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. Mediatophore was highly concentrated in presynaptic plasma membranes of electric organ, while very low contents were observed in electric nerves and electric lobes. Although some mediatophore was found in synaptic vesicle fractions, this most probably resulted from presynaptic membrane contamination as evaluated with other presynaptic membrane markers. Nerve terminals of motor end-plates were strongly stained with anti-mediatophore antibodies.     

Calcium-induced acetylcholine release and intramembrane particle occurrence in proteoliposomes equipped with mediatophore.

Proteoliposomes obtained from the mediatophore, a purified Torpedo electric organ nerve terminals protein, and endogenous lipids were used for a study of calcium-induced release of acetylcholine and freeze-fracture electron microscopy. Large intramembrane particles were induced by the influx of calcium into proteoliposomes, as previously observed for synaptosomes or stimulated electric organ nerve terminals. The involvement of mediatophore in a calcium dependent acetylcholine translocation seems therefore to be related to the occurrence of a category of intramembrane particles in the course of the release process.     

Detection with Monoclonal Antibodies of a 15-kDa Proteolipid in Both Presynaptic Plasma Membranes and Synaptic Vesicles in Torpedo Electric Organ

A protein, the mediatophore, has been purified from Torpedo electric organ presynaptic plasma membranes. This protein mediates the release of acetylcholine through artificial membranes when activated by calcium and is made up of 15-kDa proteolipid subunits. After immunization with purified delipidated mediatophore, monoclonal antibodies binding to the 15-kDa proteolipid band on Western blots of purified mediatophore were selected. A 15-kDa proteolipid antigen was also detected in cholinergic synaptic vesicles. Using an immunological assay, it was estimated that presynaptic plasma membranes and synaptic vesicles contain similar proportions of 15-kDa proteolipid antigen. Detection by immunofluorescence in the electric organ showed that only nerve endings were labeled. In electric lobes, the staining was associated with intracellular membranes of the electroneuron cell bodies and in axons. Nerve endings at Torpedo neuromuscular junctions were also labeled with anti-15-kDa proteolipid monoclonal antibodies.     

Immunolabelling of the presynaptic membrane of Torpedo electric organ nerve terminals with an antiserum towards the acetylcholine releasing protein mediatophore

Mediatophore is a nerve terminal membrane protein purified from Torpedo electric organ on its ability to translocate acetylcholine upon calcium action. An antiserum able to immunoprecipitate mediatophore activity was used to study the subcellular distribution of this protein. The presynaptic membrane exhibited a strong and discontinuous immunogold labelling, especially at the active zone where ACh is thought to be released. Two antigens were recognized on immunoblots of synaptosomal membranes: the 15-kDa subunit of mediatophore and a 14-kDa membrane protein that has a wide non-neuronal distribution. Antibodies purified from the serum on native mediatophore and monospecific towards the 15-kDa antigen still gave a high presynaptic membrane localized labelling. In addition, a few 14-kDa protein sites were present at the active zone. The Schwann cell finger interposed between the presynaptic membrane and the postsynaptic arch also exhibited the 14-kDa antigen raising the question of a possible interaction of mediatophore with the 14-kDa protein originating from the Schwann cell.     

Characterization of a polyclonal antiserum raised against mediatophore, a protein that translocates acetylcholine

A protein (the mediatophore) purified from Torpedo electric organ nerve terminals was identified by its ability to translocate acetylcholine upon calcium action. The recent large scale production of this protein led us to prepare a polyclonal antibody that was used to study the localization of the protein.     

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). The activity was more abundant in synaptosomal membranes than in mitochondrial or myelinic membranes and in cholinergic areas as compared to cerebellum.     

Mediatophore, a protein supporting quantal acetylcholine release

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.     

ASSOCIATION OF SYNTAXIN WITH SNAP 25 AND VAMP (SYNAPTOBREVIN) IN TORPEDO SYNAPTOSOMES

Two proteins of the presynaptic plasma membrane, syntaxin and SNAP 25, and VAMP/synaptobrevin, a synaptic vesicle membrane protein, form stable protein complexes which are involved in the docking and fusion of synaptic vesicles at the mammalian brain presynaptic membrane. Similar protein complexes were revealed in an homogeneous population of cholinergic synaptosomes purified from Torpedo electric organ by combining velocity sedimentation and immunoprecipitation experiments. After CHAPS solubilization, virtually all the nerve terminal syntaxin was found in the form of large 16 S complexes, in association with 65% of SNAP 25 and 15% of VAMP. Upon Triton X100 solubilization, syntaxin was still recovered in association with SNAP 25 and VAMP but in smaller 8 S complexes. A small (2–5%) percentage of the nerve terminal 15 kDa proteolipid subunit of the v-H⁺ ATPase and of mediatophore was copurified with syntaxin, using two different antisyntaxin monoclonal antibodies. The use of an homogeneous population of peripheral cholinergic nerve terminals allowed us to extend results on the composition of the brain presynaptic protein complexes to the Torpedo electric organ synapse, a model of the rapid neuromuscular synapses. Copyright © 1996 Elsevier Science Ltd     

Evoked Acetylcholine Release Expressed in Neuroblastoma Cells by Transfection of Mediatophore cDNA

Abstract: Transmitter release was elicited in two ways from cultured cells filled with acetylcholine: (a) in a biochemical assay by successive addition of a calcium ionophore and calcium and (b) electrophysiologically, by electrical stimulation of individual cells and real-time recording with an embryonic Xenopus myocyte. Glioma C6-Bu-1 cells were found to be competent for Ca²⁺-dependent and quantal release. In contrast, no release could be elicited from mouse neuroblastoma N18TG-2 cells. However, acetylcholine release could be restored when N18TG-2 cells were transfected with a plasmid coding for mediatophore. Mediatophore is a protein of nerve terminal membranes purified from the Torpedo electric organ on the basis of its acetylcholine-releasing capacity. The transfected N18TG-2 cells expressed Torpedo mediatophore in their plasma membrane. In response to an electrical stimulus, they generated in the myocyte evoked currents that were curare sensitive and calcium dependent and displayed discrete amplitude levels, like in naturally occurring synapses.     

Isolation from Cholinergic Synapses of a Protein That Binds to Membranes in a Calcium-Dependent Manner

A protein that binds to membranes in a calcium-dependent manner between calcium concentrations of 10(-5) and 10(-6) M has been isolated in large amounts (20 mg/kg tissue) from the entirely cholinergic electric organ of Torpedo marmorata. The protein bound reversibly to membrane fractions in a calcium-specific and saturable manner. The protein also bound to lipids isolated from Torpedo electric organ and to clathrin-coated vesicles prepared from pig brain. The protein bound to a Triton X-100-sensitive site. It had an apparent subunit molecular weight of 32,000 by polyacrylamide gel electrophoresis and of 35,900 by amino acid analysis; a broad isoelectric range of 4.8 to 5.5; and 27% of its amino acids after hydrolysis were observed to be aspartic and glutamic acids. Synaptosomes derived from electric organ were enriched in the protein which is probably localised within the nerve ending. It was localised in the synaptic region of the electric organ by means of immunofluorescence. In the electric lobe, discrete patches of fluorescence were seen within the cell bodies that innervate the electric organ. The protein may be involved in the recognition of membranes within the cholinergic neurone. Proteins with similar purification properties were found in all tissues investigated so far, and polypeptides of subunit molecular weight 32,000 were identified in bovine adrenal medulla and guinea pig brain synaptosomes.     

Evidence for an Association of the 15-kDa Proteolipid of Mediatophore with a 14-kDa Polypeptide

The present report shows that mediatophore, a nerve terminal membrane protein that translocates acetylcholine on calcium action, forms a complex with a 14-kDa polypeptide. The complex was identified based on the following results. (a) A polyclonal antimediatophore antiserum that immunoprecipitates activity precipitates both the 15- and 14-kDa polypeptides. (b) After HPLC purification of mediatophore, both antigens were found in the same peak. (c) After 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate solubilization of presynaptic membranes or of the purified mediatophore, an immunoaffinity column made with the anti-14-kDa antigen monoclonal antibody retained both the 14-kDa and the 15-kDa polypeptide. Similarly, immunoprecipitation experiments using protein A-coated beads sedimented an immunocomplex in which both antigens were found. (d) The 14-kDa antigen could be localized in the synaptosomal membrane where mediatophore and its 15-kDa component are found.     

Characterization of two ectocellularly oriented 67 K presynaptic proteins. Lack of effect of their removal on acetylcholine release

A presynaptic plasma membrane fraction was purified after subfractionation of pure cholinergic synaptosomes prepared from Torpedo electric organ. Two 67 kdalton proteins were highly enriched in the synaptosomal plasma membrane (SPM): the hydrophobic form of AChE and a protein against which we raised a monoclonal antibody (C1–8). These two proteins exhibit similar biochemical properties: both exist as disulphide linked dimers with the same molecular weight; they are glycoproteins binding Concanavalin A; they are exposed on the external surface of the SPM and detached as almost entire molecules by Pronase. Nevertheless, using the C1–8 monoclonal antibody, it was possible to show that they are different proteins. The C1–8 binding protein appears to be specific for the SPM in Torpedo electric organ since it was not detected in plasma membranes from the electroplaque, the electric nerve trunks or the electric lobe. The hydrophobic AChE and the C1–8 binding protein appear therefore to be useful markers of the SPM. Pronase treatment of intact synaptosomes removes most of the ectocellularly exposed proteins of the SPM, which amount to 35% of the SPM protein. Presynaptic AChE and the C1–8 binding protein are detached. But ACh release can still be induced by depolarization of the Pronase treated synaptosomes. This demonstrates that the two 67 kdalton presynaptic proteins are not directly involved in the release of the neurotransmitter.     

Effect of N,N-Dicyclohexylcarbodiimide on Acetylcholine Release from Torpedo Synaptosomes and Proteoliposomes Reconstituted with the Proteolipid Mediatophore

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.     

Calcium-Induced Desensitization of Acetylcholine Release from Synaptosomes or Proteoliposomes Equipped with Mediatophore, a Presynaptic Membrane Protein

A "fatigue" of acetylcholine (ACh) release is described in cholinergic synaptosomes stimulated with the calcium ionophore A23187 or gramicidin. A small conditioning calcium entry, which did not trigger a large ACh release, led to a decrease of transmitter release elicited by a second large calcium influx. This fatigue was half-maximal at approximately 30 microM external calcium and developed in a few minutes. In contrast, activation of release by calcium was very rapid and was half-maximal at approximately 0.5 mM external calcium. Activation and desensitization of release could be attributed to the recently identified presynaptic membrane protein, the "mediatophore." Proteoliposomes equipped with purified mediatophore showed a calcium-dependent activation and "fatigue" of ACh release similar to that of synaptosomes. It was found that the ionophore A23187 rapidly equilibrated internal and external calcium concentrations in proteoliposomes. Thus, the external calcium concentration gave the internal concentration required for activation or desensitization of proteoliposomal ACh release. The mediatophore showed remarkable calcium binding properties (20 sites/molecule) with a KD of 25 microM. The physiological implications of desensitization on the organization of release sites are discussed.     

Motor control of the jamming avoidance response of Apteronotus leptorhynchus: evolutionary changes of a behavior and its neuronal substrates

The two closely related gymnotiform fishes, Apteronotus and Eigenmannia, share many similar communication and electrolocation behaviors that require modulation of the frequency of their electric organ discharges. The premotor linkages between their electrosensory system and their medullary pacemaker nucleus, which controls the repetition rate of their electric organ discharges, appear to function differently, however. In the context of the jamming avoidance response, Eigenmannia can raise or lower its electric organ discharge frequency from its resting level. A normally quiescent input from the diencephalic prepacemaker nucleus can be recruited to raise the electric organ discharge frequency above the resting level. Another normally active input, from the sublemniscal prepacemaker nucleus, can be inhibited to lower the electric organ discharge frequency below the resting level (Metzner 1993). In contrast, during a jamming avoidance response, Apteronotus cannot lower its electric organ discharge frequency below the resting level. The sublemniscal prepacemaker is normally completely inhibited and release of this inhibition allows the electric organ discharge frequency to rise during the jamming avoidance response. Further inhibition of this nucleus cannot lower the electric organ discharge frequency below the resting level. Lesions of the diencephalic prepacemaker do not affect performance of the jamming avoidance response. Thus, in Apteronotus, the sublemniscal prepacemaker alone controls the change of the electric organ discharge frequency during the jamming avoidance response.     

A simple procedure for the preparation of highly purified (sodium + potassium) adenosinetriphosphatase from the rectal salt gland of Squalus acanthias and the electric organ of Electrophorus electricus.

A simple purification procedure for the Na,K-ATPase from membranes of the rectal gland of Squalus acanthias or crude microsomal fractions from the electric organ of Electrophorus electricus is presented here. The purification procedure consists of solubilization of the Na,K-ATPase with the nonionic detergent. Lubrol WX, chromatography of the diluted Lubrol extract on aminoethyl cellulose, and ammonium sulfate fractionation (1) of the concentrated eluate from the aminoethyl cellulose column. The yields of final purified enzyme are comparable to the earlier purification (1–4) involving the expensive and cumbersome zonal centrifugation stop. The purity of the final enzyme, as attested to by specific activity and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, is as great or greater than that previously reported for the enzyme purified by the procedure involving zonal centrifugation. The simplicity of the present procedure, coupled with the ready commercial availability of electric eels which are quite hardy on shipment, makes purification of the Na,K-ATPase widely available to workers in the field.     

Structural changes in alkaline-treated postsynaptic membranes from Torpedo marmorata are not due to lipid hydrolysis

The removal of the 43 kDa peptide from postsynaptic membranes from the electric organ of Torpedo by alkaline treatment (pH 11) or by lithium diiodosalicylate at pH 8 results in similar ultrastructural changes that cannot be produced by the action of porcine pancreatic phospholipase A₂ or lysophosphatidylcholine. Thin-layer chromatography fails to reveal significant alkaline hydrolysis of membrane lipids from postsynaptic membranes and erythrocyte ghosts under the conditions used for peptide extraction.     

Fat body cells of Amblyomma cajennense partially engorged females (Acari: Ixodidae) and their role on vitellogenesis process

During the process of Arthropoda reproduction, the synthesis and uptake of proteins, carbohydrates, and lipids by oocytes is termed vitellogenesis. These compounds that will make up the yolk may be in ticks endogenously synthesized by the oocytes and/or exogenously produced by the fat body and pedicel cells. This study examined the fat body of Amblyomma cajennense ticks at the cytochemical ultrastructural level to demonstrate the presence of lipids, proteins and carbohydrates in trophocytes. The lipids were detected in higher quantity than proteins and carbohydrates in the fat body cells, suggesting that the role of the fat body in tick is stored lipids and carbohydrates to convert them in energy, or still they could be used with cell structural purpose. The electrophoresis technique applied at A. cajennense fat body demonstrated specifically the molecular mass of proteins: about 98 kDa. By the other hands, the fat body is not the organ responsible for the synthesis of the yolk protein, role probably performed by the pedicel cells.     

A tyrosine kinase related to pp60c-src is associated with membranes of Electrophorus electricus electric organ

A tyrosine-specific protein kinase immunologically related to pp60c-src, the cellular homolog of the Rous sarcoma virus-transforming protein, was expressed at elevated levels in the electric organ of the electric eel Electrophorus electricus. The electric organ kinase phosphorylated antibodies reactive with pp60c-src at tyrosine residues in immune complex protein kinase assays and was associated with electric organ membranes enriched in acetylcholine receptors. The protein recognized by anti-pp60c-src antibodies was phosphorylated in endogenous membrane phosphorylation reactions and was shown to have a relative molecular mass of 57 kDa by two-dimensional gel electrophoresis. In immune complex protein kinase assays the 57-kDa protein was phosphorylated at threonine by a distinct threonine kinase from the electric organ. The tyrosine kinase was purified 844-fold from electric organ membranes by chromatography on omega-aminohexyl agarose, phosphocellulose, and casein-Sepharose. Threonine kinase activity in immunoprecipitates was not observed in the tyrosine kinase fractions after the first step. Incubation of the casein Sepharose fraction with [gamma-32P]ATP-Mn2+ in solution resulted in phosphorylation of only the 57-kDa protein. Phosphorylation occurred solely at tyrosine, suggesting that the kinase is capable of autophosphorylation. The structural and functional properties of the 57-kDa electric organ kinase indicate that the 57-kDa electric organ protein is a member of the src subfamily of tyrosine kinases and is closely related to pp60c-src.