lower concentrations of dihexanoylphosphatidylcholine than previously reported.     

High-level expression in Escherichia coli and rapid purification of phosphatidylinositol-specific phospholipase C from Bacillus cereus and Bacillus thuringiensis.

The construction of four vectors for high-level expression in Escherichia coli of the phosphatidylinositol-specific phospholipase C from Bacillus cereus or Bacillus thuringiensis is described. In all constructs the coding sequence for the mature phospholipase is precisely fused to the E. coli heat-stable enterotoxin II signal sequence for targeting of the protein to the periplasm. In one set of plasmids expression of the B. cereus or B. thuringiensis enzyme is under control of the E. coli alkaline phosphatase promoter, while in a second set of plasmids expression is under control of a lac-tac-tac triple tandem promoter. A simple and rapid procedure for complete purification of the phospholipase C overproduced in E. coli, involving isolation of the periplasmic proteins by osmotic shock followed by a single column chromatography step, is described. The largest quantity of purified enzyme, 40-60 mg per liter culture, is obtained with the plasmid expressing the B. cereus enzyme under control of the lac-tac-tac promoter. Lower quantities are obtained with the plasmids containing the alkaline phosphatase promoter (15-20 and 4-6 mg/liter for the B. cereus and B. thuringiensis enzymes, respectively) and with the plasmid expressing the B. thuringiensis phospholipase under control of the lac-tac-tac promoter (15-20 mg/liter). A comparison of the functional properties of the recombinant phospholipases with the native enzymes isolated from B. cereus or B. thuringiensis culture supernatant shows that they are identical with respect to their catalytic functions, viz., cleavage of phosphatidylinositol and cleavage of the glycosyl-phosphatidylinositol membrane anchor of bovine erythrocyte acetylcholinesterase.     

Bacillus cereus phospholipase C: carboxylic acid ester specificity and stereoselectivity

Thiophosphate analogs of phosphatidylcholine have been synthesized with varying structural complexity. These analogs have been used in a continuous spectrophotometric assay for phospholipase C (Bacillus cereus) to estimate the minimal structural requirements associated with the non-polar portion of the substrate phospholipid. The analogs were of three types containing zero, one or two carboxylic acid ester functionalities. The analogs with one or two ester groups acted as substrates for phospholipase C, while those without an ester functionality were not hydrolyzed. The rac-phosphatidylcholine analog with two ester functionalities gave biphasic time-course results, and was subsequently resolved into enantiomers by selective hydrolysis with a sterospecific phospholipase A2 (Crotalus atrox). The enantiomer with R absolute configuration was rapidly hydrolyzed by the phospholipase C while the enantiomer with the S configuration was slowly hydrolyzed after a long induction period. The results suggest that the B. cereus phospholipase C is specific for an ester functionality and is stereoselective for the R absolute configuration at glycerol C-2.     

High-performance liquid chromatographic assay with ultraviolet spectrometric detection for the evaluation of inhibitors of phosphatidylinositol-specific phospholipase C

A nonradioactive spectrometric assay for the evaluation of inhibitors of phosphatidylinositol-specific phospholipase C (PI-PLC) is described. l-alpha-Phosphatidylinositol from bovine liver was used as substrate in the presence of the micelle-forming detergent deoxycholic acid. PI-PLC isolated from Bacillus cereus and crude cytosol fractions from porcine brain were used as enzyme sources. PI-PLC activity was determined by measuring the release of 1-stearoyl-2-arachidonoyl-sn-glycerol with reversed-phase HPLC and UV detection at 200 nm. PI-PLC from B. cereus was not inhibited by the putative PI-PLC inhibitors U-73122 and ET-18-OCH(3) at 100 microM, whereas the isobenzofuranone derivative 5 blocked the enzyme with an IC(50) of 75 microM. PI-PLC activity present in porcine brain cytosol was decreased by all three test compounds at 100 microM to approximately 30 to 50%.     

Acetylcholinesterases from Musca domestica and Drosophila melanogaster Brain Are Linked to Membranes by a Glycophospholipid Anchor Sensitive to an Endogenous Phospholipase

The sensitivity of acetylcholinesterases (AChEs) from Musca domestica and from Drosophila melanogaster to the phosphatidylinositol-specific phospholipase C from Bacillus cereus and to the glycosylphosphatidylinositol-specific phospholipase C from Trypanosoma brucei was investigated. B. cereus phospholipase C solubilizes membrane-bound AChE, and both phospholipases convert amphiphilic AChEs into hydrophilic forms of the enzyme. The lipases uncover an immunological determinant that is found on other glycosylphosphatidylinositol-anchored membrane proteins after the same treatment. This immunological determinant is also present on the native hydrophilic form of AChE. The polypeptide bearing the active site of the membrane-bound enzyme migrates faster during sodium dodecyl sulfate-polyacrylamide gel electrophoresis than the same polypeptide from the soluble enzyme. We conclude that AChE from insect brain is attached to membranes via a glycophospholipid anchor. This anchor is covalently linked to the polypeptide bearing the active esterase site of the enzyme and can be cleaved by an endogenous lipase.     

Preparation and application of an affinity matrix for phosphatidylinositol-specific phospholipase C

A non-hydrolyzable phosphonate analogue of phosphatidyl inositol, racemic myo-inosityl-(1)-5-oxa-16-trifluoroacetamidohexadecyl phosphonate, was synthesized. This phosphonate inhibited the activity of phosphatidyl inositol-specific phospholipase C (PI-PLC) from Bacillus cereus with an IC50 of approximately 10 mM. Removal of the trifluoroacetyl blocking group followed by covalent binding of the phosphonate to cyanogen bromide activated Sepharose 4B via the amino group produced an affinity matrix specific for the PI-PLC from B. cereus. This affinity matrix was used to purify the phospholipase C from a complex mixture of proteins in a single step. Competition experiments with myo-inositol in the elution medium indicated that specific binding of the enzyme to the matrix most likely involves the enzyme active site. The inositol phosphonate derivatized matrix was stable over several months in neutral and alkaline media and was used repeatedly without loss of binding capacity. These results show that affinity matrices employing myo-inositol phosphonate ligands are useful for isolation and binding studies of PI-PLC and possibly of other enzymes interacting with phosphoinositides or myo-inositol phosphate derivatives.     

New syntheses of 1D- and 1L-1,2-anhydro-myo-inositol and assessment of their glycosidase inhibitory activities

The 1D and 1L enantiomers of 1,2-anhydro-myo-inositol (conduritol B epoxide) were synthesised from 1D-pinitol and 1L-quebrachitol, respectively, and their activities were compared in selected glycosidase inhibition assays. The 1D enantiomer was found to be the active isomer, functioning as an irreversible inhibitor of sweet almond beta-D-glucosidase. Neither isomer was active against the alpha-D-glucosidase from Bacillus stearothermophilus or the beta-D-galactosidase from Aspergillus oryzae.     

Cloning and sequencing of the gene encoding the phosphatidylcholine-preferring phospholipase C of Bacillus cereus

A synthetic oligodeoxynucleotide probe was used to clone the gene encoding the phosphatidylcholine-preferring phospholipase C of Bacillus cereus. The sequence of a 2050-bp restriction fragment containing the gene was determined. Analysis of the gene-derived amino acid (aa) sequence showed that this exoenzyme is probably synthesized as a 283-aa precursor with a 24-aa signal peptide and a 14-aa propeptide. The mature, secreted enzyme comprises 245 aa residues. Sonicates of Escherichia coli HB101 carrying the gene on a multicopy plasmid showed phospholipase C activity. This activity was inhibited by Tris, a known inhibitor of the B. cereus enzyme and also by antiserum raised against pure B. cereus phospholipase C. We conclude therefore that the gene is expressed in E. coli. The cloning and sequencing described here complete the first step toward using in vitro mutagenesis for investigations of the structure-function relationships of B. cereus phospholipase C.     

Synthetic phospholipids as substrates for phospholipase C from Bacillus cereus.

The substrate requirement of phospholipids for hydrolysis with phospholipase C from Bacillus cereus was studied with synthetic lipids well-defined in structure and configuration. For optimal activity, the glycerol molecule must contain three substituents: phosphocholine in sn-3-, an ester bond in sn-2- and an ether- or ester bond in sn-1-position. The length of the ester or ether chains is of minor importance. Any deviation from these structural requirements results in a large decrease in the hydrolysis rate. These essential structural and configurational elements for optimal activity for the B. cereus enzyme are perfectly combined in the platelet activating factor, 1-O-hexadecyl-2-acetyl-sn-glycero-3- phosphocholine. This molecule is one of the best substrates for hydrolysis with the bacterial phospholipase C.     

Characterization of soluble vs membrane-bound human placental 5'-nucleotidase

Three forms of 5'-nucleotidase purified from human placenta (two membrane-bound forms, one sensitive and one resistant to cleavage by phosphatidylinositol-specific phospholipase C, as well as a soluble form) had the same molecular weight before (73,000 Da) and after (56,000 Da) digestion with N-glycosidase F and showed similar amino acid compositions, N-terminal amino acid sequences, and KMs for IMP (9.6 to 11.9 microM). Thus, these three forms of 5'-nucleotidase appear to have very similar structures. The form sensitive to phosphatidylinositol-specific phospholipase C contained nearly 1 mol myo-inositol/mol of protein as determined by mass spectrometry, indicating a glycosyl phosphatidylinositol membrane anchor. Soluble 5'-nucleotidase contained a similar quantity of myo-inositol, suggesting that it was previously membrane-anchored via glycosyl phosphatidylinositol. The form resistant to phosphatidylinositol-specific phospholipase C contained less myo-inositol, leaving open the possibility of a third form of 5'-nucleotidase with a conventional transmembrane anchor.     

Listeria monocytogenes phosphatidylinositol (PI)-specific phospholipase C has low activity on glycosyl-PI-anchored proteins.

The ability of the phosphatidylinositol-specific phospholipase C (PI-PLC) from Listeria monocytogenes to hydrolyze glycosyl phosphatidylinositol (GPI)-anchored membrane proteins was compared with the ability of the PI-PLC from Bacillus thuringiensis to hydrolyze such proteins. The L. monocytogenes enzyme produced no detectable release of acetylcholinesterase from bovine, sheep, and human erythrocytes. The cleavage of the GPI anchors of alkaline phosphatase from rat and rabbit kidney slices was less than 10% of the cleavage seen with the PI-PLC from B. thuringiensis. Activity for release of Fc gamma receptor IIIB (CD16) on human granulocytes was also low. Variations in pH and salt concentration had little effect on the release of GPI-anchored proteins. Our data show that L. monocytogenes PI-PLC has low activity on GPI-anchored proteins.     

Enzymatic hydrolysis by bacterial phospolipases C and D of immobilized radioactive sphingomyelin and phosphatidylcholine

An assay system for phospholipases C has been described with sphingomyelin immobilized to octyl-Sepharose CL-4B as substrate. The immobilization procedure was further developed and used with [14 C-choline]-sphingomyelin and [14C-choline] phosphatidylcholine (lecithin). These immobilized radioactive phospholipids made the enzymatic assays easier to perform and made it possible to increase the sensitivity. Furthermore, since release of the choline part instead of the phosphate part of the substrate molecule was measured, it was possible to use this assay for phospholipase D as well. The enzyme characteristics of phospholipase D from Corynebacterium ovis were compared in this test system with those of three phospholipases C (from Clostridium perfringens, Bacillus cereus and Staphylococcus aureus) with respect to hydrolysing capacities and optimal ion concentrations.     

Role of tryptophan residues in interfacial binding of phosphatidylinositol-specific phospholipase C

The phosphatidylinositol-specific phospholipase C (PI-PLC) from Bacillus thuringiensis exhibits several types of interfacial activation. In the crystal structure of the closely related Bacillus cereus PI-PLC, the rim of the active site is flanked by a short helix B and a loop that show an unusual clustering of hydrophobic amino acids. Two of the seven tryptophans in PI-PLC are among the exposed residues. To test the importance of these residues in substrate and activator binding, we prepared several mutants of Trp-47 (in helix B) and Trp-242 (in the loop). Two other tryptophans, Trp-178 and Trp-280, which are not near the rim, were mutated as controls. Kinetic (both phosphotransferase and cyclic phosphodiesterase activities), fluorescence, and vesicle binding analyses showed that both Trp-47 and Trp-242 residues are important for the enzyme to bind to interfaces, both activating zwitterionic and substrate anionic surfaces. Partitioning of the enzyme to vesicles is decreased more than 10-fold for either W47A or W242A, and removal of both tryptophans (W47A/W242A) yields enzyme with virtually no affinity for phospholipid surfaces. Replacement of either tryptophan with phenylalanine or isoleucine has moderate effects on enzyme affinity for surfaces but yields a fully active enzyme. These results are used to describe how the enzyme is activated by interfaces.     

Increase in osmotic fragility of bovine erythrocytes induced by bacterial phospholipases C

Bovine erythrocytes were treated with each of three bacterial phospholipases C; phosphatidylcholine-hydrolyzing phospholipase C (PCase) of Clostridium perfringens, sphingomyelinase C (SMase) of Bacillus cereus and phosphatidylinositol-specific phospholipase C (PIase) of Bacillus thuringiensis. An increase in osmotic fragility was detected by means of a coil planet centrifugation (CPC) apparatus (Biomedical Systems Co., Tokyo) after the treatment with these enzymes. The peak of hemolysis normally observed in the untreated erythrocytes at the range between 50 and 100 mOsM shifted to 160 to 200 mOsM with the progress of sphingomyelin hydrolysis by phospholipase C of C. perfringens. Sphingomyelinase C of B. cereus showed two different effects on bovine erythrocytes: In the absence of divalent cations or in the presence of Ca2+ alone, the peak of hemolysis shifted to the region from 130 to 160 mOsM, without appreciable hydrolysis of sphingomyelin, while in the presence of Mg2+ or Mg2+ plus Ca2+, the peak of hemolysis further shifted to the region from 160 to 200 mOsM with the hydrolysis of sphingomyelin. Abrupt shift in osmotic fragility to a much higher region around 250 mOsM was produced by treatment with increasing amounts of phosphatidylinositol-specific phospholipase C. In this case, a significant amount of acetylcholinesterase was released from the erythrocyte membrane without hot or hot-cold hemolysis. The mechanism of alteration of osmotic fragility of bovine erythrocytes by treatment with phospholipases C seems to differ from case to case, depending upon the specific action of each enzyme toward the membrane phospholipids.     

Identification and characterization of toxigenic Bacillus cereus isolates responsible for two food-poisoning outbreaks

The epidemiology of Bacillus cereus strains responsible for food poisoning is scantly known, mostly because the genotypic and toxigenic properties of the B. cereus strains isolated during food-poisoning outbreaks have been never catalogued. The occurrence of two simultaneous food-poisoning outbreaks gave us the opportunity to wonder whether (i) the identity of individual strains isolated from clinical, environmental, and food samples could be established by random amplified polymorphic DNA (RAPD)-PCR and multiplex RAPD-PCR, and (ii) the toxigenic potential of the isolates could be determined by testing their ability to secrete hemolysin BL, phosphatidylcholine-specific phospholipase C, and cereulide, as well as by determining the presence of the genes encoding enterotoxins NHE, T, and FM/S, cytotoxin K, sphingomyelinase, and phosphatidylinositol-specific phospholipase C. This is the first report demonstrating that the combination of several phenotypic and genotypic traits provides a powerful tool for tracing the source of infection of toxigenic B. cereus strains relevant for epidemiological survey.     

Attachment of human placental-type alkaline phosphatase via phosphatidylinositol to syncytiotrophoblast and tumour cell plasma membranes

Phosphatidylinositol anchors human placental-type alkaline phosphatase (PLAP) to both syncytiotrophoblast and tumour cell plasma membranes. PLAP activity was released from isolated human placental syncytiotrophoblast plasma membranes and the surface of tumour cells with a phospholipase C from Bacillus cereus. This was a specific event, not the result of proteolysis or membrane perturbation, but the action of a phosphatidylinositol-specific phospholipase C in the preparation. Soluble PLAP, released with B. cereus phospholipase C and purified by immunoaffinity chromatography, ran on SDS-PAGE as a 66-kDa band. This corresponded to intact PLAP molecules. The protease bromelain cleaved lower-molecular-mass PLAP (64 kDa) from the membranes. Flow cytometry demonstrated that B. cereus phospholipase C released human tumour cell membrane PLAP in preference to other cell-surface molecules. This was in contrast to the non-specific proteolytic action of bromelain or Clostridium perfringens phospholipase C, which had no effect on membrane PLAP expression. Radiolabelling of tumour cells with fatty acids indicated PLAP to be labelled with both [3H]myristic and [3H]palmitic acid. This fatty-acid--PLAP bond was sensitive to pH 10 hydroxylamine treatment indicating an O-ester linkage.