SIMILARITIES OF β‐BUNGAROTOXIN AND PHOSPHOLIPASE A<sub>2</sub> AND THEIR MECHANISM OF ACTION

Sen, Indira; Cooper, Joel

doi:10.1111/j.1471-4159.1978.tb10468.x

Cited by 43 publications

(33 citation statements)

References 24 publications

(1 reference statement)

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“…Additionally, the ability of either f3-bungarotoxin or crotoxin to stimulate release was abolished when Ca2+ was removed from the perfusing medium. Other studies (13,14) suggest that the Ca2+-dependent release of these two toxins may be explained by phospholipase A activity. Extracts of the venom glands of the black widow do stimulate high frequencies of mepps at the neuromuscular junction, but they also fail to generate biphasic release in a single experiment.…”

Section: Methodsmentioning

confidence: 95%

Leptinotarsin: A presynaptic neurotoxin that stimulates release of acetylcholine

McClure

Abbott

Baxter

et al. 1980

Proc. Natl. Acad. Sci. U.S.A.

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Leptinotarsin, a toxin found in the hemolymph of the beetle Leptinotarsa haidemani, can stimulate release of acetylcholine from synaptic termini. Leptinotarsin causes an increase in the frequency of miniature end plate potentials (mepps) of the rat phrenic nerve-diaphragm preparation. The increase in the frequency of mepps induced by leptinotarsin is biphasic: about 10% of the total mepps are released in an initial burst that lasts about 90 sec, after which the remaining mepps are released over a period of 10-20 min. Tetrodotoxin has no effect upon the release induced by leptinotarsin, but low-Ca2+ conditions abolish the first phase. The two phases of release may represent two presynaptic pools of acetylcholine, both of which can be released in quantized form. In a second study, rat brain synaptosomes were incubated with [3H]choline and were immobilized on Millipore filters. Leptinotarsin induced release of [3H]acetylcholine from this preparation, confirming the release seen by using neurophysiological methods. The ability of leptinotarsin to induce release from either intact nerve terminals or synaptosomes was abolished when the toxin was heated. The releasing activity of leptinotarsin from synaptosomes was also partially dependent upon the presence of Ca2+ in the perfusing solution. Release from synaptosomes followed first-order kinetics, and was not inhibited by commercial antibodies to black widow spider antigens. The data suggest that leptinotarsin acts as a presynaptic neurotoxin and may be of value as a mechanistic probe in understanding the storage and release of neurotransmitters.The mechanisms mediating the release of neurotransmitter have not been resolved. Very little is known about the sequence of events after the entrance of Ca2+ into the presynaptic terminal and the eventual release of neurotransmitter. One approach to this problem has been the identification of toxins that interact with the presynaptic terminal to modify the process of release. By examining the mechanism of action of the toxin, as well as the mechanism of the step(s) upon which the toxin acts, a more detailed description of the process of release may be obtained.Extracts of black widow spider venom glands (BWGE) will stimulate a massive increase in the frequency of miniature end plate potentials (mepps) from the frog neuromuscular junction (1). When Ca2+ is removed from the perfusing medium, BWGE were still able to stimulate release, although at reduced rates, in a variety of preparations (1-4). These data suggest that the action of BWGE was not simply to depolarize the membrane, thereby allowing Ca2+ to enter and elicit release of acetylcholine (AcCho), but was a more specific interaction with the release mechanism. Purification (5,6) indicates that the protein responsible for the releasing activity in BWGE could not be recovered in large amounts, and suggests that the lack of availability of pure protein may limit the usefulness of the toxin as a mechanistic probe. The present study was directed towards the search for a...

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Section: Methodsmentioning

confidence: 95%

Leptinotarsin: A presynaptic neurotoxin that stimulates release of acetylcholine

McClure

Abbott

Baxter

et al. 1980

Proc. Natl. Acad. Sci. U.S.A.

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“…In addition, phospholipase activity towards radiolabelled membrane phospholipids of synaptosomes was measured essentially as described by Sen and Cooper [37] with the following modifications. Purified synaptosomes of 5-week-old rats were used rather than synaptic plasma membranes from 16-day-old animals; 400 pCi of [3H]olcic acid (80 nmol) were injected intracerebrally using stereotaxic equipment positioned at L-4.0, S-(-0.2) and D-2.5 mm.…”

Section: Assuj9 Of Phospliolipase Act Ivitjmentioning

confidence: 99%

“…injection; the value in parentheses is for 3H-/-bungarotoxin after isoelectric focussing and was calculated using the specific radioactivity of the unfocussed material. Phospholipase activity was measured in the presence of deoxycholate at 37 labelled toxin contained negligible amounts of unlabelled toxin prior to electrofocussing.…”

Section: Labelling Of /?-Bungaroroxin With N-suc~inirnidyl-[23-~h]prmentioning

confidence: 99%

Preparation of Neurotoxic 3H-β-Bungarotoxin: Demonstration of Saturable Binding to Brain Synapses and Its Inhibition by Toxin I

Othman

Spokes

Dolly

2005

European Journal of Biochemistry

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Homogeneous β‐bungarotoxin, isolated from the venom of Bungarus multicinctus was radiolabelled with N‐Succinimidyl‐[2,3‐3H]propionate. Stable, di‐propionylated material was obtained which was tritiated on both subunits and had a specific radioactivity of 102 Ci/mmol. After separation from unlabelled toxin by isoelectric focussing, it was shown to exhibit significant biological activity in both the peripheral and central nervous systems but had negligible phospholipase A2 activity towards lecithin or cerebrocortical synaptosomes. The labeled neurotoxin binds specifically to a single class of non‐interacting sites of high affinity (Kd= 0.6 nM) on rat cerebral cortex synaptosomes; the content of sites is about 150 fmol/mg protein. This binding was inhibited by unlabelled β‐bungarotoxin with a potency which indicates that tritiation does not alter the affinity significantly. The association of toxin with its binding component and its dissociation were monophasic; rate constants observed were 7.8 × 105 M−1 s−1 and 5.6 × 10−4 s−1 at 37°C, respectively. β‐Bungarotoxin whose phospholipase activity had been inactivated with p‐bromophenacyl bromide inhibited to some extent the binding of tritiated toxin but with low efficacy. Taipoxin and phospholipase A2 from bee venom, but not Naja melanoleuca, inhibited the synaptosomal binding of toxin with low potencies in the presence, but not the absence, of Ca2+. Toxin I, a single‐chain protein from Dendroaspis polylepis known to potentiate transmitter release at chick neuromuscular junction, completely inhibited the binding of 3H‐β‐bungarotoxin with a Ki of 0.07 nM; this explains its ability to antagonise the neuroparalytic action of β‐bungarotoxin. Other pure presynaptic neurotoxins, α‐latrotoxin and botulinum neurotoxin failed to antagonise the observed binding; likewise tityustoxin, which is known to affect sodium channels, had no effect on 3H‐β‐bungarotoxin binding. Trypsinization of synaptosomes completely destroyed the binding activity, suggesting that the binding component is a protein; the functional role of the latter is discussed in relation to the specificity of toxin binding.

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“…Although it remains to be shown by direct means that fl-Btx causes a sustained increase of cytosolic Ca2+, the triphasic effects of /3-Btx on ACh release (Abe et al 1976) and the toxin's stimulation of ACh synthesis might develop in the following manner. The early, phospholipase-independent inhibition of ACh release is probably correlated with toxin binding (Abe et al 1976;Kelly et al 1979); then the toxin-mediated depolarization of nerve endings (Sen & Cooper, 1978;Ng & Howard, 1979) initiates the rise of nerve terminal Ca2+ leading to the period of enhanced transmitter release. We propose that this second phase begins the period of enhanced synthesis and accumulation of tissue ACh.…”

Section: Fl-btx Stimulates Ach Synthesismentioning

confidence: 99%

“…The resulting depletion of energy stores was presumed to block ACh release. Alternatively, Sen & Cooper (1978) proposed that fl-Btx disrupts membrane phospholipids, thereby depolarizing the nerve ending and inhibiting high affinity choline transport. A reduction of choline uptake would lead to a drop in ACh content and an inhibition of ACh release.…”

Section: Introductionmentioning

confidence: 99%

Beta‐bungarotoxin stimulates the synthesis and accumulation of acetylcholine in rat phrenic nerve diaphragm preparations.

Gundersen¹,

Jenden²,

Newton³

1981

The Journal of Physiology

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SUMMARY1. The effects of f-bungarotoxin on acetylcholine (ACh) synthesis, tissue content and release have been studied in the rat diaphragm. A gas chromatographic mass spectrometric assay was used to measure ACh and choline. 5. fl-Bungarotoxin did not directly activate choline acetyltransferase in muscle homogenates.6. The toxin-induced rise in tissue ACh was largely absent in Ca2+-free solutions which contained either EGTA (1 mM) or SrCl2 (2 or 10 mM).7. Non-neurotoxic phospholipases A2, fatty acids and the neurotoxic phospholipase A2, notexin, did not cause ACh accumulation in the diaphragm.8. fl-Bungarotoxin did not stimulate ACh synthesis in denervated muscle. 9. The extra ACh which accumulated after fi-bungarotoxin did not contribute to enhanced release by nerve impulses even when 4-aminopyridine was added to the medium. High K+ solution and black widow spider venom were also ineffective in increasing output from toxin-treated diaphragms relative to controls that had not been treated with f3-bungarotoxin.10. Prior injection of a rat with botulinum toxin prevented the accumulation of ACh due to f3-bungarotoxin. Tubocurarine, however, did not antagonize fl-bungarotoxin.11. These data indicate that fi-bungarotoxin has a unique capacity to inhibit ACh release and stimulate ACh synthesis in diaphragm nerve endings. The results are discussed in terms of a possible action of f3-bungarotoxin to raise the level of ionized Ca in the nerve terminal cytosol.

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SIMILARITIES OF β‐BUNGAROTOXIN AND PHOSPHOLIPASE A₂ AND THEIR MECHANISM OF ACTION

Cited by 43 publications

References 24 publications

Leptinotarsin: A presynaptic neurotoxin that stimulates release of acetylcholine

Leptinotarsin: A presynaptic neurotoxin that stimulates release of acetylcholine

Preparation of Neurotoxic 3H-β-Bungarotoxin: Demonstration of Saturable Binding to Brain Synapses and Its Inhibition by Toxin I

Beta‐bungarotoxin stimulates the synthesis and accumulation of acetylcholine in rat phrenic nerve diaphragm preparations.

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SIMILARITIES OF β‐BUNGAROTOXIN AND PHOSPHOLIPASE A2 AND THEIR MECHANISM OF ACTION

Cited by 43 publications

References 24 publications

Leptinotarsin: A presynaptic neurotoxin that stimulates release of acetylcholine

Leptinotarsin: A presynaptic neurotoxin that stimulates release of acetylcholine

Preparation of Neurotoxic 3H-β-Bungarotoxin: Demonstration of Saturable Binding to Brain Synapses and Its Inhibition by Toxin I

Beta‐bungarotoxin stimulates the synthesis and accumulation of acetylcholine in rat phrenic nerve diaphragm preparations.

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SIMILARITIES OF β‐BUNGAROTOXIN AND PHOSPHOLIPASE A₂ AND THEIR MECHANISM OF ACTION