Binding of a Glycera convoluta neurotoxin to cholinergic nerve terminal plasma membranes.

The crude extract of venom glands of the polychaete annelid Glycera convoluta triggers a large Ca2+-dependent acetylcholine release from both frog motor nerve terminals and Torpedo electric organ synaptosomes. This extract was partially purified by Concanavalin A affinity chromatography. The biological activity was correlated in both preparations to a 300,000-dalton band, as shown by gel electrophoresis. This confirmed previous determinations obtained with chromatographic methods. This glycoprotein binds to presynaptic but not postsynaptic plasma membranes isolated from Torpedo electric organ. Pretreatment of intact synaptosomes by pronase abolished both the binding and the venom-induced acetylcholine release without impairing the high K+-induced acetylcholine release. Pretreatment of nerve terminal membranes by Concanavalin A similarly prevented the binding and the biological response. Binding to Torpedo membranes was still observed in the presence of EGTA. An antiserum directed to venom glycoproteins inhibited the neurotoxin so we could directly follow its binding to the presynaptic membrane. Glycera convoluta neurotoxin has to bind to a ectocellularly oriented protein of the presynaptic terminal to induce transmitter release.

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Large-scale purification of presynaptic plasma membranes from Torpedo marmorata electric organ.

The Journal of Cell Biology ◽

10.1083/jcb.101.5.1757 ◽

1985 ◽

Vol 101 (5) ◽

pp. 1757-1762 ◽

Cited By ~ 29

Author(s):

N Morel ◽

J Marsal ◽

R Manaranche ◽

S Lazereg ◽

J C Mazie ◽

...

Keyword(s):

Plasma Membrane ◽

Large Scale ◽

Plasma Membranes ◽

Electric Organ ◽

Acetylcholinesterase Activity ◽

Cholinergic Nerve Terminals ◽

Membrane Bound ◽

Torpedo Electric Organ ◽

Presynaptic Plasma Membrane ◽

Glycera Convoluta

The presynaptic plasma membrane (PSPM) of cholinergic nerve terminals was purified from Torpedo electric organ using a large-scale procedure. Up to 500 g of frozen electric organ were fractioned in a single run, leading to the isolation of greater than 100 mg of PSPM proteins. The purity of the fraction is similar to that of the synaptosomal plasma membrane obtained after subfractionation of Torpedo synaptosomes as judged by its membrane-bound acetylcholinesterase activity, the number of Glycera convoluta neurotoxin binding sites, and the binding of two monoclonal antibodies directed against PSPM. The specificity of these antibodies for the PSPM is demonstrated by immunofluorescence microscopy.

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Diadenosine polyphosphate hydrolase from presynaptic plasma membranes of Torpedo electric organ

Biochemical Journal ◽

10.1042/bj3230677 ◽

1997 ◽

Vol 323 (3) ◽

pp. 677-684 ◽

Cited By ~ 21

Author(s):

Jesús MATEO ◽

Pedro ROTLLAN ◽

Eulalia MARTI ◽

Inmaculada GOMEZ DE ARANDA ◽

Carles SOLSONA ◽

...

Keyword(s):

Plasma Membranes ◽

Pyridoxal Phosphate ◽

Electric Organ ◽

Competitive Inhibitor ◽

Inhibitory Effect ◽

Ic50 Value ◽

Neural Origin ◽

Disulphonic Acid ◽

Torpedo Electric Organ ◽

Enzyme Activators

The diadenosine polyphosphate hydrolase present in presynaptic plasma membranes from the Torpedo electric organ has been characterized using fluorogenic substrates of the form di-(1,N6-ethenoadenosine) 5´,5‴-P1,Pn-polyphosphate. The enzyme hydrolyses diadenosine polyphosphates (Apn A, where n = 3–5), producing AMP and the corresponding adenosine (n-1) 5´-phosphate, Ap(n-1). The Km values of the enzyme were 0.543± 0.015, 0.478±0.043 and 0.520±0.026 μM, and the Vmax values were 633±4, 592±18 and 576±45 pmol/min per mg of protein, for the etheno derivatives of Ap3A (adenosine 5´,5‴-P1,P3-triphosphate), Ap4A (adenosine 5´,5‴-P1,P4 -tetraphosphate) and Ap5A (adenosine 5´,5‴-P1,P5-pentaphosphate) respectively. Ca2+, Mg2+ and Mn2+ are enzyme activators, with EC50 values of 0.86±0.11, 1.35±0.24 and 0.58±0.10 mM respectively. The fluoride ion is an inhibitor with an IC50 value of 1.38±0.19 mM. The ATP analogues adenosine 5´-tetraphosphate and adenosine 5´-[γ-thio]triphosphate are potent competitive inhibitors and adenosine 5´-[α,β-methylene]diphosphate is a less potent competitive inhibitor, the Ki values being 0.29±0.03, 0.43±0.05 and 7.18±0.8 μM respectively. The P2-receptor antagonist pyridoxal phosphate 6-azophenyl-2´,4´-disulphonic acid behaves as a non-competitive inhibitor with a Ki value of 29.7±3.1 μM, and also exhibits a significant inhibitory effect on Torpedo apyrase activity. The effect of pH on the Km and Vmax values, together with inhibition by diethyl pyrocarbonate, strongly suggests the presence of functional histidine residues in Torpedo diadenosine polyphosphate hydrolase. The enzyme from Torpedo shows similarities with that of neural origin from neurochromaffin cells, and significant differences compared with that from endothelial vascular cells.

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Nerve Terminal Sacs from Torpedo Electric Organ: A New Preparation for the Study of Presynaptic Cholinergic Mechanisms at the Molecular Level

Cholinergic Mechanisms and Psychopharmacology - Advances in Behavioral Biology ◽

10.1007/978-1-4684-3096-7_25 ◽

1978 ◽

pp. 359-375 ◽

Cited By ~ 5

Author(s):

M. J. Dowdall

Keyword(s):

Nerve Terminal ◽

Molecular Level ◽

Electric Organ ◽

Cholinergic Mechanisms ◽

Torpedo Electric Organ

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Quantitative description of acetylcholine release and fluctuations in nerve terminals of torpedo electric organ submitted to stimulation

Pflügers Archiv - European Journal of Physiology ◽

10.1007/bf00584383 ◽

1978 ◽

Vol 377 (1) ◽

pp. 117-118 ◽

Cited By ~ 1

Author(s):

M. Israel ◽

B. Lesbats ◽

R. Manaranche

Keyword(s):

Acetylcholine Release ◽

Quantitative Description ◽

Electric Organ ◽

Nerve Terminals ◽

Torpedo Electric Organ

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Mediatophore, a protein supporting quantal acetylcholine release

Canadian Journal of Physiology and Pharmacology ◽

10.1139/y99-080 ◽

1999 ◽

Vol 77 (9) ◽

pp. 689-698 ◽

Cited By ~ 5

Author(s):

Maurice Israël ◽

Yves Dunant

Keyword(s):

Acetylcholine Release ◽

Calcium Influx ◽

Electric Organ ◽

Release Mechanism ◽

Genomic Locus ◽

Calcium Dependent ◽

High Calcium ◽

Essential Properties ◽

Quantal Acetylcholine Release ◽

Torpedo Electric Organ

After having reconstituted in artificial membranes the calcium-dependent acetylcholine release step, and shown that essential properties of the mechanism were preserved, we purified from Torpedo electric organ nerve terminals a protein, the mediatophore, able to release acetylcholine upon calcium action. A plasmid encoding for Torpedo mediatophore was introduced into cells deficient for acetylcholine release and for the expression of the cholinergic genomic locus defined by the co-regulated choline acetyltransferase and vesicular transporter genes. The transfected cells became able to release acetylcholine in response to a calcium influx in the form of quanta. The cells had to be loaded with acetylcholine since they did not synthesize it, and without transporter they could not concentrate it in vesicles. We may then attribute the observed quanta to mediatophores. We know from previous works that like the release mechanism, mediatophore is activated at high calcium concentrations and desensitized at low calcium concentrations. Therefore only the mediatophores localized within the calcium microdomain would be activated synchronously. Synaptic vesicles have been shown to take up calcium and those of the active zone are well situated to control the diffusion of the calcium microdomain and consequently the synchronization of mediatophores. If this was the case, synchronization of mediatophores would depend on vesicular docking and on proteins ensuring this process.Key words: acetylcholine release, presynaptic proteins, quantal release, mediatophore, transfection.

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