Biomedical subjects

M F Roberts

Publications and source records attributed to M F Roberts.

At least 37 records · Page 2Linked to original sources

PubMed1998

Cloning and expression of the inositol monophosphatase gene from Methanococcus jannaschii and characterization of the enzyme.

Inositol monophosphatase (EC 3.1.3.25) plays a pivotal role in the biosynthesis of di-myo-inositol-1,1'-phosphate, an osmolyte found in hyperthermophilic archaeal. Given the sequence homology between the MJ109 gene product of Methanococcus jannaschii and human inositol monophosphatase, the MJ109 gene was cloned and expressed in Escherichia coli and examined for inositol monophosphatase activity. The purified MJ109 gene product showed inositol monophosphatase activity with kinetic parameters (K(m) = 0.091 +/- 0.016 mM; Vmax = 9.3 +/- 0.45 mumol of Pi min-1 mg of protein-1) comparable to those of mammalian and E. coli enzymes. Its substrate specificity, Mg2+ requirement, Li+ inhibition, subunit association (dimerization), and heat stability were studied and compared to those of other inositol monophosphatases. The lack of inhibition by low concentrations of Li+ and high concentrations of Mg2+ and the high rates of hydrolysis of glucose-1-phosphate and p-nitrophenylphosphate are the most pronounced differences between the archaeal inositol monophosphatase and those from other sources. The possible causes of these kinetic differences are discussed, based on the active site sequence alignment between M. jannaschii and human inositol monophosphatase and the crystal structure of the mammalian enzyme.

Amino Acid Sequence↗

PubMed1998

Biosynthesis of Di-myo-inositol-1,1'-phosphate, a novel osmolyte in hyperthermophilic archaea.

Biosynthesis of di-myo-inositol-1,1'-phosphate (DIP) is proposed to occur with myo-inositol and myo-inositol 1-phosphate (I-1-P) used as precursors. Activation of the I-1-P with CTP and condensation of the resultant CDP-inositol (CDP-I) with myo-inositol then generates DIP. The sole known biosynthetic pathway of inositol in all organisms is the conversion of D-glucose-6-phosphate to myo-inositol. This conversion requires two key enzymes: L-I-1-P synthase and I-1-P phosphatase. Enzymatic assays using 31P nuclear magnetic resonance spectroscopy as well as a colorimetric assay for inorganic phosphate have confirmed the occurrence of L-I-1-P synthase and a moderately specific I-1-P phosphatase. The enzymatic reaction that couples CDP-I with myo-inositol to generate DIP has also been detected in Methanococcus igneus. 13C labeling studies with [2,3-13C]pyruvate and [3-13C]pyruvate were used to examine this pathway in M. igneus. Label distribution in DIP was consistent with inositol units formed from glucose-6-phosphate, but the label in the glucose moiety was scrambled via transketolase and transaldolase activities of the pentose phosphate pathway.

Cell-Free System↗

PubMed1997

Short-chain phosphatidylinositol conformation and its relevance to phosphatidylinositol-specific phospholipase C.

The solution conformation of chiral diheptanoylphosphatidylinositol (D- and L-inositol isomers) has been characterized by NMR spectroscopy. A positive NOE between the inositol C2 proton and an sn-3 glycerol CH2 proton has been observed in the D- but not in the L-inositol isomer of diheptanoylphosphatidylinositol (PI). Computer modeling using QUANTA constrained by this NOE and ring coupling constants suggests that the inositol ring is nearly parallel to the chain packing direction, leaving the phosphate ester accessible to attack by phosphatidylinositol-specific phospholipase C enzymes. In this model, the hydroxyl groups in the 2- and 6-positions of inositol form hydrogen bonds with the pro-R and ester oxygens, respectively. Chemical shifts and 13C spin-lattice relaxation times were also used to assess conformation and lipid dynamics in monomer and micelle states. The 13C T1's of inositol C2 and C6 in monomeric phosphatidylinositol were markedly less than for other inositol ring carbons. These results are consistent with the hydrogen bonds to the phosphate constraining the motions of C2 and C6. Diheptanoylphosphatidyl-2-O-methylinositol is a good inhibitor of PI-specific phospholipase C because it blocks the initial phosphotransferase step in PI hydrolysis. Introduction of the methyl group on the C-2 hydroxyl group lowers the CMC of the derivative compared to diheptanoylphosphatidylinositol. However, an NOE between an sn-3 glycerol proton and the inositol C2 proton constrains the orientation of the inositol ring with respect to the glycerol backbone in a conformation similar to diheptanoylphosphatidylinositol. Modeling of the 2-O-methylinositol derivative suggests that the methyl group blocks one side of the phosphate, consistent with the observation that nonspecific phospholipase C enzymes which are able to hydrolyze PI, albeit poorly, are unable to hydrolyze diheptanoylphosphatidyl-2-O-methylinositol.

Bacillus thuringiensis↗

PubMed1997

Diacylglycerol partitioning and mixing in detergent micelles: relevance to enzyme kinetics.

For many of the enzymes that utilize or produce diacylglycerols, detergent mixed micelles are often used in assay systems to solubilize the lipophilic substrates or products. The assumption is often made that the diacylglycerol (DAG) is solubilized and well mixed throughout the population of micelles during the time course of the assay. In the present work the partitioning and exchange dynamics of diacylglycerols (from dihexanoyl-DAG to didecanoyl-DAG) in a variety of detergent micelles have been studied by NMR and fluorescence methods. In all detergents, the longer the DAG chain lengths, the more detergent is required for solubilization. However, efficiency of solubilization varies tremendously with Triton X-100 the most efficient (i.e. the least detergent is required), and deoxycholate the least efficient in solubilizing DAG. The mixing and exchange dynamics of pyrene-labeled DAG molecules in these micelles (measured by stopped-flow fluorescence) were fastest for Triton X-100 and slowest with charged bile salt micelles. Of the detergent systems characterized, Triton X-100 appears to be the optimal detergent for use in assays of enzymes that interact with DAG (beta-octylglucoside and diheptanoylphosphatidylcholine have good exchange dynamics, but higher amounts of these detergents are needed to solubilize DAG). Bile salt micelles provide the least solubilization and the slowest exchange kinetics (so slow that this could be a significant problem in some enzyme assays). This information on DAG behavior in micelles is discussed with respect to assays of an enzyme that generates DAG as product (phospholipase C) and one that uses DAG as substrate (DAG kinase). Although slow exchange of DAG occurs in some micelle systems, this does not appear to be a rate-limiting step in the kinetics for either of these enzymes.

Bacillus cereus↗

PubMed1997

Phosphoinositide-specific phospholipase C delta1 activity toward micellar substrates, inositol 1,2-cyclic phosphate, and other water-soluble substrates: a sequential mechanism and allosteric activation.

The kinetics of full-length and PH domain truncated cloned PI-PLC delta1 from rat toward soluble substrates [inositol 1, 2-(cyclic)-phosphate (cIP) and glycerophosphoinositol phosphates (GPIPx)] as well as PI in detergent micelles provide the following insights into the mechanism of this enzyme. (i) That cIP is a substrate for the enzyme implies a two-step mechanism for PI hydrolysis [intramolecular phosphotransferase reaction to form cIP followed by cyclic phosphodiesterase activity to form inositol-1-phosphate (I-1-P)]. The dependence of enzyme activity on cIP is sigmoidal, suggesting a transition between less active and more active forms of the enzyme that is affected by substrate. (ii) Interfaces increase the kcat for cIP (but do not affect the cooperativity), and this allosteric activation requires an intact PH domain. (iii) Phosphorylation of the soluble inositol phosphodiesters GPI, GPIP, and GPIP2 enhances PI-PLC delta1 activity by dramatically increasing kcat and decreasing Km. For these phosphodiesters, the substrate saturation curve is no longer sigmoidal but hyperbolic, indicating the phosphorylated substrate can shift the enzyme to the activated form. (iv) Given the kinetic parameters for cIP hydrolysis and the constant ratio of cIP/I-1-P generated during PI hydrolysis, the cIP produced in situ is either released (and not readily rebound since its concentration is well below Km) or attacked by a water molecule for the generation of the acyclic product.

Allosteric Regulation↗

PubMed1997

Allosteric activation of phosphatidylinositol-specific phospholipase C: specific phospholipid binding anchors the enzyme to the interface.

Phosphatidylinositol-specific phospholipase C (PI-PLC) from Bacillus thuringiensis exhibits 'interfacial activation' toward the water-soluble substrate myo-inositol 1,2-(cyclic)phosphate [Zhou et al. (1997) Biochemistry 36, 347-355]. The activation of PI-PLC enzyme is optimal with PC or PE interfaces. NMR experiments (TRNOE and 31P line width analyses) were carried out to investigate the interaction of PI-PLC with activator amphiphiles. These studies showed that the enzyme had high affinity for phosphatidylcholine (or PE) molecules with dissociation constants of 0.5 and 0.3 mM for diC6PC and diC7PC, respectively. TRNOE cross-peaks of bound PC were confirmed to represent intramolecular relaxation pathways using partially perdeuterated PC molecules consistent with a single molecule binding tightly. The large activation by a PC interface can be explained by a single PC molecule binding specifically to PI-PLC and anchoring the enzyme-lipid complex to the interface. Other interfaces, such as micellar diC8PS, can activate PI-PLC about 2-3-fold; however, the monomers of these detergents showed little affinity for the enzyme as measured by TRNOE or 31P NMR line widths. The 3.6-fold activation produced by polymerized vesicles of 1,2-bis[12-(lipoyloxy)dodecanoyl]-sn-glycero-3-phosphocholine (compared to the 15-fold activation generated by nonpolymerized PC vesicles) was comparable to the nonspecific activation of other detergents. This confirmed that single-PC molecule binding was allosteric and anchored the enzyme in the interface. The conformation of interfacially activated enzyme is discussed in term of the stabilization of a critical surface loop and helix B observed with weak intensity in the X-ray crystal structure.

Allosteric Regulation↗

PubMed1997

Phosphatidylinositol-specific phospholipase C cyclic phosphodiesterase activity depends on solvent polarity.

Large enhancements (maximum of 82-fold in terms of enzyme efficiency, Vmax/Km) of bacterial PI-PLC cyclic phosphodiesterase activity were observed in the presence of organic solvents miscible in water (dimethyl sulfoxide, dimethylformamide, and 2-propanol). In general, organic solvents lowered the Km for myo-inositol 1,2-cyclic phosphate (cIP) and increased Vmax substantially. This kinetic effect was similar to that obtained with phosphatidylcholine micelles and bilayers in an aqueous assay system for cyclic inositol phosphate hydrolysis [Zhou, C., et al. (1997) Biochemistry 36, 347-355]. Solvent properties were examined to determine which ones correlated with the activation of PI-PLC toward cIP in each solvent. Activation correlated best with the solvent polarity as measured by ET(30); no significant correlation was observed with solution surface tension, the bulk dielectric constant (epsilon), 1/epsilon (a measure of the strength of charge interactions), or the Hildebrand solubility parameter. The sigmoidal curve of the enzyme activity versus solvent polarity was consistent with the solvent promoting a transition in the enzyme from a low-activity to a high-activity form. Possible candidates for this change, including enzyme dimerization, helix B/loop stabilization, and dehydration of the active site, are discussed.

1-Propanol↗

PubMed1997

Structural mapping of the catalytic mechanism for a mammalian phosphoinositide-specific phospholipase C.

The crystal structures of various ternary complexes of phosphoinositide-specific phospholipase C-delta 1 from rat with calcium and inositol phosphates have been determined at 2.30-2.95 A resolution. The inositol phosphates used in this study mimic the binding of substrates and the reaction intermediate and include D-myo-inositol-1,4,5-trisphosphate, D-myo-inositol-2,4, 5-trisphosphate. D-myo-inositol-4,5-bisphosphate, and D,1-myo-inositol-2-methylene-1,2-cyclićmonophosphonate. The complexes exhibit an almost invariant mode of binding in the active site, each fitting edge-on into the active site and interacting with both the enzyme and the catalytic calcium at the bottom of the active site. Most of the active site residues do not undergo conformational changes upon binding either calcium or inositol phosphates. The structures are consistent with bidentate liganding of the catalytic calcium to the inositol phosphate intermediate and transition state. The complexes suggest explanations for substrate preference, pH optima, and ratio of cyclic to acyclic reaction products. A reaction mechanism is derived that supports general acid/base catalysis in a sequential mechanism involving a cyclic phosphate intermediate and rules out a parallel mechanism where acyclic and cyclic products are simultaneously generated.

Animals↗

PubMed1997

Activation of phosphatidylinositol-specific phospholipase C toward inositol 1,2-(cyclic)-phosphate.

Phosphatidylinositol-specific phospholipase C (PI-PLC) from Bacillus thuringiensis catalyzes the hydrolysis of phosphatidylinositol (PI) in discrete steps: (i) an intramolecular phosphotransferase reaction to form inositol 1,2-(cyclic)-phosphate (cIP), followed by (ii) a cyclic phosphodiesterase activity that converts cIP to inositol 1-phosphate. Water-soluble cIP was used as the substrate to study the cyclic phosphodiesterase activity and interfacial behavior of PI-PLC. Different detergent micelles and phospholipid vesicles were used to examine if "interfacial activation" of the enzyme could occur toward a soluble substrate. Almost all detergents examined activated the enzyme at least 2-fold, with PC species yielding the largest increases in PI-PLC specific activity. Kinetic parameters were measured in the absence and presence of several representative detergents (e.g., Triton X-100 and diheptanoylphosphatidylcholine (diC7PC)). Gel filtration experiments showed that, under these conditions, the cIP did not partition to any measurable extent with these detergent micelles. The concentration at which half the maximum activation was observed occurred near the detergent CMC. Both Km and Vmax were altered by the presence of a surface: Km decreased to different degrees depending on the detergent, while Vmax increased substantially. The Km for cIP was 90 mM without detergent and decreased to 29 mM with diC7PC micelles added; Vmax increased almost 7-fold in the presence of diC7PC micelles. The enzyme efficiency (Vmax/Km) in the presence of diC7PC increased more than 21-fold, but it was still 20-fold lower than initial phosphotransferase activity for monomeric dihexanoylphosphatidylinositol. The poor efficiency of the cyclic phosphodiesterase activity is largely due to substrate binding affinity. The dependence of rate on substrate concentration exhibits cooperative behavior, especially without detergent. This cooperativity is discussed in terms of protein aggregation and ligand binding sites on the enzyme.

Bacillus subtilis↗

PubMed1997

Stereocontrolled syntheses of water-soluble inhibitors of phosphatidylinositol-specific phospholipase C: inhibition enhanced by an interface.

Three inositol 1,2-(cyclic)-phosphate analogs, inositol cyclic phosphonates with different stereochemistry at the C-2 position of the inositol ring, have been synthesized as water-soluble inhibitors of phosphatidylinositol-specific phospholipase C (PI-PLC). Their inhibition of both phosphotransferase and cyclic phosphodiesterase activities has been studied in the absence and presence of an interface. Key results include the following. (i) Only the analog with the same stereochemistry at the C-2 position of the inositol ring as the natural substrate, myo-inositol 1,2-(cyclic)-phosphate (cIP), exhibits effective inhibition of PI-PLC. (ii) The inhibition of the PI-PLC cyclic phosphodiesterase activity by this cIP analog is enhanced by the presence of an interface (Triton X-100 or diC7PC micelles). This is the first observation of detergent enhancing the effectiveness of a water-soluble inhibitor competing with a water-soluble substrate. (iii) For the cyclic phosphodiesterase activity measured in the presence of 8 mM of the best (e.g., most activating) interface, diC7PC, myo-inositol 1,2-(cyclic)-2-methylenephosphonate (cICH2P) was shown to be a competitive inhibitor with a Ki of 12.3 mM. (iv) The IC50 obtained for the same compound inhibiting the PI-PLC hydrolysis of PI dispersed in DiC7PC micelles was consistent with a Ki approximately 10 mM for the phosphotransferase activity. The similarity of Ki for both PI and cIP processing by PI-PLC suggests both reactions occur at the same site on the enzyme.

Bacillus subtilis↗

PubMed1997

Cloning, overexpression, refolding, and purification of the nonspecific phospholipase C from Bacillus cereus.

Bacillus cereus secretes a nonspecific phospholipase C (PLC) that catalyzes the hydrolysis of phospholipids to yield diacylglycerol and a phosphate monoester. B. cereus PLC has been overexpressed with its signal sequence in Escherichia coli using a T7 expression system. The expressed enzyme formed intracellular inclusion bodies which were solubilized in the presence of 8 M urea. Renaturation was initiated by gradual removal of urea and addition of zinc ions. The signal peptide was specifically cleaved by a protease, clostripain, added when the urea concentration was 1.5 M. Factors that led to protein reaggregation included rapid removal of urea, use of Tris instead of barbital buffer, and presence of the signal peptide when the urea concentration was below 1.5 M. The folded protein was purified by Q-Sepharose Fast flow chromatography to yield a preparation > 99% pure. The final yield of active enzyme was 30-40 mg per liter of culture. The recombinant PLC exhibited biochemical and kinetic properties identical to those of extracellularly produced PLC from B. cereus. Site-specific mutagenesis of Asn-134 was carried out as a test of the general effectiveness of the refolding procedure.

Bacillus cereus↗

PubMed1997

Organic osmolytes in aerobic bacteria from mono lake, an alkaline, moderately hypersaline environment.

The identity and concentrations of intracellular organic solutes were determined by nuclear magnetic resonance spectroscopy for two strains of aerobic, gram-negative bacteria isolated from Mono Lake, Calif., an alkaline, moderately hypersaline lake. Ectoine (1,4,5,6-tetrahydro-2-methyl-4-pyrimidinecarboxylic acid) was the major endogenous solute in both organisms. Concentrations of ectoine varied with external NaCl levels in strain ML-D but not in strain ML-G, where the level was high but invariant from 1.5 to 3.0 M NaCl. Hydroxyectoine also occurred in strain ML-D, especially at elevated NaCl concentrations (2.5 and 3.0 M), but at levels lower than those of ectoine. Exogenous organic solutes that might occur in Mono Lake were examined for their effects on the de novo synthesis of ectoine. Dimethylsulfoniopropionate (DMSP) (0.1 or 1 mM) did not significantly lower ectoine levels in either isolate, and only strain ML-G showed any capacity for DMSP accumulation. With nitrogen limitation, however, DMSP (0.1 mM) substituted for ectoine in strain ML-G and became the main organic solute. Glycine betaine (GB) was more effective than DMSP in affecting ectoine levels, principally in strain ML-D. Strain ML-D accumulated GB to 50 or 67% of its organic solute pool at 2.5 M NaCl, at an external level of 0.1 or 1 mM GB, respectively. Strain ML-D also accumulated arsenobetaine. The methylated zwitterionic compounds, probably metabolic products of phytoplankton (DMSP and GB) or brine shrimps (arsenobetaine) in Mono Lake, may function as osmolytes for indigenous bacteria when present at high concentrations or under conditions of nitrogen limitation or salt stress.

Journal Article↗

PubMed1997

Effects of Osmolyte Precursors on the Distribution of Compatible Solutes in Methanohalophilus portucalensis.

The halophilic methanogen Methanohalophilus portucalensis synthesizes three distinct zwitterions, (beta)-glutamine, N(sup(epsilon))-acetyl-(beta)-lysine (NA(beta)Lys), and glycine betaine, as osmolytes when it is grown at high concentrations of external NaCl. The selective distribution of these three species was determined by growing cells in the presence of osmolyte biosynthetic precursors. Glycine betaine is formed by the stepwise methylation of glycine. Exogenous glycine (10 mM) and sarcosine (10 mM), although internalized, do not bias the cells to accumulate any more betaine. However, exogenous N,N-dimethylglycine (10 mM) is available to the appropriate methyltransferase and the betaine generated from it suppresses the synthesis of other osmolytes. Precursors of the two zwitterionic (beta)-amino acids ((beta)-glutamate for (beta)-glutamine and (alpha)-lysine and diaminopimelate for NA(beta)Lys) have only small effects on (beta)-amino acid accumulation. The largest effect is provided by L-(alpha)-glutamine, suggesting that nitrogen assimilation is a key factor in osmolyte distribution.

Journal Article↗

PubMed1997

2-Sulfotrehalose, a novel osmolyte in haloalkaliphilic archaea.

A novel 1-->1 alpha-linked glucose disaccharide with sulfate at C-2 of one of the glucose moieties, 1-(2-O-sulfo-alpha-D-glucopyranosyl)-alpha-D-glycopyranose, was found to be the major organic solute accumulated by a Natronococcus sp. and several Natronobacterium species. The concentration of this novel disaccharide, termed sulfotrehalose, increased with increasing concentrations of external NaCl, behavior consistent with its identity as an osmolyte. A variety of noncharged disaccharides (trehalose, sucrose, cellobiose, and maltose) were added to the growth medium to see if they could suppress synthesis and accumulation of sulfotrehalose. Sucrose was the most effective in suppressing biosynthesis and accumulation of sulfotrehalose, with levels as low as 0.1 mM being able to significantly replace the novel charged osmolyte. Other common osmolytes (glycine betaine, glutamate, and proline) were not accumulated or used for osmotic balance in place of the sulfotrehalose by the halophilic archaeons.

Disaccharides↗

PubMed1996

Vanadate is a potent competitive inhibitor of phospholipase C from Bacillus cereus.

Monomeric vanadate is a potent competitive inhibitor of phospholipase C from Bacillus cereus, much better than other oxyanions (e.g., phosphate or iodate). The apparent efficiency of inhibition depends on the substrate aggregate structure. The measured inhibition constant with respect to monomeric phosphatidylcholine substrate is 0.21 mM under conditions where the K(m) is 0.12 mM; for micellar substrate the apparent Ki appears much lower and in fact tracks the apparent K(m) which decreases 10-fold. Vanadate inhibition is removed by addition of exogenous diacylglycerol, which by itself is an inhibitor. In contrast to its effect with monomeric or micellar substrate, vanadate does not strongly inhibit the PLC-catalyzed hydrolysis of small unilamellar vesicles of phosphatidylcholine. These results are interpreted in terms of the surface binding of the enzyme. Because of its ability to mimic the transition state of phosphate ester hydrolysis vanadate is also used to investigate the constraints on the occurrence of strained cyclic intermediates in phospholipid hydrolysis by PLC.

Bacillus cereus↗

PubMed1996

Conversion of lysophospholipids to cyclic lysophosphatidic acid by phospholipase D.

Phospholipase D from Streptomyces chromofuscus hydrolyzes lysophosphatidylcholine or lysophosphatidylethanolamine in aqueous 1% Triton X-100 solution. In situ monitoring of this reaction by 31P NMR revealed the formation of cyclic lysophosphatidic acid (1-acyl 2,3-cyclic glycerophosphate) as an intermediate which was hydrolyzed further by the enzyme at a functionally distinct active site to lysophosphatidic acid (lyso-PA). Synthetic cyclic lyso-PA (1-octanoyl 2,3-cyclic glycerophosphate) was found to be stable in aqueous neutral solutions at room temperature. It was hydrolyzed by the bacterial phospholipase D to lyso-PA at a rate which was approximately 4-fold slower than the rate of formation of cyclic lyso-PA. The addition of 5-10 mM sodium vanadate could partially inhibit the ring opening reaction and thus increase substantially the cyclic lyso-PA accumulation. Cyclic lyso-PA may act as a dormant configuration of the physiologically active lyso-PA or may even possess specific activities which await verification.

Hydrolysis↗

PubMed1996

Antifungal properties of yam (Dioscorea alata) peel extract.

The extraction of natural antifungal compounds from the peels of yam (Dioscorea alata) and the effect of these compounds on both the vegetative and reproductive structures of some yam not pathogens were studied. Four prominent antifungal components were obtained; one of the components was fully characterized and identified as beta-sitosterol. The antifungal activity of the compounds toward the germination of spores of two yam pathogens showed an inhibition of less than 57% at a concentration of 50 mg/L while inhibition on the elongation of germ-tubes of Fusarium moniliforme was as high as 82% at the same concentration. However, the ED50 for inhibition of germ-tube elongation in the yam compounds for the same organism was below 32 mg/L. The role of the yam compounds at high concentrations in disease resistance is discussed.

Antifungal Agents↗

PubMed1996

Mechanism and structure based inhibitors of phospholipase C enzymes.

PI-specific PLC enzymes are a key component of phosphatidylinositol-mediated signaling pathways since the hydrophobic product, diacylglycerol, activates protein kinase C and the water-soluble product, inositol trisphosphate, is involved in Ca2+ mobilization. Nonspecific, or PC-PLC, enzymes can generate diacylglycerol without Ca2+ mobilization. A series of inhibitors, both lipophilic and water-soluble, have been synthesized to target each of these two classes of PLC enzymes. Design of the inhibitors was based on proposed enzyme mechanisms and available crystal structures. The solution conformations of the lipophilic phospholipid analogs, (diheptanoylphosphatidyl(2-O-methyl)inositol for PI-PLC and a dihexanoyl-sn-(3-N-benzylaminoglycero)phosphoramidocholine for PC-PLC, have been determined using NMR methodology and the interaction of these compounds with bacterial enzymes has been examined. Water-soluble inhibitors include strained cyclic phosphonates for PI-PLC and vanadate for PC-PLC. An eventual goal of this work is to generate compounds that specifically target each type of intracellular PLC activity.

Bacillus cereus↗