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  • 1
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Abstract: The mediatophore is a presynaptic membrane protein that has been shown to translocate acetylcholine (ACh) under calcium stimulation when reconstituted into artificial membranes. The mediatophore subunit, a 15-kDa proteolipid, presents a very high sequence homology with the N,N′-dicyclohexylcarbodiimide (DCCD)-binding proteolipid subunit of the vacuolar-type H+-ATPase. This prompted us to study the effect of DCCD, a potent blocker of proton translocation, on calcium-dependent ACh release. The present work shows that DCCD has no effect on ACh translocation either from Torpedo synaptosomes or from proteoliposomes reconstituted with purified mediatophore. However, using [14C]DCCD, we were able to demonstrate that the drug does bind to the 15-kDa proteolipid subunit of the mediatophore. These results suggest that although the 15-kDa proteolipid subunits of the mediatophore and the vacuolar H+-ATPase may be identical, different domains of these proteins are involved in proton translocation and calcium-dependent ACh release and that the two proteins have a different membrane organization.
    Type of Medium: Electronic Resource
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  • 2
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Abstract: In previous work, it was shown that cytoplasmic acetylcholine decreased on stimulation of Torpedo electric organ or synaptosomes in a strictly calcium-dependent manner. This led to the hypothesis that the presynaptic membrane contained an element translocating acetylcholine when activated by calcium. To test this hypothesis, the presynaptic membrane constituents were incorporated into the membranes of liposomes filled with acetylcholine. The proteoliposomes thus obtained released the transmitter in response to a calcium influx. The kinetics and calcium dependency of acetylcholine release were comparable for proteoliposomes and synaptosomes. The presynaptic membrane element ensuring calcium-dependent acetylcholine release is most probably a protein, since it was susceptible to Pronase, but only when the protease had access to the intracellular face of the presynaptic membrane. Postsynaptic membrane fractions contained very low amounts of this protein. It was extracted from the presynaptic membrane under alkaline conditions in the form of a protein-lipid complex of large size and low density which was partially purified. The specificity of the calcium-dependent release for acetylcholine was tested with proteoliposomes filled with equal amounts of acetylcholine and choline or acetylcholine and ATP. In both cases, acetylcholine was released preferentially. After cholate solubilization and gel filtration, the protein ensuring the calcium-dependent acetylcholine release was recovered at a high apparent molecular weight (between 600,000 and 200,000 daltons), its apparent sedimentation coefficient being 17S after cholate elimination. This protein is probably an essential coin of the transmitter release mechanism. We propose to name it mediatophore.
    Type of Medium: Electronic Resource
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  • 3
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Publishing Ltd
    Journal of neurochemistry 55 (1990), S. 0 
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Abstract: The neuromuscular synapses of the rat sternomastoid muscles contain a membrane protein, mediatophore, that endows artificial membranes with a calcium-dependent acetylcholine release mechanism. Mediatophore and choline acetylase had similar distributions along the muscle. Sciatic nerve membranes contain mediatophore, and a purified preparation was obtained from the nerve.
    Type of Medium: Electronic Resource
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  • 4
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Science Ltd
    Journal of neurochemistry 71 (1998), S. 0 
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Abstract: Torpedo electric organ synaptosomes possess a typical vacuolar H+-ATPase (V-ATPase), inhibited by concanamycin A and insensitive to vanadate, made of the association of a catalytic soluble sector V1 to a membrane domain V0. In the electric nerves, the 57-kDa subunit B of the V1 sector was transported to the nerve endings by the slow axonal flow and did not accumulate upstream from an axonal block. In contrast, a 500% accumulation of the 15-kDa subunit c of the V0 membrane domain was observed, demonstrating that this subunit is conveyed by the fast axonal anterograde transport. After velocity sedimentation of solubilized nerve proteins, the 57- and 15-kDa subunits were recovered in different complexes corresponding, respectively, to the V1 and V0 domains. No fully assembled V-ATPase was detected. It is concluded that V1 and V0 domains of V-ATPase are transported separately in axons, at different rates, and that they only associate once arrived in nerve endings to form the active V-ATPase.
    Type of Medium: Electronic Resource
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  • 5
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Science Ltd
    Journal of neurochemistry 68 (1997), S. 0 
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Abstract: During the process of docking and fusion of synaptic vesicles to the presynaptic membrane, several presynaptic proteins bind sequentially to a core complex associating two proteins of the presynaptic membrane, syntaxin and SNAP 25, and a protein of synaptic vesicles, VAMP/synaptobrevin. We have immunoprecipitated this core complex after CHAPS solubilization of pure cholinergic synaptosomes of Torpedo electric organ, using anti-syntaxin or anti-VAMP immunobeads. In parallel, we studied syntaxin and VAMP, which are transported by the rapid axonal flow to the nerve endings. We found that syntaxin and VAMP accumulating at the proximal end of an electric nerve ligature were already engaged in complexes, as in synaptosomes. In unligated nerves also, significant amounts of VAMP associate with syntaxin. The possibility that these complexes form after solubilization was eliminated because added VAMP was unable to associate with syntaxin in solubilized control nerves and because similar amounts of complex were obtained after sodium dodecyl sulfate or CHAPS solubilization. Hence, syntaxin is already associated with SNAP 25 and VAMP during axonal transport, before reaching nerve endings.
    Type of Medium: Electronic Resource
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  • 6
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Publishing Ltd
    Journal of neurochemistry 36 (1981), S. 0 
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Abstract: The release of acetylcholine (ACh) and ATP from pure cholinergic synaptosomes isolated from the electric organ of Torpedo was studied in the same perfused sample. A presynaptic ATP release was demonstrated either by depolarization with KCl or after the action of a venom extracted from the annelid Glycera convoluta (GV). The release of ATP exhibited similar kinetics to that of ACh release and was therefore probably closely related to the latter. The ACh/ATP ratio in perfusates after KCl depolarization was 45; this was much higher than the ACh/ATP ratio in cholinergic synaptic vesicles, which was 5. The ACh/ATP ratio released after the action of GV was also higher than that of synaptic vesicles. These differences are discussed. The stoichiometry of ACh and ATP release is not consistent with the view that the whole synaptic vesicle content is released by exocytosis after KCl depolarization, as is the case for chromatin cells in the adrenal medulla.
    Type of Medium: Electronic Resource
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  • 7
    ISSN: 1460-9568
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Complexins are presynaptic proteins whose functional roles in synaptic transmission are still unclear. In cultured rat hippocampal neurons, complexins are distributed throughout the cell bodies, dendrites and axons, whereas synaptotagmin I and synaptobrevin/VAMP-2, essential proteins for neurotransmitter release, accumulated in the synaptic-releasing sites as early as 1 week in culture. With a maturation of synapses in vitro, complexins also accumulated in the synaptic release sites and co-localized with synaptotagmin I and synaptobrevin/VAMP-2 after 3–4 weeks in culture. Complexins I and II were expressed in more than 90 and 70% of the cultured neurons, respectively; however, they were largely distributed in different populations of synaptic terminals. In the developing rat brain, complexins were distributed in neuronal cell bodies in the early stage of postnatal development, but gradually accumulated in the synapse-enriched regions with development. In mature presynaptic neurons of Aplysia buccal ganglia, injection of anticomplexin II antibody caused a stimulation of neurotransmitter release. Injection of recombinant complexin II and αSNAP caused depression and facilitation of neurotransmitter release from nerve terminals, respectively. The effect of complexin was reversed by a subsequent injection of recombinant αSNAP, and vice versa. These results suggest that complexins are not essential but have some regulatory roles in neurotransmitter release from presynaptic terminals of mature neurons.
    Type of Medium: Electronic Resource
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  • 8
    ISSN: 1460-9568
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Antisense oligodesoxynucleotides were used to determine whether the mediatophore proteolipid is necessary for the Ca2+-dependent release of the neurotransmitter acetylcholine. Xenopus laevis oocytes were injected with poly(A)+ mRNAs extracted from the electric lobes of Torpedo marmorata. The electric lobes contain an homogeneous population of cholinergic neurons homologous to motoneurons. Addition of antisense probes hybridizing to the mediatophore 15 kDa subunit inhibited the expression of both the mediatophore proteolipid in oocyte membranes and the Ca2+-dependent acetylcholine release. Expression of other neuronal functions such as synthesis of [14C]acetylcholine from [14C]acetate was not inhibited. Another antisense probe specific for the sequence of a related proteolipid cDNA (the 15 kDa subunit of the chromaffin granule protonophore) was used as a control. It did not hybridize with the Torpedo mediatophore mRNA and, injected in addition to electric lobe mRNAs, it did not inhibit either mediatophore expression or acetylcholine release. We showed in addition that the mRNA primed oocytes did not contain a vesicular pool of acetylcholine. It was concluded (i) that the mediatophore proteolipid is essential for Ca2+-dependent acetylcholine release and (ii) that the cytosolic pool of neurotransmitter seems to be preferentially used in this system.
    Type of Medium: Electronic Resource
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  • 9
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Publishing Ltd
    Annals of the New York Academy of Sciences 493 (1987), S. 0 
    ISSN: 1749-6632
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Natural Sciences in General
    Type of Medium: Electronic Resource
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