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M Wictome

Publications and source records attributed to M Wictome.

15 recordsLinked to original sources
PubMed1999

Development of in vitro assays for the detection of botulinum toxins in foods.

Currently the only accepted method for the detection of botulinum neurotoxin in contaminated samples is the mouse bioassay. Although highly sensitive this test has a number of drawbacks: it is expensive to perform, lacks specificity and involves the use of animals. With increasing resistance to such animal tests there is a need to replace the bioassay with a reliable in vitro test. Over the past six years it has been demonstrated that all the botulinum neurotoxins act intracellularly as highly specific zinc endoproteases, cleaving proteins involved in the control of secretion of neurotransmitters. In the work described, this enzymatic activity has been utilised in assay formats for the detection in foods of neurotoxin of the serotypes involved in food-borne outbreaks in man. These assays have been shown to have a greater sensitivity, speed and specificity than the mouse bioassay. It is envisaged that such assays will prove realistic alternatives to animal-based tests.

Animals↗
PubMed1999

Development of an in vitro bioassay for Clostridium botulinum type B neurotoxin in foods that is more sensitive than the mouse bioassay.

A novel, in vitro bioassay for detection of the botulinum type B neurotoxin in a range of media was developed. The assay is amplified by the enzymic activity of the neurotoxin's light chain and includes the following three stages: first, a small, monoclonal antibody-based immunoaffinity column captures the toxin; second, a peptide substrate is cleaved by using the endopeptidase activity of the type B neurotoxin; and finally, a modified enzyme-linked immunoassay system detects the peptide cleavage products. The assay is highly specific for type B neurotoxin and is capable of detecting type B toxin at a concentration of 5 pg ml(-1) (0.5 mouse 50% lethal dose ml(-1)) in approximately 5 h. The format of the test was found to be suitable for detecting botulinum type B toxin in a range of foodstuffs with a sensitivity that exceeds the sensitivity of the mouse assay. Using highly specific monoclonal antibodies as the capture phase, we found that the endopeptidase assay was capable of differentiating between the type B neurotoxins produced by proteolytic and nonproteolytic strains of Clostridium botulinum type B.

Animals↗
PubMed1999

Novel assays for the detection of botulinum toxins in foods.

Currently the only accepted method for the detection of botulinum neurotoxin in contaminated samples is the mouse bio-assay. Although highly sensitive this test has a number of drawbacks: it is expensive to perform, lacks specificity and involves the use of animals. With increasing resistance to such animal tests there is a need to replace the bio-assay with a reliable in vitro test. Over the past six years it has been demonstrated that all the botulinum neurotoxins act intracellularly as highly specific zinc endoproteases, cleaving proteins involved in the control of secretion of neurotransmitters. In the work described, this enzymatic activity has been utilised in assay formats for the detection in foods of neurotoxin from the serotypes involved in food-borne outbreaks in man. These assays have been shown to have a greater sensitivity, speed and specificity than the mouse bio-assay. It is envisaged that such assays will prove realistic alternatives to animal based tests.

Animals↗
PubMed1996

Labelling the Ca(2+)-ATPase of skeletal-muscle sarcoplasmic reticulum with the cross-linker o-phthalaldehyde.

The Ca(2+)-ATPase in the sarcoplasmic reticulum of skeletal muscle reacts with o-phthalaldehyde (OPA) to form a fluorescent isoindole product. The stoichiometry of labelling of the ATPase is 9 nmol of isoindole/mg of ATPase, corresponding to a 1:1 molar ratio of isoindole: ATPase. There is no evidence for any intermolecular cross-linking. Isoindole formation is faster in the presence of methylamine, but the stoichiometry of labelling is unchanged, whereas in the presence of 2-mercaptoethanol the level of labelling is much higher. It is concluded that OPA reacts with a single Cys residue (defining the specificity of the reaction) in a fast step, subsequent reaction with a Lys residue to form the isoindole being rate-controlling. Labelling the ATPase with OPA in the absence of methylamine leads to total loss of ATPase activity, whereas in the presence of methylamine, the decrease in ATPase activity on reaction is small. We conclude that the loss of ATPase activity probably follows from formation of the intramolecular cross-link rather than from the initial modification of the Cys residue. Reaction with OPA is not affected by the presence of ATP, ADP or Ca2+, so that the reactive Cys is not part of a ligand-binding site. The fluorescence emission spectrum of the labelled ATPase indicates a hydrophobic environment for the isoindole ring.

Adenosine Diphosphate↗
PubMed1996

Substrate residues N-terminal to the cleavage site of botulinum type B neurotoxin play a role in determining the specificity of its endopeptidase activity.

Clostridium botulinum type B neurotoxin is a highly specific zinc-endopeptidase which cleaves vesicle-associated membrane protein (VAMP/synaptobrevin), a critical component of the vesicle docking/fusion mechanism. In this study, substrate residues flanking the N-terminal side of the cleavage site are shown to play a key role in enzyme substrate recognition. Two aspartate residues in this region are identified as critical determinants of the neurotoxin's specificity. These findings are discussed in relation to the mechanism by which botulinum type B neurotoxin cleaves its substrate.

Amino Acid Sequence↗
PubMed1995

Interactions of dihydroxybenzenes with the Ca(2+)-ATPase: separate binding sites for dihydroxybenzenes and sesquiterpene lactones.

The Ca(2+)-ATPase of skeletal muscle sarcoplasmic reticulum is inhibited by 2,5-di-tert-butyl-1,4-dihydroxybenzene (BHQ) and other hydrophobic 1,4-dihydroxybenzenes. Inhibitory potency increases on increasing substituent chain length from 2,5-dipropyl-1,4-dihydroxybenzene to 2,5-di-tert-amyl-1,4-dihydroxybenzene, the most potent inhibitor, but then decreases for 2,5-bis(7-methylheptyl)-1,4-dihydroxybenzene. Kinetic measurements are consistent with isomerization following the initial binding of BHQ to the ATPase to give a modified E2 conformation, E2AI, as for the binding of sesquiterpene lactones, such as thapsigargin. Binding of BHQ to the ATPase shifts the E1-E2 equilibrium toward E2 because of the formation of E2AI. Measurements of Ca2+ binding as a function of BHQ concentration suggest that BHQ can bind to the E1 conformation of the ATPase (but without the subsequent conformational change observed on binding to E2) and that the binding constants of E1 for Ca2+ are unaffected by binding of BHQ. Binding of BHQ to the ATPase in the presence of substoichiometric amounts of thapsivillosin A and effects of mixtures of BHQ and thapsivillosin A show that these two inhibitors have separate binding sites on the ATPase.

Animals↗
PubMed1995

Binding of sesquiterpene lactone inhibitors to the Ca(2+)-ATPase.

The mechanism of inhibition of the Ca(2+)-ATPase from sarcoplasmic reticulum by the sesquiterpene lactones thapsigargin, trilobolide and thapsivillosin A (TvA) has been determined. A decrease in the affinity of the ATPase for Ca2+ is observed in the presence of the inhibitors (I), consistent with a shift in the E1/E2 equilibrium for the ATPase towards E2 forms. Amounts of inhibitor beyond a 1:1 molar ratio with ATPase produce no further decrease in affinity for Ca2+, inconsistent with the formation of a dead-end complex. Measurements of the rate of quenching of the tryptophan fluorescence of the ATPase by TvA are consistent with an association step to give E2I followed by an isomerization to a modified state E2AI. The kinetics of the reversal of the effects of TvA by Ca2+ at sub-stoichiometric amounts of TvA are bi-exponential, with a fast component whose rate is independent of TvA concentration and equal to the rate observed in the absence of TvA, and a slow component whose rate decreases with increasing TvA concentration. These observations are also consistent with the formation of a modified state E2AI following the initial binding of I to E2. The equilibrium constant E2AI/E2I increases in the order TvA < trilobolide < thapsigargin. The results suggest that the effects of the inhibitors on the overall ratio of E2 to E1 forms of the ATPase follow largely from the formation of E2AI from E2I, and that binding constants are very similar for E1Ca2, E1 and E2.

Animals↗
PubMed1994

Characterisation of a novel Ca2+ pump inhibitor (bis-phenol) and its effects on intracellular Ca2+ mobilization.

Bis-phenol, a phenolic antioxidant, is an inhibitor of sarcoplasmic reticulum (SR), endoplasmic reticulum (ER) and plasma membrane Ca2+ ATPases. The concentration of bis-phenol giving half-maximal inhibition of the SR Ca(2+)-ATPase is 2 microM. On binding to the SR Ca(2+)-ATPase it shifts the E2 to E1 transition towards the E2 state and slows the transition between E2 to E1. Bis-phenol completely inhibits Ca(2+)-dependent ATP hydrolysis and Ca2+ uptake by rat cerebellar microsomes at a concentration of 30 microM. The plasma membrane Ca(2+)-ATPase is also completely inhibited at similar concentrations, however, the Na+/K(+)-ATPase is only marginally affected. Other inhibitors of the ER Ca(2+)-ATPases, thapsigargin and 2,5-di-(tert-butyl)-1,4-benzohydroquinone (BHQ), inhibit Ca2+ uptake by approximately 75%. Bis-phenol therefore inhibits all types of ER Ca(2+)-ATPases present in cerebellum. This inhibitor is also able to mobilize Ca2+ from intracellular Ca2+ stores, including those sensitive to InsP3, in intact HL-60 cells.

Adenosine Triphosphate↗
PubMed1994

The importance of the hydroxyl moieties for inhibition of the Ca(2+)-ATPase by trilobolide and 2,5-di(tert-butyl)-1,4-benzohydroquinone.

Trilobolide and 2,5-di(tert-butyl)-1,4-benzohydroquinone (BHQ) are potent inhibitors of the Ca(2+)-ATPase of skeletal muscle sarcoplasmic reticulum. Desoxytrilobolide and 2,5-di(tert-butyl)-1,4-diacetylphenol (acetyl-BHQ) have much lower potencies than their parent compounds and 2,5-di(tert-butyl)-1,4-benzoquinone (BQ) has no effect on ATPase activity. Studies using the ATPase labelled with 4-nitrobenzo-2-oxa-1,3-diazole (NBD) suggest that both trilobolide and BHQ bind more strongly to the E2 conformation of the ATPase than to the E1 conformation. Desoxytrilobolide, acetyl-BHQ and BQ have little effect on the E1/E2 equilibrium. Studies with mixtures of trilobolide and desoxytrilobolide suggest that the inactive derivatives are unable to bind to the ATPase.

Adenosine Triphosphatases↗
PubMed1994

Binding of Ca2+ to the (Ca(2+)-Mg2+)-ATPase of sarcoplasmic reticulum: kinetic studies.

Stop-flow fluorescence and rapid-filtration methods have been used to establish the kinetics of Ca2+ binding to, and dissociation from, the (Ca(2+)-Mg2+)-ATPase of skeletal-muscle sarcoplasmic reticulum and to define the effects of H+ and Mg2+ on Ca2+ binding and dissociation rates. The kinetics have been interpreted in terms of the scheme: E2 E2<==>E1<==>E1Ca<==>E1'Ca<==>E1'Ca2. The kinetics of the E2<==>E1 E1 transition have been determined by measuring the rate of change of the fluorescence of the ATPase labelled with 4-nitrobenzo-2-oxa-1,3-diazole after a pH jump or the addition of Ca2+ to the labelled ATPase in the presence of thapsigargin or thapsivillosin A. It has been shown that Mg2+ has a marked effect on Ca2+ dissociation at pH 7.2 and that changes in the tryptophan fluorescence of the ATPase follow the same time course as the dissociation of 45Ca2+. It is proposed that the effect of Mg2+ follows from binding to a 'gating' site, as detected by changes in the fluorescence of the ATPase labelled with 4-(bromomethyl)-6,7-dimethoxycoumarin. The rate of dissociation of Ca2+ from the ATPase increases with increasing pH. The rate of dissociation of Ca2+ decreases with increasing Ca2+ concentration in the medium, with an apparent affinity for Ca2+ greater than that seen for the change in fluorescence amplitude. It is shown that this follows if the first, inner, Ca(2+)-binding site on the ATPase has a lower affinity for Ca2+ than the second, outer, site. Effects of H+ and Mg2+ on Ca2+ dissociation can be treated by the quasiequilibrium approach. Mg2+ and H+ also affect the rate of Ca2+ binding to the ATPase, and effects of H+ and Mg2+ on the E2<==>E1 equilibrium explain the results of experiments in which the concentrations of H+ and Mg2+ are jumped.

Animals↗
PubMed1992

The inhibitors thapsigargin and 2,5-di(tert-butyl)-1,4-benzohydroquinone favour the E2 form of the Ca2+,Mg(2+)-ATPase.

2,5-Di(tert-butyl)-1,4-benzohydroquinone has been shown to inhibit the Ca2+,M(2+)-ATPase of sarcoplasmic reticulum with an affinity of 0.4 microM. It has been shown to shift the E2-E1 equilibrium for the ATPase towards E2, as shown previously for the inhibitor thapsigargin. The shift towards E2 results in a decrease in affinity for Ca2+, as also observed for thapsigargin. A marked decrease in the rate of the E2-E1 transition is observed for both BHQ and thapsigargin. A decrease in the equilibrium level of phosphorylation by Pi and of the steady-state level of phosphorylation by ATP are consistent with a decrease in the equilibrium constant for phosphorylation by Pi and an increase in the rate of dephosphorylation.

Ca(2+) Mg(2+)-ATPase↗
PubMed1992

Mechanism of inhibition of the calcium pump of sarcoplasmic reticulum by thapsigargin.

The steady-state ATPase activity of sarcoplasmic-reticulum (Ca(2+)-Mg2+)-ATPase is inhibited by thapsigargin at a molar ratio of 1:1, with a dissociation constant for thapsigargin estimated to be in the sub-nanomolar range. In the presence of thapsigargin, only a single Ca2+ ion binds to the ATPase. Similarly, addition of thapsigargin to the ATPase incubated in the presence of Ca2+ results in the release of one of the two originally bound Ca2+ ions. As monitored by the fluorescence of nitrobenzo-2-oxa-1,3-diazole-labelled ATPase, thapsigargin appears to shift the transition between E1 and E2 conformations towards E2. Addition of thapsigargin prevents phosphorylation of the ATPase by P(i) and results in a very low steady-state level of phosphorylation of the ATPase by ATP, as observed previously for nonylphenol.

Animals↗