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J J Volwerk

Publications and source records attributed to J J Volwerk.

At least 19 recordsLinked to original sources
PubMed1994

Phosphatidylinositol-specific phospholipase C from Bacillus cereus at the lipid-water interface: interfacial binding, catalysis, and activation.

Binding characteristics of phosphatidylinositol-specific phospholipase C (PI-PLC) from Bacillus cereus binding to the phospholipid-water interface were determined by spectroscopic methods and correlated with PI-PLC's catalytic properties. Binding of the enzyme to micelles and bilayers of zwitterionic phosphocholines is accompanied by an increase in the fluorescence emission from tryptophan, whereas a decrease in the emission is observed with synthetic anionic lipids containing a phosphomethanol head group. A similar decrease in the tryptophan emission is observed with phosphatidylinositol (PI) analogues containing the phospho-D-1-myo-inositol head group, but not with the enantiomeric L-1-myo-inositol. In covesicles of PI and phosphatidylcholine (PC), the rate of cleavage of PI is reduced because, as a neutral diluent, PC effectively reduces the surface concentration of PI that the bound enzyme "sees" in the interface. This permits determination of the interfacial Michaelis constant (KM*) as 0.26 mol fraction for PI as substrate. On the other hand, ditetradecylglycerophosphomethanol (DTPM) acts as a kinetic competitive inhibitor in the covesicles. The spectroscopic and catalytic activity data taken together show that PI-PLC binds to the interface of aqueous dispersions of phospholipids with an apparent Kd (in terms of the lipid monomers) of about 10-50 microM. However, only lipids with an anionic head group, such as phosphomethanol and phospho-D-1-myo-inositol, are able to bind as single molecules into the active site of the enzyme at the interface. Enantiomeric phospho-L-1-myo-inositol or the zwitterionic phosphocholine head group has little affinity for the enzyme at the interface. Thus, PI-PLC appears to obey the two-stage, Michaelis-Menten adaptation of interfacial catalysis, according to which the binding of the enzyme to the interface precedes the steps of the catalytic turnover at the interface. Limit estimates suggest that on PI or PI/PC vesicles the catalysis occurs in the "scooting" mode with a moderate processivity. DTPM vesicles also inhibit the activity of PI-PLC toward the synthetic water-soluble substrate myo-inositol 1-(4-nitrophenyl phosphate), but the activity is enhanced severalfold in the presence of vesicles of zwitterionic phosphatidylcholine. Several possible explanations of this interfacial activation are considered within the general context of the kinetic scheme for interfacial catalysis. The kinetic results for the action of PI-PLC bound to vesicles are consistent with a model in which the interface acts as an "allosteric" effector of the catalytic rate constant, kcat, without affecting the substrate binding.

Bacillus cereus↗
PubMed1993

A chemiluminescent substrate for the detection of phosphatidylinositol-specific phospholipase C.

A water-soluble, chemiluminescent substrate for sensitive detection of bacterial phosphatidylinositol-specific phospholipase C (PI-PLC) activity was synthesized. This was achieved by replacing the diacylglycerol moiety found in the natural substrate, phosphatidylinositol, by a dioxetane-containing moiety, resulting in the synthetic substrate (+/-)-3-(4-methoxyspiro[1,2-dioxetane-3,2'- tricyclo[3.3.1.1(3,7]decan]-4-yl)phenyl myo-inositol-1-O-hydrogen phosphate (LUMI-PI). PI-PLC-catalyzed cleavage of the phosphodiester bond of LUMI-PI results in the formation of a chemiluminescent precursor, the labile anion AMP-D, which decays with emission of light. An assay procedure was developed using 96-well microtiter plates with detection of the chemiluminescence on autoradiography film. With this procedure the detection limit of purified phospholipase C was about 10 pg. Furthermore, PI-PLC activity was readily detected in situ using small quantities (< 1 microliters) of liquid cultures of PI-PLC-producing bacterial strains.

Adamantane↗
PubMed1993

Differential expression of phospholipase C specific for inositol phospholipids at the cell surface of rat glial cells and REF52 rat embryo fibroblasts.

Phosphatidylinositol(PI)-specific phospholipase C activity was detected on the surface of rat astrocytes, rat C6 glioma cells, and rat embryo (REF52) fibroblasts. The cell surface phospholipase C (ecto-PLC) activity was calcium-dependent, did not result from secreted phospholipase C, and was not released from the cell surface by bacterial PI-specific phospholipase C. Agents known to stimulate intracellular PI turnover, including carbachol, L-glutamic acid, acetylcholine, and orthovanadate, did not induce measurable alterations in the activity of the ecto-PLC. The expression of ecto-PLC activity by REF52 fibroblasts was density-dependent: subconfluent cultures of REF52 exhibited low levels of activity (less than 80 pmol of inositol phosphate formed/min/10(6) cells), whereas in confluent cultures ecto-PLC activity increased to approximately 300 pmol/min/10(6) cells. In contrast to this behavior and that exhibited by previously reported ecto-PLC-positive cell types, the ecto-PLC activity exhibited by astrocytes (approximately 1,000 pmol/min/10(6) cells) and by C6 glioma cells (approximately 100 pmol/min/10(6) cells) was independent of cell culture density up to confluence. The constitutive expression of ecto-PLC activity of astroglial cells may be related to their function as accessory cells in close association with neurons.

Animals↗
PubMed1992

Substrate stereospecificity of phosphatidylinositol-specific phospholipase C from Bacillus cereus examined using the resolved enantiomers of synthetic myo-inositol 1-(4-nitrophenyl phosphate).

The substrate stereospecificity of phosphatidylinositol-specific phospholipase C from Bacillus cereus is examined using the resolved optical isomers of synthetic myo-inositol 1-(4-nitrophenyl phosphate), a chromogenic substrate for the phospholipase. The synthetic route employs mild acid-labile protecting groups and separation of the substituted myo-inositol enantiomers as the (-)-camphanyl ester diastereomers. Measurements of the initial rates of cleavage of the D and L enantiomers of the nitrophenyl substrate by phosphatidylinositol-specific phospholipase C from B. cereus show that this enzyme is essentially stereospecific for the D enantiomer. Under identical conditions, the rate of cleavage of the L isomer is less than 0.2% of that observed for the D isomer. The same is observed for the highly homologous enzyme from Bacillus thuringiensis. There is no measurable inhibition by the L enantiomer of the B. cereus enzyme acting on the D enantiomer, even when the molar ratio of L:D is 5, indicating that binding of the L enantiomer to the phospholipase is negligible. Thus, the enzyme active site is exquisitely sensitive to the stereochemistry of the myo-inositol group of the substrate.

Bacillus cereus↗
PubMed1992

A high level of cell surface phosphatidylinositol-specific phospholipase C activity is characteristic of growth-arrested 3T3 fibroblasts but not of transformed variants.

Confluent monolayers of four contact-inhibited mouse fibroblast lines (Swiss 3T3, Balb/c 3T3, NIH 3T3, and C3H10T1/2) were found to have substantial levels of a cell surface phosphatidylinositol-specific phospholipase C (ecto-PLC). In contrast, confluent cultures of virally, chemically, or spontaneously transformed variants derived from these cell lines expressed undetectable or negligible levels of this enzyme activity. A simple and rapid assay, using lysophosphatidylinositol radio-labeled in the inositol group ([3H]-lysoPI) as the substrate was developed to provide a quantitative measure of the phospholipase C activity present at the external cell surface. For cells testing positive for ecto-PLC activity, rapid uptake of [3H]-lysoPI is accompanied by the simultaneous appearance of [3H]-inositol phosphate in the external medium. Confluent monolayers of the four mouse fibroblast lines exhibiting density-dependent growth inhibition had levels of ecto-PLC activity in the range of 50-800 pmol/min/10(6) cells (i.e., about 20-50 times greater than the activity observed for the transformed variants). The expression of ecto-PLC activity at the cell surface of the Swiss or Balb/c cells was dependent on the state of cell proliferation. Cultures which had become quiescent through attainment of confluence displayed a tenfold increased activity over that of subconfluent, growing cultures of these cells. Similarly, subconfluent Swiss 3T3 cells which had become quiescent following exposure to low serum conditions also showed increased activity. These results indicate that there may exist a correlation between the control of cell proliferation in contact-inhibited mouse fibroblasts and the expression of inositol phospholipid-specific phospholipase C activity at the external cell surface.

3T3 Cells↗
PubMed1991

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.

Bacillus cereus↗
PubMed1991

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.

Chromogenic Compounds↗
PubMed1991

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.

Amino Acid Sequence↗
PubMed1990

Inhibition of the phosphatidylinositol-specific phospholipase C from Bacillus cereus by a monoclonal antibody binding to a region with sequence similarity to eukaryotic phospholipases.

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 acetylcholinesterase 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.

Acetylcholinesterase↗
PubMed1990

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.

Bacillus cereus↗
PubMed1990

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.

Bacillus cereus↗
PubMed1990

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.

Chromatography, Affinity↗
PubMed1990

Synthesis of phosphonate derivatives of myo-inositol for use in biochemical studies of inositol-binding proteins.

Phospholipids containing the inositol headgroup (phosphoinositides) serve as membrane storage forms of a family of messenger molecules that transmit signals in cells. In this study a general synthesis of myo-inositol phosphate derivatives in which the phosphorus oxygen bond is replaced with a phosphorus carbon bond (i.e. phosphonates) is presented. Four specific examples of phosphonate analogs of phosphatidylinositol (PI) are prepared which have a single alkyl chain in place of the diacylglycerol. These derivatives are stable in neutral and alkaline solutions and are designed for use in biochemical studies of PI-specific phospholipases C and other enzymes involved in the phosphoinositide signal transduction pathway.

Chemical Phenomena↗
PubMed1989

Functional characteristics of phosphatidylinositol-specific phospholipases C from Bacillus cereus and Bacillus thuringiensis.

Phosphatidylinositol-specific phospholipase C was purified from the culture medium of B. thuringiensis to high specific activity using a procedure we recently described for purification of PI-PLC from B. cereus (Volwerk et al. (1989) J. Cell. Biochem. 39, 315-325). The purified enzymes from B. thuringiensis and B. cereus have similar specific activities towards hydrolysis of the membrane lipid phosphatidylinositol, and also towards hydrolysis of the glycosyl-phosphatidylinositol-containing membrane anchor of bovine erythrocyte acetylcholinesterase. These results indicate very similar catalytic properties for the structurally homologous PI-specific phospholipases C secreted by these bacilli.

Acetylcholinesterase↗
PubMed1989

Phosphatidylinositol-specific phospholipase C from Bacillus cereus: improved purification, amino acid composition, and amino-terminal sequence.

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.

Amino Acid Sequence↗
PubMed1988

Rotational dynamics of the single tryptophan of porcine pancreatic phospholipase A2, its zymogen, and an enzyme/micelle complex. A steady-state and time-resolved anisotropy study.

The rotational dynamics of the single tryptophan of porcine pancreatic phospholipase A2 and its zymogen (prophospholipase A2) have been studied by polarized fluorescence using steady-state and time-resolved single-photon counting techniques. The motion of Trp-3 in phospholipase A2 consists of a rapid subnanosecond wobble of the indole ring with an amplitude of about +/- 20 degrees accompanied by slower isotropic rotation of the entire protein. The rotational correlation times for overall particle rotational diffusion are consistent with conventional hydrodynamic theory. When phospholipase A2 binds to micelles of n-hexadecylphosphocholine, the amplitude of the fast ring rotation decreases. The whole particle rotational correlation time of the enzyme/micelle complex is smaller than the minimum value calculated from hydrodynamic theory. A similar result is obtained for the micelle itself by using the lipophilic probe transparinaric acid. These low values for the particle correlation times can be understood by postulating that an isotropic motion of the fluorophore in the small detergent particles contributes to the angular reorientation of the fluorophore. The internal reorientational motion of the tryptophan in the zymogen, prophospholipase A2, is of larger amplitude than that observed for the enzyme; specifically, the proenzyme exhibits a motion with a significant amplitude on the nanosecond time scale. This additional freedom of motion is attributed to segmental mobility of the N-terminal residues of prophospholipase A2. This demonstrates that this region of the protein is flexible in the zymogen but not in the processed enzyme. The implications of these findings for the mechanism of surface activation of phospholipase A2 are discussed by analogy with a trypsinogen-trypsin activation model.

Animals↗
PubMed1988

A novel approach to the ultrastructural localization of cell surface receptors: affinity-gold labeling of Na, K-ATPase.

We describe here a novel method of affinity-gold labeling for the ultrastructural localization and biochemical characterization of functional cell surface receptors. This approach combines the widely used colloidal gold technique, a previously published method for coating the gold with a matrix of derivatized dextran, small receptor-specific ligands, and a photoactivatable cross-linker. The resulting gold-affinity probe directed to a selected receptor by the ligand, is subsequently attached to the receptor by light activation of the cross-linker. As a specific example, a gold affinity probe prepared with ouabain, a selective inhibitor of Na,K-ATPase, as the directing ligand was used to investigate the ultrastructural localization of this enzyme complex in cell membranes. The biological activity of ouabain covalently linked to derivatized dextran containing the photoactivatable cross-linker was examined by its action on ion transport across dog trachea epithelium and on the enzyme activity of Na,K-ATPase preparations obtained from the rectal gland of the elasmobranch, Mustelus californicus. By these tests the probe mimics the effects of free ouabain. Electron micrographs of labeled human erythrocytes and cultured human foreskin fibroblasts showed an apparent random distribution of Na,K-ATPase on the plasma membranes of these cells. Binding of the probe was blocked in the presence of excess ouabain, a result demonstrating that the affinity probe binds at the same sites as free ouabain. Covalent attachment of probe by light activation of the nitroarylazido groups greatly enhanced retention during washing and standard procedures of fixation and dehydration. The high density of the gold probe was utilized to isolate the covalently attached membrane components from labeled human foreskin fibroblasts.(ABSTRACT TRUNCATED AT 250 WORDS)

Affinity Labels↗