Membrane-bound choline acetyltransferase in Torpedo electric organ: a marker for synaptosomal plasma membranes?

The enzyme choline-O-acetyltransferase catalyses the biosynthesis of acetylcholine from acetyl coenzyme A and choline and is considered as one of the best markers for cholinergic nerve endings. The distribution of this enzymatic activity was analysed during the purification of plasma membranes of purely cholinergic nerve endings isolated from the electric organ of the fish Torpedo marmorata. This tissue, which receives a profuse and purely cholinergic innervation, can be considered as being a "giant" neuromuscular synapse. The isolated nerve endings (synaptosomes) were first osmotically disrupted and their plasma membranes isolated by equilibrium density centrifugation (discontinuous followed by continuous sucrose gradients). Choline acetyltransferase activity was found to exist in three forms: (1) a soluble form (the major one) present in the cytoplasm of the nerve endings, (2) a form which is ionically associated with membranes and which can be solubilized by washing exhaustively the membrane fraction with solutions of high ionic strength (0.5 M NaCl) and (iii) a form which is non-ionically bound to membranes and cannot be solubilized with high salt solution. The soluble and the non-ionically bound activities exhibited very similar affinities for choline (1.34 and 1.64 mM, respectively). The non-ionically membrane-associated form of choline acetyltransferase was found to "copurify" with the cholinergic synaptosomal plasma membranes of Torpedo, its specific activity being increased from 122 (crude fraction) to 475 (purified membrane fraction) nmol/h/mg protein. An enrichment was also observed for another cholinergic marker, the enzyme acetylcholinesterase, but not for the nicotinic receptor to acetylcholine, a marker for postsynaptic membranes. No choline acetyltransferase activity could be detected in preparations of synaptic vesicles that were highly purified from the electric organ. Also, the non-ionically associated form of choline acetyltransferase activity was hardly detectable (2.4 nmol/h/mg protein) in fractions enriched in axonal membranes prepared from the cholinergic electric nerves innervating the electric organ. The partition into soluble and membrane-bound activity was also analysed for choline acetyltransferase present in human placenta, a rich source for the enzyme but a non-innervated tissue. In this case the great majority of the enzyme appeared as soluble activity. Very low levels of non-ionically membrane-bound activity were found to be present in a crude membrane fraction from human placenta (2.8 nmol/h/mg protein).(ABSTRACT TRUNCATED AT 400 WORDS)

Amphiphilic and hydrophilic forms of choline-O-acetyltransferase in cholinergic nerve endings of the Torpedo.

In the purely cholinergic nerve endings isolated (i.e. synaptosomes) from the electric organ of the fish Torpedo, the enzyme choline acetyltransferase was found to exist not solely in its well-known soluble form but also in a form which is non-ionically bound to the plasma membrane; this activity could not be solubilized in solutions of high ionic strength (0.5 M NaCl). The non-ionic detergent Triton X-114 was used to solubilize synaptosomes isolated from either the electric organ of Torpedo or rat brain. This detergent allows to separate hydrophilic from amphiphilic proteins of cells or subcellular fractions. Twelve per cent of the synaptosomal choline acetyltransferase partitioned as amphiphilic and 80-97% as hydrophilic activity. The percentage of amphiphilic activity present in synaptosomes was significantly higher than that of the form of activity (4.4%) extracted from samples containing only the soluble form of choline acetyltransferase but was significantly lower than the percentage of amphiphilic enzyme present in preparations of synaptosomal plasma membrane (20-22%) which were enriched in the non-ionically membrane-bound form of choline acetyltransferase. These results indicate that the soluble and the non-ionically membrane-bound enzymes differ in their capacity to interact with non-ionic detergents. The preparations of synaptosomal plasma membranes contained significantly higher proportions of detergent-insoluble choline acetyltransferase activity than did the whole synaptosomes; the difference was more striking for the Torpedo than for the rat enzyme. This detergent-insoluble activity was not due to aggregates of the enzyme. Some properties of the hydrophilic and amphiphilic choline acetyltransferase of Torpedo were analyzed. The two forms of the enzyme did not exhibit different affinities for their substrates; they were found to differ with respect to their sensitivity to inhibition by increasing concentrations of the two products of the reaction, acetylcholine and coenzyme A and heat inactivation at 45 degrees C. Most probably the hydrophilic and amphiphilic activities correspond to what was referred to as soluble and non-ionically membrane-bound choline acetyltransferase, respectively. The amphiphilic form may be an integral enzyme of the plasma membrane of cholinergic nerve endings or may be tightly bound to a specific protein in this membrane which may act as a "receptor" for choline acetyltransferase.

Modulation of choline acetyltransferase synthesis by okadaic acid, a phosphatase inhibitor, and KN-62, a CaM kinase inhibitor, in NS-20Y neuroblastoma.

Choline-O-acetyltransferase (ChAT) is the enzyme which catalyses the biosynthesis of the neurotransmitter acetylcholine in cholinergic neurons. Here we show that in mouse cholinergic NS-20Y neuroblastoma cells cultured in the presence of either okadaic acid (serine/threonine phosphatases 1 and 2A inhibitor) or KN-62 (CaM kinase inhibitor) ChAT activity and mRNA either increased or decreased as a function of the drug concentration, respectively. After 24 h exposure, okadaic acid exerted a dramatic effect on cell morphology; cells became round and had no more neurites. On the contrary, KN-62 induced a slight morphological differentiation of the cells. The present results suggest that phosphatases 1 and 2A and CaM kinase could mediate regulation of ChAT gene expression.

Specific antibodies to bovine choline acetyltransferase raised in mice immunised with small amounts of partially purified enzyme.

Choline acetyltransferase was purified approximately 18,000-fold from 300 g of bovine caudate nuclei to a specific activity of 21 ?mol min mg protein. The overall procedure used was: extraction of the enzyme by high salt concentration, chromatography on carboxy-methyl-Sephadex, precipitation by ammonium sulphate, affinity chromatography on Blue-Sepharose and, finally absorption on hydroxylapatite. When the enzyme absorbed on hydroxylapatite was injected into mice, it provoked reproducibly a transient production of 'inhibitory' antibodies, followed by higher antibody titres mainly of 'non-inhibitory' type. These responses were elicited by injecting less than a total of 20 ?g of immunogen. The highest antibody titre was obtained less than 2 months following the initial immunisation. Species cross reactivity was investigated. This procedure should prove to be of value in the production of monoclonal antibodies to choline acetyltransferase.

Direct inhibition of choline acetyltransferase activity by a monoclonal antibody raised against the plasma membrane of cholinergic nerve terminals.

A monoclonal antibody raised against cholinergic synaptosomal plasma membranes isolated from Torpedo electric organ, inhibited completely amphiphilic and hydrophilic choline acetyltransferase (ChAT) activities extracted and separated by using Triton X-114 phase partition of synaptosomes. We tested whether ChAT inhibition was direct or not. We found that the antibody was inhibiting ChAT in preparations of very low purity as well as ChAT that was immunoprecipitated by using a non-inhibitory anti-ChAT polyclonal antibody. Also, inclusion of acetylcoenzyme A at 20 times its Km during incubation of ChAT and antibody, completely prevented ChAT inhibition. This same concentration of the ChAT substrate could significantly but not completely dissociate the complex enzyme-antibody. These results spoke in favour of a direct inhibition of ChAT; the antibody most probably binds to an epitope that may be located at or near the acetylcoenzyme A binding site. The inhibitory effect on hydrophilic and amphiphilic ChAT was dependent on the antibody concentration, but amphiphilic activity required higher concentrations to be affected to the same extent as hydrophilic activity. This was found not only with Torpedo, but also with rat and human ChAT activities. Thus, the antibody appears to be able to distinguish the two forms of ChAT activity.

Membrane-bound choline acetyltransferase of the torpedo has characteristics of an integral membrane protein and can be solubilized by proteolysis.

Due to Triton X-114 fractionation of synaptosomes isolated from the electric organ of the fish Torpedo, the existence of a hydrophilic and an amphiphilic form of the enzyme choline-O-acetyltransferase (ChAT) was revealed. Amphiphilic ChAT which represents about 10% of total enzyme activity in synaptosomes, reached 40% of ChAT activity measured in preparations of synaptosomal plasma membranes (SPM) which were washed with solutions of increasing ionic strength. ChAT activity bound to washed SPM could be partially solubilized using proteinase K but not phospholipase C. No ChAT solubilization occurred by treating intact synaptosomes with proteinase K. Water/Triton X-114 partition coefficients of hydrophilic and amphiphilic ChAT were found to be 6.5 and 0.17, respectively. Sedimentation coefficients determined by centrifugation in linear density gradients of sucrose containing Triton X-100, were 4.2S and 4.4S for amphiphilic and hydrophilic ChAT, respectively. On the other hand, removal of Triton X-114 from the detergent phase containing amphiphilic ChAT activity led to enzyme aggregation. Finally, amphiphilic ChAT was slightly more acidic (pH 6.6) than was hydrophilic enzyme (6.8-7.0). We conclude that in Torpedo synaptosomes two forms of ChAT activity, a soluble and a membrane-bound form, are indeed present which differ in their hydrophobicity. The soluble form is hydrophilic. The membrane-bound form is amphiphilic and it aggregates upon removal of detergent. These are two characteristics of integral membrane proteins. Membrane-bound ChAT is most probably intracellularly oriented and not bound to membrane through a 'receptor' protein.

The proportion of amphiphilic choline acetyltransferase in Drosophila melanogaster is higher than in rat or Torpedo and is developmentally regulated.

We show that in the central nervous system of the fly, Drosophila melanogaster, choline acetyltransferase (ChAT) activity exists under two molecular forms, a soluble, hydrophilic form and a membrane-bound, amphiphilic form. This is based on the following demonstrations of differential solubilization and interaction with non-denaturing detergents: sequential extraction of Drosophila heads produced low-salt-soluble (83-87%) and detergent-soluble (6-7%) ChAT activity. Sedimentation in sucrose gradients of detergent-soluble ChAT was found to be influenced by the type of detergent present in the gradient (Triton X-100 and Brij 96). This was not the case for low-salt-soluble ChAT. To further confirm these findings, we subjected Drosophila heads to Triton X-114 fractionation. This method, which yielded 12% of amphiphilic ChAT activity, separates hydrophilic from amphiphilic proteins. Compared to central nervous tissue of rat and Torpedo electric lobes, Drosophila head contained the highest proportion of amphiphilic ChAT activity. Synaptosomes isolated from Torpedo electric organ exhibited higher levels of amphiphilic ChAT than did electric lobes. Of the three animal species analyzed here, the Torpedo amphiphilic enzyme was the most hydrophobic and the rat enzyme the least hydrophobic. The proportion of amphiphilic ChAT was analyzed during Drosophila development. The percentage of this activity increased about 7 times from embryo to larva and then remained constant until the adult fly age.

Hydrophilic and amphiphilic forms of Drosophila choline acetyltransferase are encoded by a single mRNA.

We have previously shown that the enzyme choline-O-acetyltransferase (ChAT) exists in a hydrophilic and an amphiphilic form in Drosophila head. A complementary DNA clone of 4.2 kb containing the entire coding region of ChAT was isolated from a cDNA library of Drosophila heads. The cDNA was subcloned in an expression vector and injected into the nucleus of Xenopus oocytes. Injected oocytes expressed high levels of ChAT activity. This activity was inhibited by bromoacetylcholine, a specific inhibitor of the enzyme. In the present study the non-ionic detergent Triton X-114 was used to analyse whether the expression of hydrophilic and amphiphilic ChAT was or was not directed by a single cDNA. The two forms of ChAT were found to be synthesized in injected oocytes. Approximately 9% of the recombinant enzyme partitioned as amphiphilic activity. This value was similar to that found for native amphiphilic ChAT in Drosophila heads. Sedimentation in sucrose gradients of amphiphilic enzyme was found to be influenced by the type of detergent present in the gradient whereas this was not the case for hydrophilic ChAT. Hydrophilic and amphiphilic enzyme activities differed in some of their biochemical properties. Amphiphilic ChAT was less sensitive to inhibition by the product acetylcholine than was hydrophilic ChAT. Moreover, amphiphilic ChAT was found to be more resistant than hydrophilic ChAT to heat inactivation at 45 degrees C. These properties were observed for the native as well as for recombinant ChAT. These results demonstrate that the hydrophilic and amphiphilic forms of ChAT are derived from one mRNA.

A monoclonal antibody raised against plasma membrane of cholinergic nerve terminals of the Torpedo inhibits choline acetyltransferase activity and acetylcholine release.

Monoclonal antibodies were raised against the synaptosomal plasma membranes (SPMs) purified from the electric organ of the Torpedo. One antibody that reacts preferentially with SPMs rather than with other membrane fractions isolated from this tissue was previously found to inhibit hydrophilic and amphiphilic choline-O-acetyltransferase (ChAT) activity. On immunoblots of SPMs, this antibody recognizes two polypeptides of 135 and 66 kilodaltons that are related; the 66-kilodalton polypeptide appears to exist as a monomer and as a dimer in SPMs. The antibody was also able to inhibit the calcium-dependent release of acetylcholine in Torpedo synaptosomes without affecting the total neurotransmitter content. This inhibition was dependent on the antibody concentration and was observed when the release was elicited by either KCl depolarization or the calcium ionophore A23187; this suggests that inhibition was not mediated by a blockage of the depolarization-activated calcium influx. The inhibition could not be prevented by atropine, a result indicating that the antibody does not block release by mimicking the action of acetylcholine on presynaptic muscarinic autoreceptors. Thus, the antigen recognized by this antibody appeared to be involved in acetylcholine release; this antigen could be membrane-bound ChAT, another protein of the SPMs, or both.

Pulsatile release of acetylcholine by nerve terminals (synaptosomes) isolated from Torpedo electric organ.

1. Electrophysiological detection of acetylcholine (ACh) release by synaptosomes from the electric organ of Torpedo was searched for by laying the isolated nerve terminals on a culture of Xenopus embryonic muscle cells (myocytes), and by recording the ACh-induced inward currents in the myocytes. 2. Whole-cell recording in one of the myocytes revealed rapid inward currents that where generated soon after synaptosome application. These pulsatile events strongly resembled those occurring normally during the early phase of synaptogenesis after nerve-muscle contact in Xenopus cell cultures. They were called spontaneous synaptic currents (SSCs). 3. The SSCs produced by the synaptosomes had a rapid time course, with mean time-to-peak and half-decay times of 2.6 +/- 0.4 ms and 6.0 +/- 1.1 ms, respectively. Most events had a falling phase that could be fitted with a single exponential. The mean time constant of decay was 6.2 +/- 1.1 ms. More than half of the SSCs (approximately 60%) constituted a rather homogenous population in which the time-to-peak versus amplitude showed a positive relationship, the smallest events displaying a shorter time course. The rest of the SSCs had a more variable and slower time course. Such events are also observed in young and mature junctions in situ. 4. The amplitudes of SSCs had a wide distribution which was skewed towards the smallest values. The mean amplitude was 65.2 +/- 16.1 pA. 5. During the minutes following an application of synaptosomes, the frequency of the SSCs tended to decrease, but their mean amplitude remained constant. Such behaviour could be reproduced during several successive additions of synaptosomes while recording in the same myocyte. 6. Just after synaptosome application, the SSCs were superposed to a noisy inward current that lasted for 20-60 s. Noise analysis of this current gave the values of 0.7 +/- 0.1 pA for the mean amplitude of the elementary event, and 4.7 +/- 0.2 ms for its mean duration, values that compare well with those reported for the activation of frog embryonic nicotinic receptor. This suggests that the noisy current was due to ACh molecules set free by synaptosomes which were either damaged or which released ACh at some distance. This view was strengthened by biochemical analysis of ACh release by synaptosomes in vitro. 7. Tubocurarine reversibly abolished the appearance of both the noise and the synaptosome-generated SSCs, showing that these currents were due to the action of ACh.(ABSTRACT TRUNCATED AT 400 WORDS)

Release of acetylcholine by Xenopus oocytes injected with mRNAs from cholinergic neurons.

Xenopus laevis oocytes were injected with poly(A)+ mRNAs extracted from the electric lobes of Torpedo marmorata. The electric lobes contain the perikarya of approximately 120,000 cholinergic neurons that innervate the electric organs and are homologous to motor neurons. The injected oocytes accumulated acetylcholine and were able to synthesize [14C]acetylcholine from 1-[14C]acetate. With KCl depolarization and upon treatment with a Ca2+ ionophore, they released their endogenous as well as the radiolabelled neurotransmitter in a Ca(2+)-dependent manner. No synthesis or release were obtained from control oocytes. With respect to their dependency upon Ca2+ concentration, the oocytes injected with Torpedo electric lobe mRNAs released acetylcholine in a manner which closely resembled that found in the native synapses. In contrast to the controls, primed oocytes were also able to release [14C]acetylcholine that was injected a few hours prior to the release trial. Immunoblot analysis demonstrated that the 15 kd proteolipid antigen of the purified mediatophore, a 200 kd presynaptic protein able to translocate acetylcholine, was expressed in the ACh-releasing oocytes but not in the controls. The present observation may provide a useful approach for investigating the proteins involved in the release of acetylcholine and of other neurotransmitter substances.

Study of subcellular localization of membrane-bound choline acetyltransferase in Drosophila central nervous system and its association with membranes.

Choline acetyltransferase (ChAT), the enzyme which catalyses the biosynthesis of the neurotransmitter acetylcholine, exists in a soluble and membrane-bound form in cholinergic nerve terminals of different animal species. This study was performed on the enzyme present in Drosophila central nervous system. We show that the two forms of the enzyme have the same apparent molecular weight (75 kDa) when analysed by immunoblotting using an antibody we raised against the recombinant enzyme. According to different authors, membrane-bound enzyme might be associated with synaptic vesicles or plasma membrane. Subfractionation of Drosophila head homogenates in linear glycerol gradients showed that ChAT does not associate with synaptic vesicles. Analysis of ChAT activity and immunoreactivity showed that two peaks of ChAT were produced. One peak was present in fractions containing soluble components and the other was associated with rapidly sedimenting membranes containing plasma membranes. ChAT in the first peak was mainly hydrophilic. A large proportion of ChAT associated with rapidly sedimenting membranes was amphiphilic. Further fractionation of these membranes by flotation in sucrose gradients showed that membrane-associated ChAT sedimented in fractions containing plasma membrane marker. Membrane-bound ChAT was neither solubilized nor converted to hydrophilic enzyme after membrane treatment with 1 M hydroxylamine, suggesting that the enzyme is not palmitoylated and therefore not anchored to membrane through thioester-linked long chain fatty acid. Partial solubilization of ChAT present on membranes with urea and carbonate suggests that this form of ChAT is a peripheral membrane protein. Carbonate solubilization of membrane-bound ChAT converted the enzyme from hydrophobic to hydrophilic protein.

Evoked acetylcholine release expressed in neuroblastoma cells by transfection of mediatophore cDNA.

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 Ca(2+)-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.

Cell lines expressing an acetylcholine release mechanism; correction of a release-deficient cell by mediatophore transfection.

Several neuronal and non-neuronal cell lines express a Ca(2+)-dependent mechanism of transmitter release that can be demonstrated after loading the cells with acetylcholine during culture. In contrast, a particular cell line, the neuroblastoma N18TG-2, was found to be deficient for release. We transfected N18TG-2 cells with a plasmid encoding Torpedo mediatophore, a protein able to translocate acetylcholine in response to calcium. The N18TG-2 cells expressed the Torpedo protein which reached their plasma membrane. At the same time, these cells acquired a Ca(2+)-dependent quantal release mechanism similar to the one naturally expressed by other cell lines. Hence, the presence of mediatophore in the plasma membrane seems essential for quantal release.

Quantal acetylcholine release induced by mediatophore transfection.

Mediatophore is a protein of approximately 200 kDa able to translocate acetylcholine in response to calcium. It was purified from the presynaptic plasma membranes of the electric organ nerve terminals. Mediatophore is a homooligomer of a 16-kDa subunit, homologous to the proteolipid of V-ATPase. Cells of the N18TG-2 neuronal line are not able to produce quantal acetylcholine release. We show here that transfection of N18TG-2 cells with a plasmid encoding the mediatophore subunit restored calcium-dependent release. The essential feature of such a release was its quantal nature, similar to what is observed in situ in cholinergic synapses from which mediatophore was purified.