Is the acetylcholine releasing protein mediatophore present in rat brain?

Mediatophore is a protein purified from the nerve terminal membranes of Torpedo electric organ. It confers to artificial membranes a calcium-dependent mechanism that translocates acetylcholine. When similar reconstitution experiments are applied to rat brain synaptosomal membranes they reveal the presence of mediatophore activity with properties close to those described for the Torpedo protein (extractability, sensitivity to calcium, and effect of the drug cetiedil).

Immunological detection of mediatophore in motor end-plates and electric organ subcellular fractions of torpedo marmorata.

A rabbit antiserum to mediatophore, a nerve terminal membrane protein involved in calcium dependent ACh release, was raised after immunization with the purified protein. An immunological assay for mediatophore was then developed and the subcellular distribution of this protein in Torpedo electric organ fractions was studied. A good agreement was obtained between the distribution in the different fractions of the antigen and of mediatophore related acetylcholine releasing activity as determined by reconstitution in proteoliposomes.

The lipid requirements of mediatophore for acetylcholine release activity. Large-scale purification of this protein in a reactive form.

The mediatophore, a protein which translocates acetylcholine, has been recently purified from the plasma membrane of the Torpedo electric organ nerve terminals. The functional integrity of the mediatophore requires the presence of lipids. Removal of associated lipids led to an irreversible loss of activity when obtained by ether precipitation of the protein.

Effect of cetiedil on acetylcholine release and intramembrane particles in cholinergic synaptosomes.

The release of acetylcholine (ACh) from instantly frozen Torpedo electric organ synaptosomes in the course of stimulation is systematically associated with an increase in the number of large intramembrane particles counted on freeze-fracture replicas. The drug cetiedil, which is a potent inhibitor of ACh release, also blocks the increase in the number of large particles. The blockage was studied either after ionophore A 23187 or Glycera neurotoxin action in the presence of calcium.

Purification of a presynaptic membrane protein that mediates a calcium-dependent translocation of acetylcholine.

A protein, which we call "mediatophore," that mediates calcium-dependent release of acetylcholine from proteoliposomes has been purified from the presynaptic plasma membrane. About 250 micrograms of this material was obtained from 500 g of Torpedo marmorata electric organ. Precipitation of the protein and subsequent removal of associated lipids inactivated the protein, which then became water soluble; this permitted evaluation of its Stokes radius (52 A) and its sedimentation coefficient (9.

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

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.

Large-scale purification of Torpedo electric organ synaptosomes.

A procedure for the large-scale purification of Torpedo electric organ synaptosomes is described. The synaptosomal fraction obtained is very pure as judged from biochemical and morphological data. In addition, acetylcholine (ACh) release was demonstrated after KCl depolarization of synaptosomes in the presence of calcium.

Partial purification of ?-glycerotoxin, a presynaptic neurotoxin from the venom glands of the polychaete annelid glycera convoluta.

The venom secreted from glands appended to the jaws of Glycera convoluta, a Polychaete Annelid, increases the spontaneous quantal release of transmitter from nerve terminals. The component that is biologically active on vertebrate cholinergic nerve terminals has recently been shown to be a high molecular weight protein. In the present work, the crude extract from the venom apparatus was shown to be toxic for mammals and crustaceans.

The release of acetylcholine: from a cellular towards a molecular mechanism.

The isolation of synaptic vesicles rich in acetylcholine (ACh) from the electric organ of Torpedo has indeed strengthened the hypothesis of transmitter exocytosis, but soon after it was found that non-vesicular free ACh was released and renewed upon stimulation. In contrast, vesicular ACh and the number of vesicles remained stable during physiological stimulations. In addition free ACh variations (representing the cytoplasmic pool) were correlated to the release kinetics as measured by the electroplaque discharge.

Reconstitution of a functional synaptosomal membrane possessing the protein constituents involved in acetylcholine translocation.

Reconstitution of a functional presynaptic membrane possessing calcium-dependent acetylcholine release properties has been achieved. The proteoliposomal membrane obtained gains its acetylcholine-releasing capabilities from presynaptic membrane proteins. At the peak of acetylcholine release, intramembrane particles became more numerous in one of the proteoliposomal membrane faces.

Binding of a Glycera convoluta neurotoxin to cholinergic nerve terminals triggers a Ca-dependent acetylcholine release.

The venom glands of the annelid Glycera convoluta contain a neurotoxin which triggers ACh release from frog motor terminals and Torpedo synaptosomes. This neurotoxin binds to presynaptic, but not postsynaptic plasma membranes prepared from Torpedo electric organ. The binding site is an ectocellularly oriented protein.

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.

Acetylcholine release from proteoliposomes equipped with synaptosomal membrane constituents.

A lyophilized presynaptic membrane powder prepared from Torpedo electric organ synaptosomes was incorporated into liposomes. These proteoliposomes had a large internal volume. The P and E faces of their membrane showed particles which were comparable to the presynaptic membrane ones.

Isolation of a presynaptic plasma membrane fraction from Torpedo cholinergic synaptosomes: evidence for a specific protein.

Synaptosomal plasma membranes were isolated from Torpedo cholinergic synaptosomes which had been purified as previously described or repurified by equilibrium centrifugation. The synaptosomal plasma membrane could be distinguished from postsynaptic membranes by the absence of postsynaptic specific markers (nicotinic AChR) and by its low intramembrane particle complement after freeze fracture. In addition, the presynaptic membrane fraction contained acetylcholinesterase.

Evidence for a specific protein associated to the plasma membrane of cholinergic synaptosomes.

Synaptosomal plasma membrane fractions were prepared by fractionation of pure Torpedo cholinergic synaptosomes. A 67 000 dalton peptide was shown to be a major component of the presynaptic membrane. It appears specific for this membrane since (1) it copurifies with the synaptosomal plasma membrane; (2) it was not present in similar plasma membranes but prepared from Torpedo electric lobes or electric nerves, and since (3) rabbit antibodies to presynaptic antigens which were mainly directed to this 67 000 dalton peptide band were shown to bind to the nerve terminal network in Torpedo electric organ.

Partial purification of the Glycera convoluta venom components responsible for its presynaptic effects.

The crude extract of glands appended to the jaws of the polychaete annelid Glycera convoluta induces an important increase in the spontaneous quantal transmitter release on frog and crayfish neuromuscular junctions and on Torpedo nerve-electroplaque junctions. The venom similarly triggers acetylcholine (ACh) release from synaptosomes purified from Torpedo electric organ. At the frog neuromuscular junction, the reproducibility, the reversibility and the dose-dependence of the venom action permit a quantitative evaluation of the effect.

Rearrangement of intramembrane particles as a possible mechanism for the release of acetylcholine.

1. A chemiluminescent procedure for measuring acetylcholine (ACh) has recently been described. The procedure is based on the hydrolysis of ACh by acetylcholinesterase and on the oxidation of choline to betaine and H2O2 by choline oxidase.

Calcium uptake by cholinergic synaptic vesicles.

Pure synaptic vesicles have been isolated in sucrose-KCl media. They are able to take up calcium in the presence of ATP and Mg. This is based on the following evidence.

[Changes in the number and distribution of intramembranous particles of electric organ synaptosomes of Torpedo during synaptic activity].

Cholinergic synaptosomes were depolarized with KCl or treated with a venom extracted from the annelid Glycera convoluta. This venom was shown to increase considerably the frequency of the miniature endplate potentials at neuromuscular junctions. The synaptosomes were frozen and fractured in the absence of any fixative or cryoprotectant.

ATP-dependent calcium uptake by cholinergic synaptic vesicles isolated from Torpedo electric organ.

Cholinergic synaptic vesicles were purified from Torpedo electric organ to near morphological homogeneity. They were isolated in a K+ environment. A method is described for the preparation of concentrated synaptic vesicles that allows uptake studies by conventional techniques.

Effect of the venom of Glycera convoluta on the spontaneous quantal release of transmitter.

A neurotoxin able to increase the spontaneous release of transmitter was found in the venom glands of the polychaete annelid Glycera convoluta. We studied the effect of this venom on the frog cutaneous pectoris muscle, where its application produced a prolonged (20-h), high-frequency discharge of miniature potentials. After 5 h of action, the initial store was renewed several times but no detectable ultrastructural changes were observed.