Choline acetyltransferase in mammalian synaptosomes: Evidence for an integral membrane-bound form

Choline acetyltransferase activity was detected in extensively washed membranes prepared from rat and guinea pig synaptosomes. When these preparations were treated with the non-ionic detergent Triton X-114 and heated to 37°C to cause phase separation, a significant percentage was found to associate with the detergent-rich phase, indicating that the enzyme might be an integral membrane-bound protein. In guinea pigs receiving septal lesions, a large reduction in both total and in Triton X-114-extractable choline acetyltransferase in hippocampal synaptosomes was observed indicating that the detergent-extracted form is associated with cholinergic nerve terminals. When membrane-bound choline acetyltransferase from lysed, washed synaptosomes was incubated in Triton X-114, 30% of the membrane-associated enzyme could be extracted into the detergent-rich phase. This extraction could be improved by reducing the chloride content of the extraction medium. When the chloride content of synaptosomes, prepared from rat cerebral cortex, was manipulated, by either exposure to γ-aminobutyric acid, muscimol or to a medium containing reduced levels of chloride, the ability of antibodies against choline acetyltransferase to specifically immunolyse (in the presence of complement) the cholinergic synaptosome population was enhanced. These results suggest that the choline acetyltransferase found in the nerve terminal region exists in at least two forms (a soluble and a lipophilic form) which are partially interconvertible. The conversion between the two forms can be influenced by chloride ions.     

SURFACE CHARGE OF CHOLINE ACETYLTRANSFERASE FROM DIFFERENT SPECIES

—The adsorption of partially purified choline acetyltransferase (ChAc) from cat, rat, guinea-pig and pigeon brains by the cation exchange resins, CM-Sephadex (C-50) and Amberlite CG-50 II, was studied at various pH values and ionic strengths. ChAc from cat and rat were more strongly adsorbed by cation exchangers and therefore have a stronger net positive surface charge than those from guinea pig and pigeon. Experiments showed that the difference in adsorption between these two groups of enzymes could not be explained by overloading of the resin, by competitive effect of other proteins present in the enzyme preparations or by the presence of any component suppressing the adsorption of ChAc in any of the enzyme preparations. The adsorption of ChAc by a cation exchanger is very similar to its binding to synaptosome membranes. The significance of the positive surface charge of ChAc in studies on the compartmentation of ChAc in synaptosomes is discussed.     

Synaptosomal "Membrane-Bound" Choline Acetyltransferase Is Most Sensitive to Inhibition by Choline Mustard

The objectives of the present study were to validate the presence of cytoplasmic and membrane-associated pools of choline acetyltransferase (ChAT) in rat brain synaptosomes, and to evaluate inhibition of these different forms of the enzyme by the nitrogen mustard analogue of choline, choline mustard aziridinium ion (ChM Az). The relative distribution of ChAT and lactate dehydrogenase (LDH) was followed in subfractions of synaptosomes to establish whether ChAT activity associated with salt-washed presynaptic membranes represents membrane-bound protein rather than cytosolic enzyme trapped within undisrupted synaptosomes or revesiculated membrane fragments. The percentage of total synaptosomal ChAT activity (14%) recovered in the final membrane pellet always exceeded that of LDH (6%), lending support to the hypothesis that much of the ChAT associated with the membranes was a membrane bound form of the enzyme. Incubation of purified synaptosomes with ChM Az led to irreversible inhibition of ChAT activity; this loss of enzyme activity could not be accounted for by lysis of nerve terminals during incubation in the presence of the mustard analogue. Subfractionation of the ChM Az-treated nerve terminals revealed that the membrane-bound form of ChAT was inhibited to the greatest extent, followed by the ionically membrane-associated enzyme, with the activity of the water-solubilized enzyme not differing significantly from control. Preparation of the synaptosomal ChAT subfractions from untreated nerve terminals prior to incubation with varying concentrations of ChM Az or naphthylvinylpyridine revealed that under these conditions water-solubilized, ionically membrane-associated, and detergent-solubilized membrane-bound pools of ChAT were not differentially inhibited by either compound.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cytosolic Choline Acetyltransferase Binds Specifically to Cholinergic Plasma Membrane of Rat Brain Synaptosomes to Generate Membrane-Bound Enzyme

Uncovering the way membrane-bound choline acetyltransferase (ChAT) interacts with membranes and with which membrane in cholinergic neurons may help in understanding its role in acetylcholine metabolism. Subfractionation of rat hippocampal synaptosomes aiming to separate synaptic vesicles from plasma membranes shows that membrane-bound ChAT is bound to plasma membrane. Either detergents or urea and alkali can solubilize membrane-bound enzyme. Detergent-solubilized enzyme has a higher sedimentation rate than urea-alkali solubilized or cytosolic ChAT. Once dissociated, membrane-bound ChAT reassociates specifically with cholinergic plasma membranes, a process that was abolished by previous treatment of membranes with trypsin. Cytosolic ChAT behaves similarly. Thus, in cholinergic synaptosomes, ChAT exists as cytosolic and peripheral activity. Cytosolic ChAT generates peripheral enzyme most probably by interacting with a protein of plasma membrane of cholinergic nerve terminals. This receptor protein might regulate the amount of membrane-bound ChAT in cholinergic neurons.     

Acetylcholine in the brain of rats exposed to acute irradiation

A two-fold increase in acetylcholine, that can randomly be released by brain synaptosomes, is registered 60 min following whole-body X-irradiation of rats with a dose of 0.21 C/kg; depolarization of the synaptosome membranes by potassium chloride increases the release of acetylcholine the augmentation of the release in this case being lower than that in the control. The initial rate of spontaneous neuromediator release from synaptosomes grows by 80 per cent whereas after depolarization of synaptosome membranes by potassium chloride, by 15 per cent. There is a 2.5-fold increase in the maximum rate of a highly specific uptake of choline with Km value being constant. Acetylcholine content of gray substance of irradiated rat brain is invariable.     

Quantitation of Cholinergic Synaptosomes from Guinea Pig Brain

An antiserum raised to nerve terminal sacs derived from the electric organ and Torpedo marmorata was used to lyse guinea pig brain synaptosomes in the presence of complement. From the release of the cytoplasmic enzymes choline acetyltransferase, lactate dehydrogenase, tyrosine hydroxylase and glutamate decarboxylase it appears that the antiserum binds specifically to cholinergic terminals. The amount of lactate dehydrogenase released was used to estimate the proportion of cholinergic nerve terminals in different synaptosome preparations.     

Functional characterization of phosphorylation of 69-kDa human choline acetyltransferase at serine 440 by protein kinase C

Choline acetyltransferase, the enzyme that synthesizes the transmitter acetylcholine in cholinergic neurons, is a substrate for protein kinase C. In the present study, we used mass spectrometry to identify serine 440 in recombinant human 69-kDa choline acetyltransferase as a protein kinase C phosphorylation site, and site-directed mutagenesis to determine that phosphorylation of this residue is involved in regulation of the enzyme's catalytic activity and binding to subcellular membranes. Incubation of HEK293 cells stably expressing wild-type 69-kDa choline acetyltransferase with the protein kinase C activator phorbol 12-myristate 13-acetate showed time- and dose-related increases in specific activity of the enzyme; in control and phorbol ester-treated cells, the enzyme was distributed predominantly in cytoplasm (about 88%) with the remainder (about 12%) bound to cellular membranes. Mutation of serine 440 to alanine resulted in localization of the enzyme entirely in cytoplasm, and this was unchanged by phorbol ester treatment. Furthermore, activation of mutant enzyme in phorbol ester-treated HEK293 cells was about 50% that observed for wild-type enzyme. Incubation of immunoaffinity purified wild-type and mutant choline acetyltransferase with protein kinase C under phosphorylating conditions led to incorporation of [(32)P]phosphate, with radiolabeling of mutant enzyme being about one-half that of wild-type, indicating that another residue is phosphorylated by protein kinase C. Acetylcholine synthesis in HEK293 cells expressing wild-type choline acetyltransferase, but not mutant enzyme, was increased by about 17% by phorbol ester treatment.     

Induction of choline acetyltransferase in the neuroblastoma x glioma cell line NG108-15

The mechanism of the induction of choline acetyltransferase activity in the hybrid cell line NG108-15 was studied. Induction by cyclic AMP analogs, forskolin, and prostaglandin E₁ + theophylline was found to be rapid with an increase in choline acetyltransferase specific activity detectable within 8 hrs and maximal after 24 hrs. Immunoblot analysis was used to demonstrate that the increase in choline acetyltransferase specific activity induced by prostaglandin E₁ + theophylline was due to an increase in enzyme protein. Cycloheximide effectively blocked the induction of choline acetyltransferase by prostaglandin E₁ + theophylline. These results demonstrate that the induction of choline acetyltransferase activity involves the synthesis of new enzyme protein. Attempts to measure choline acetyltransferase turnover by blocking its synthesis with cycloheximide indicated that this enzyme is a relatively stable protein with a half-life of greater than 24 hrs.     

Homocholine and Short-Chain N-Alkyl Choline Analogues as Substrates for Torpedo Choline Acetyltransferase

Abstract: The kinetic parameters, K_m and V_max, for the acetylation of choline and several close analogues were determined by using (a) purified choline acetyltransferase and (b) a hypotonically lysed synaptosomal extract prepared from the electric organ of Torpedo marmorata. Whereas the K_m for choline was similar in both cases (0.51 and 0.42 m m ), the crude enzyme showed a three- to fivefold greater affinity for its analogues than the purified enzyme, the activity decreasing rapidly with increased N -alkyl substitution. Homocholine was a poor substrate, but was clearly acetylated by both preparations. The effect of salt on analogue acetylation by the crude enzyme was studied by increasing NaCl concentration from zero to 150 m m . There was an increase in both K_m and V_max for all substrates; choline, N,N,N -dimethylmonoethylaminoethanol, -monomethyldiethylaminoethanol and -dimethylmonobutylaminoethanol showed the greatest changes, whilst N,N,N -triethylaminoethanol and -dimethylmonopropylaminoethanol and homocholine were much less affected. However, in all cases, the kinetic parameter V_max / K_m remained unchanged. The maximal velocities of the different substrates varied more under conditions of high than of low salt. Sodium chloride up to 300 m m had no effect on the amount of enzyme which was bound to membranes in the synaptosomal extract. It is concluded that choline acetyltransferase has a high degree of substrate specificity, which is slightly altered by purification. The effects of salt cannot be explained as a consequence of nonspecific ionic association with membranes.     

EFFECTS OF BOTULINUM AND TETANUS TOXINS ON CHOLINE ACETYLTRANSFERASE ACTIVITY IN SKELETAL MUSCLE IN THE MOUSE

Abstract— The effects of botulinum and tetanus toxins on the activity of choline acetyltransferase present in the motor nerve terminals of fast and slow skeletal muscle in the mouse were investigated. There was no change in the activities of choline acetyltransferase in either muscle after the injection of botulinum toxin but tetanus toxin caused a rise in the activity of the enzyme in fast muscle. Botulinum toxin is known to inhibit the release of acetylcholine and whilst neuromuscular transmission is blocked the motor nerves sprout and form new end-plates. Tetanus toxin has been shown to cause hyperactivity of motor neurons. The nerve growth caused by the botulinum toxin did not result in increased choline acetyltransferase levels in the muscles, whereas the synaptic hyperactivity caused by tetanus was associated with increased enzyme levels.     

DISTRIBUTION OF ACETYLCHOLINE, CHOLINE, CHOLINE ACETYLTRANSFERASE AND ACETYLCHOLINESTERASE IN REGIONS AND SINGLE IDENTIFIED AXONS OF THE LOBSTER NERVOUS SYSTEM

Abstract— Acetylcholine, its precursor (choline), and the enzymes of its biosynthesis and degradation (choline acetyltransferase and acetylcholinesterase, respectively) have been studied and quantified in extracts of several regions of the nervous system of the lobster and in single, isolated axons of identified efferent excitatory, efferent inhibitory and afferent sensory neurons. The choline acetyltransferase is a soluble enzyme similar to that from other species. The predominant acetylcholine-hydrolysing enzyme is largely membrane-bound and has been characterized as a specific acetylcholinesterase. A single peak of acetylcholinesterase activity can be detected upon velocity sedimentation analysis of Triton X-100-treated extracts of all regions of the nervous system. Choline acetyltransferase distribution parallels that of sensory neural elements, and its specific activity shows nearly a 500-fold difference from the richest to the poorest neural source. Acetylcholinesterase levels span only a 23-fold range, and activity is found in all neural regions, including those free of known sensory components. A radiochemical microassay for choline and acetylcholine in the range of 20–2000 pmol is described in detail. All 3 types of axons contain comparable levels of choline ( ca. 2 pmol/μg protein), but acetylcholine is asymmetrically distributed. Efferent axons contain no detectable acetylcholine, while sensory axons from abdominal muscle receptor organs have an average of 1·9 pmol/μg protein. Choline acetyltransferase is similarly distributed; sensory axons show at least 500-fold greater activity than efferent axons. Acetylcholinesterase is nearly uniformly distributed among the three types of fibres. These results are discussed in terms of a general view of transmitter accumulation in single neurons.     

Effects of Choline and Quiescence on Drosophila Choline Acetyltransferase Expression and Acetylcholine Production by Transduced Rat Fibroblasts

Rat-1 fibroblasts were transduced to express Drosophila choline acetyltransferase. The presence of an active enzyme in these cells (Rat-1/dChAT) was confirmed using various methods. Rat-1/dChAT fibroblasts released acetylcholine (ACh) into the culture medium. Moreover, intra- and extracellular levels of ACh could be increased by adding exogenous choline chloride. In addition, serum starvation or confluence-induced quiescence caused an 80% decrease in recombinant choline acetyltransferase activity (compared with actively growing cells). ACh release was also repressed in quiescent fibroblast cultures. Exogenous choline could mitigate the decrease in ACh release. These results indicate that Rat-1 fibroblasts can be genetically modified to produce ACh and that ACh release can be controlled by introducing choline into the culture medium. Furthermore, these data demonstrate that the expression of the retroviral promoter used in this study decreases with the onset of quiescence; however, exogenous choline can increase the amount of ACh released by quiescent fibroblasts.     

Isolation of Hippocampal Synaptosomes on Percoll Gradients: Cholinergic Markers and Ligand Binding Sites

The S1 Percoll procedure, devised empirically for cortical tissue, provides highly purified, functionally viable synaptosomes on a four-step Percoll gradient. Here, for the first time, the procedure has been applied to rat hippocampus, and the gradient fractions have been analysed with respect to cholinergic markers and the synaptosomal index, lactate dehydrogenase. The presynaptic cholinergic markers choline acetyltransferase and [3H]choline uptake were most enriched in fraction 4. In contrast, acetylcholinesterase activity was broadly distributed across the gradient, consistent with the separation of synaptic plasma membranes (in fractions 1 and 2) from synaptosomes (in fractions 3 and 4). This is supported by the recovery of muscarinic binding sites labelled with [3H]quinuclidinylbenzilate in fractions 1 and 2. (-)-[3H]-Nicotine binding sites, however, were most enriched in fraction 4, consistent with their predominantly presynaptic localisation in the CNS. These results demonstrate the applicability of the S1 Percoll method to discrete brain regions for the recovery of homogeneous and viable synaptosome fractions. The separation of presynaptic terminals from post-synaptic membranes is a further advantage of this technique.     

Multiple forms of choline acetyltransferase in several species demonstrated by isoelectric focusing

1. The behaviour of choline acetyltransferase from pigeon, guinea-pig, rat and cat brain on isoelectric focusing was studied. 2. Choline acetyltransferase from pigeon and guinea-pig brain showed single peaks with isoelectric points at pH6.6 and 6.8 respectively. Only one molecular form of the enzyme was therefore detected in these species. 3. Three peaks of choline acetyltransferase activities with isoelectric points 7.3-7.6, 7.7-7.9 and 8.3 were obtained with enzyme preparations from rat brain. 4. The separate identities of each of the three forms were confirmed by refocusing. 5. Choline acetyltransferase activity from a high-speed supernatant of rat brain homogenate was distributed similarly to a partially purified enzyme preparation from rat brain in the isoelectric gradient. 6. The enzyme activities from cat brain were separated into two distinct peaks with isoelectric points 7.0 and 8.4, and a possible third peak with isoelectric point 7.6. 7. The two main peaks showed considerable differences in stability on storage, and their identities were confirmed by refocusing. 8. The distribution of the enzyme activities was unaltered by isoelectric focusing in the presence of 3m-urea. 9. The apparent K(m) for choline of choline acetyltransferase from rat, cat and guinea-pig brain was 0.8mm, whereas for the pigeon enzyme it was 0.4mm.     

Phosphorylation of Rat Brain Choline Acetyltransferase and Its Relationship to Enzyme Activity

Abstract— Choline acetyltransferase catalyzes the formation of acetylcholine from choline and acetyl-CoA in cholin-ergic neurons. The present study examined conditions for modulation of kinase-mediated phosphorylation of this enzyme. By using a monospecific polyclonal rabbit anti-human choline acetyltransferase antibody to immunoprecipi-tate cytosolic and membrane-associated subcellular pools of enzyme from rat hippocampal synaptosomes, we determined that only the cytosolic fraction of the enzyme (67,000 ± 730 daltons) was phosphorylated under basal, unstimulated conditions. The quantity of this endogenous phosphoprotein was dependent, in part, upon the level of intracellular calcium, with ³²P_i incorporation into the enzyme in nerve terminals incubated in nominally calcium-free medium only 43 ± 7% of control. The corresponding enzymatic activity of cytosolic choline acetyltransferase did not appear to be altered by lowered cytosolic calcium, whereas membrane-associated choline acetyltransferase activity was decreased to 58 ± 11 % of control. Depolarization of synaptosomes with 50 μ M veratridine neither altered the extent of phosphorylation or specific activity of cytosolic choline acetyltransferase, nor induced detectable phosphorylation of membrane-associated choline acetyltransferase, although the specific activity of the membrane-associated enzyme was increased to 132 ± 5% of control. In summary, phosphorylation of choline acetyltransferase does not appear to regulate cholinergic neurotransmission by a direct action on catalytic activity of the enzyme.     

Presynaptic Distribution of the Cholinergic-Specific Antigen Chol-1 and 5''-Nucleotidase in Rat Brain, as Determined by Complement-Mediated Release of Neurotransmitters

Nerve terminals prepared from rat cortex and hippocampus were loaded with seven radioactive putative neurotransmitters (serotonin, noradrenaline, dopamine, gamma-aminobutyric acid, aspartate, glutamate, and taurine). The release of these transmitters, choline acetyltransferase, 3,4-dihydroxyphenylalanine decarboxylase, enolase, and lactate dehydrogenase was monitored during complement-mediated lysis. Three antisera were used: anti-5'-nucleotidase, anti-Chol-1, and anti-rat cerebrum. Anti-5'-nucleotidase serum did not cause the release of any labelled transmitter or of any of the enzymes studied. Anti-Chol-1 serum released choline acetyltransferase and small amounts of enolase and lactate dehydrogenase. Anti-rat cerebrum caused the release of all seven transmitters, choline acetyltransferase, and small amounts of the other three enzymes. It was concluded that 5'-nucleotidase was not present on any of the terminals studied, and that Chol-1 is only present on cholinergic terminals.     

Changes in the calcium metabolism of cerebral cortical structures in anoxia in vitro

Changes in membrane-bound calcium (Ca2+(b)) content in the brain cortex membrane structures were studied on subcellular fractions (synaptosomes, microsomes, mitochondria) during in vitro anoxia. The changes in Ca2+ content in hydrophobic domains of intracellular membranes were assessed, using chlorotetracycline fluorescent probe. It has been found that membranes of different neuronal compartments are not equally vulnerable to anoxia. A decrease in Ca2+9(b) content in response to anoxia occurs in synaptosomes and microsomes much sooner than in mitochondria. Therefore, Ca2+ release from intracellular membrane compartments, preceding the massive inward flow of extracellular Ca2+, seems to be one of those mechanisms initiating a complex range of intracellular reactions to disturbed oxygen supply in brain cortex neurons.     

INHIBITION OF CHOLINE ACETYLTRANSFERASE AND ITS HISTOCHEMICAL LOCALIZATION

Abstract— A method for the histochemical identification of choline acetyltransferase has been investigated further by studying the effects of certain inhibitors of the enzyme both on rat brain homogenates and on the localization of the enzyme in tissue sections.
It was confirmed that acetyl-CoA hydrolase activity both in homogenates and in tissue sections is inhibited by preincubation in 1 mM-DFP. The effects of the choline acetyltransferase inhibitors chloro- and bromoacetylcholine on the appearance of histochemical staining were related to their activity in homogenates and tissue slices. Bromoketone was found to inhibit choline acetyltransferase in homogenates and, less efficiently, in tissue sections but it also inhibited the hydrolysis of acetyl-CoA by some other unknown enzyme which is inactivated by 1 mM-DFP.
The results obtained with the choline acetyltransferase inhibitors provide support for the specificity of the histochemical method.     

The action of purified lysophospholipases on microsomal membrane-bound lysophosphatidylcholine

Rat liver membranes were labelled by intraperitoneal injection of [Me-14C]choline chloride. Isolated microsomal membranes were briefly treated with pancreatic phospholipase A2 to produce different levels of membrane-bound lysophosphatidylcholine. The hydrolysis of this lysophosphatidylcholine by two purified lysophospholipases from beef liver was studied. The specific activity of enzyme I at saturating membrane concentrations appeared to increase linearly with the lysophosphatidylcholine level in the membranes until the lysoderivative represented 15% of the original phosphatidylcholine. In contrast, the specific activity of enzyme II was independent of the lysophosphatidylcholine level, at least in the range of 4.9-34.0% tested. These different kinetics are discussed in terms of the possible functions of both enzymes in liver.     

The `compartmentation'' of choline acetyltransferase within the synaptosome

1. Choline acetyltransferase may be isolated in either a bound or soluble form after hypo-osmotic treatment of a crude synaptosome fraction, depending on the conditions. 2. In the bound form, the enzyme appears to be associated with the larger membrane fragments rather than with synaptic vesicles. 3. The bound form is predominant at slightly acid pH values and low ionic strength, the soluble form under more physiological conditions of pH and ionic strength. 4. Sodium chloride, potassium chloride, magnesium chloride and calcium chloride at similar ionic strengths solubilize the enzyme. 5. Choline acetyltransferase was found to be soluble under these conditions after release from synaptosomes from rat and pigeon cerebra, guinea-pig cortex and rabbit cortex, caudate nuclei, diencephalon and midbrain. 6. Certain isoenzymes of lactate dehydrogenase behaved similarly.