Mutations Affecting Acetylcholine Levels in the Nematode Caenorhabditis elegans

Gene cha-1.unc-17 of the nematode Caenorhabditis elegans is a complex gene, consisting of at least two complementation groups. One part (cha-1 region) of the gene encodes the enzyme choline acetyltransferase (ChAT), but the function of the other part (unc-17 region) is still unclear. We measured the ChAT activity and ACh levels of the cha-1 and unc-17 complex gene mutants. We show here that alterations in ACh levels, rather than the ChAT activity, reflect abnormal phenotypes accompanying cha-1.unc-17 mutations, that is, the decreased ACh levels in cha-1 mutations and abnormal accumulation in unc-17 mutations. Our results suggest that the unc-17 region may encode functions necessary for storage and/or release of ACh at the presynaptic level.     

Monoclonal Antibodies to and Immunoaffinity Purification of Choline Acetyltransferase from Bovine Brain

Three hybridomas producing monoclonal antibodies to bovine brain choline acetyltransferase (ChAT) have been established by fusion of the spleen cells from a mouse immunized with purified enzyme with myeloma NS-1 cells. All three clones produced IgGl antibodies that reacted with enzyme protein denatured with sodium dodecyl sulfate. By using one of the monoclonal antibodies, a rapid and efficient immunoaffinity purification procedure of bovine ChAT has been established. Immunoblot analysis and immunoaffinity purification indicated that bovine ChAT is a single 68-kilodalton protein. The monoclonal antibodies will offer us a good tool to isolate the cDNA clones encoding ChAT.     

Immunoaffinity Purification of Human Choline Acetyltransferase: Comparison of the Brain and Placental Enzymes

A rapid and efficient immunoaffinity purification procedure has been developed for human placental choline acetyltransferase (ChAT). Using this procedure, human placental ChAT was purified to homogeneity with high recovery of enzyme activity (50-60%). Purified ChAT was used to raise a monospecific anti-human ChAT polyclonal antibody in rabbits. A comparison of the physical properties of ChAT was made between the enzymes purified from human brain and human placenta. Only one form of the enzyme exists in either tissue, having identical molecular weights of 68,000 and a single apparent pI of 8.1. A more detailed comparison of the two enzymes using peptide mapping and epitope mapping indicates identity between the brain and placental enzymes.     

Purification of Choline Acetyltransferase from the Locust Schistocerca gregaria and Production of Serum Antibodies to This Enzyme

Choline acetyltransferase (ChAT; EC 2.3.1.6) was purified from the heads of Schistocerca gregaria to a final specific activity of 1.61 mumol acetylcholine (ACh) formed min-1 mg-1 protein. The molecular mass of the enzyme as determined by gel filtration is 66,800 daltons. The final enzyme preparation showed one major band at 65,000 daltons on sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, which corresponds with the native molecular mass of the enzyme, a band at 56,000 daltons, and two bands at 40,500 and 38,000 daltons. Antibodies raised against ChAT in rabbit react only with the active band on native gel after Western blotting. They strongly react with the 65,000-dalton polypeptide band on Western blots of SDS gel separation of pure preparation of enzyme and with both the 65,000- and 56,000-dalton bands after SDS gel separation of crude extract.     

Studies on the Alkylation of Choline Acetyltransferase by Choline Mustard Aziridinium Ion

Although a potent irreversible inhibitor of high-affinity choline transport in rat brain synaptosomes, choline mustard aziridinium ion (ChM Az) appeared to be a relatively weak inhibitor of choline acetyltransferase (ChAT) in rat brain homogenates, and evidence for irreversible binding of this compound to the enzyme had not been established. Accordingly, the irreversible inactivation of partially purified rat brain ChAT by ChM Az was studied. This compound is a rather weak inhibitor of the enzyme, with 50% inhibition of ChAT activity achieved following 30 min incubation at 37 degrees C with 0.6 mM ChM Az. This result indicates that although ChM Az has affinity for many nucleophiles there was little diluting effect of the inhibitor in the crude brain homogenate which could be attributed to such reactions (50% inhibition caused by 1.8 mM ChM Az following 10 min incubation). Although the initial binding of ChM Az to ChAT may be of a competitive nature, irreversible bond formation resulted. The time-dependent alkylation reaction conformed to pseudo-first-order kinetics with an observed forward rate constant (kobs) of 0.173 min-1; the half-time (t 1/2) for irreversible binding was about 4 min. The irreversible inactivation of ChAT by ChM Az would appear to be slower than the alkylation of high-affinity choline carriers in synaptosomes by this compound, and the relatively weak inhibitory action of ChM Az against either partially purified ChAT or ChAT activity in crude rat brain homogenates is in striking contrast to previous evidence that ChAT in intact synaptosomes was inhibited irreversibly by lower concentrations of the inhibitor.     

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)     

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.     

Choline Acetyltransferase and Cholinesterases in the Developing Xenopus Retina

To understand the developmental regulation of acetylcholine (ACh) synthesis in the Xenopus retina, the properties of choline acetyltransferase (CAT) and cholinesterase (ChE), as well as histochemical localization of ChE in the retina, were studied during development. CAT activity first became detectable in the developing eyecup at stages 35/36. This was followed by a rapid, 50-fold rise in specific activity between stages 35/36 and 44. Since this rapid rise coincided with an almost identical increase in total ACh synthesis in whole retinae found in previous studies, it is suggested that this increase was sufficient to account for the rapid increase in total ACh synthesis. Moreover, it also correlated with increased rates of synaptogenesis in both the inner and the outer plexiform layers. Total ChE was resolved into specific and nonspecific ChE by the use of tetraisopropylpyrophosphoramide. Total ChE activities first became detectable at stages 35/36. Specific ChE [acetylcholinesterase (AChE)] increased from 50% at stage 39 to 95% of total ChE activities at stage 66. Again, the most rapid increase in both total ChE and AChE activities occurred between stages 35/36 and 44. Histochemical studies showed that AChE was localized predominantly in the two plexiform layers, with the inner plexiform layer more heavily stained at all stages. Moreover, a stratified staining pattern, clearly discerned in the inner plexiform layer, also correlated with synaptogenesis during this early period of retinal development.     

Functional Regulation of Choline Acetyltransferase by Phosphorylation

Choline acetyltransferase (ChAT) catalyzes synthesis of acetylcholine (ACh) in cholinergic neurons. ACh synthesis is regulated by availability of precursors choline and acetyl coenzyme A or by activity of ChAT; ChAT regulates ACh synthesis under some conditions. Posttranslational phosphorylation is a common mechanism for regulating the function of proteins. Analysis of the primary sequence of 69-kD human ChAT indicates that it has putative phosphorylation consensus sequences for multiple protein kinases. ChAT is phosphorylated on serine-440 and threonine-456 by protein kinase C and CaM kinase II, respectively. These phosphorylation events regulate activity of the enzyme, as well as its binding to plasma membrane and interaction with other cellular proteins. It is relevant to investigate differences in constitutive and inducible patterns of phosphorylation of ChAT under physiological conditions and in response to challenges that cholinergic neurons may be exposed to, and to determine how changes in phosphorylation relate to changes in neurochemical transmission.     

Sequence Analysis of a Proteolyzed Site in Drosophila Choline Acetyltransferase

The amino terminal sequence of the 13-kilodalton (kD) polypeptide present in purified Drosophila acetyl-CoA: choline-O-acetyltransferase (EC 2.3.1.6) was determined, and its position in the sequence of the intact enzyme was located. Enzyme polypeptides for sequencing were obtained from native enzyme protein by denaturation, followed by fractionation on reverse-phase HPLC. The 13-, 54-, and 67-kD polypeptides recovered from the separation were subjected to amino terminal sequencing. Only the 13-kD fragment yielded a sequence. The 67- and 54-kD polypeptides appeared completely blocked to gas-phase Edman sequencing. The location of the amino terminal sequence from the 13-kD polypeptide in the cDNA-deduced enzyme sequence indicated that this fragment represents the carboxyl portion of the 67-kD enzyme, with the 54-kD polypeptide providing the amino terminal portion. The proteolysis that gave rise to the 13-kD polypeptide occurred at the carboxyl side of a monobasic lysine residue. An earlier comparison of the enzyme from Drosophila and pigs indicated that the cleaved lysine may be a conserved residue in the porcine enzyme. The cleaved enzyme region characterized in this study does not coincide with the regions of high homology found in the two enzymes, but hydrophilicity profiles generated for this area showed similarities.     

Choline Acetyltransferase Activity in the Rat Trigeminal System

Choline acetyltransferase activity was investigated in the superior cervical ganglia and in six microdissected regions of the medulla oblongata of the rat ipsilateral and contralateral to electrolytic lesions of the trigeminal sensory ganglia (Gasserian). Electrolytic lesions of the Gasserian ganglia failed to modify levels of enzymatic activity in all structures studied. This result would be an argument against the existence of a major cholinergic population of sensory neurones in the trigeminal system.     

Conservation of Amino Acid Sequences Between Human and Porcine Choline Acetyltransferase

Abstract: The amino acid sequence of 11 peptides generated from human placental choline acetyltransferase was compared to the corresponding amino acid sequences predicted from the nucleotide sequence of a recently cloned porcine choline acetyltransferase cDNA. These peptides, which were generated by cyanogen bromide cleavage or tryptic digestion, accounted for 23% of the amino acids in the enzyme. Of the 145 amino acids sequenced eight differed between the two species, yielding an identity of 94% over the regions sampled.
Of the eight amino acids that differed six could represent single base changes in the DNA sequence. These findings demonstrate strong sequence similarity between porcine and human choline acetyltransferase and indicate that they are closely related evolutionarily.     

Activation of Choline Acetyltransferase by Vasoactive Intestinal Peptide

Addition of vasoactive intestinal peptide (VIP) to brain homogenates increased the activity of choline acetyltransferase (ChAT) but not that of acetylcholinesterase or glucose-6-phosphate dehydrogenase. Activity of ChAT was increased in the anterior hypothalamus and in the dorsal and ventral hippocampus, but not in the parietal cortex or posterior hypothalamus. Increased activity occurred rapidly after VIP addition to homogenates and was maximal at 10(-7)M concentration. Kinetic analysis indicates that the Vmax of the enzyme is increased and the Km for choline, but not acetyl-coenzyme A, is decreased in the presence of VIP. Results support a possible VIP-cholinergic interaction in the CNS.     

Choline Acetyltransferase Activities in Single Spinal Motor Neurons from Patients with Amyotrophic Lateral Sclerosis

Activities of choline acetyltransferase (ChAT) were microassayed in individual cell bodies of motor neurons, isolated from freeze-dried sections after autopsy of lumbar spinal cords from four patients with sporadic amyotrophic lateral sclerosis (ALS) and four control patients with nonneurological diseases. Numerous large neurons were found in the anterior horn at the early degeneration stage of ALS, but the cell bodies atrophied and decreased in number at the late advanced stage. The small, atrophied neurons were very fragile and were easily destroyed during the isolation procedure with a microknife. The average activity, expressed on a dry weight basis, of 58 ALS neurons was lower than that of 67 control neurons. The large, well-preserved neurons at the early nonadvanced stage had markedly lower ChAT activities than control neurons. The specific activity gradually increased with the progress of atrophy but did not return to the control level.     

Calcium-Independent Release of Acetylcholine from Stable Cell Lines Expressing Mouse Choline Acetyltransferase cDNA

Abstract: Stably transfected cells expressing mouse choline acetyltransferase (ChAT) cDNA were established, and the synthesis and release of acetylcholine (ACh) were examined. A cDNA clone coding for mouse ChAT was inserted into an expression vector (pEF321) containing a promoter for human elongation factor 1α to construct pEFmChAT. Neuronal (NG108-15, NS20Y, N1E115, and Neuro2A) and nonneuronal cell lines (L cells and NIH3T3) were transfected with pEFmChAT, and the cell lines that stably expressed high ChAT activity were selected. These cells expressed the 66-kDa ChAT protein and accumulated ACh mostly in the cytosol. The concentration of intracellular ACh in the cells increased upon raising the choline level in the medium. The cells continuously released ACh in a Ca²⁺-independent fashion. Neither high K⁺ nor calcium ionophore stimulated release of ACh from the cells.     

In Vivo Production and Release of Acetylcholine from Primary Fibroblasts Genetically Modified to Express Choline Acetyltransferase

Abstract: Primary rat fibroblasts genetically modified to express Drosophila choline acetyltransferase (dChAT) synthesize and release acetylcholine (ACh) in vitro. The ACh produced from the transduced fibroblasts was found to be enhanced by increasing amounts of choline chloride in the culture media. These dChAT-expressing cells were then implanted into the intact hippocampus of adult rats and in vivo microdialysis was performed 7–10 days after grafting to assess the ability of the cells to produce ACh and respond to exogenous choline in vivo. Samples collected from anesthetized rats revealed fourfold higher levels of ACh around dChAT grafts than from either non-grafted or control-grafted hippocampi. Localized choline infusion (200 μ) through the dialysis probes was found to induce a selective twofold increase in ACh release only from the dChAT-expressing fibroblasts. These results indicate not only that dChAT-expressing fibroblasts continue to synthesize and secrete ACh for at least 10 days after intracerebral grafting, but that the levels of ACh can be manipulated in vivo. The ability to regulate products within genetically modified cells in vivo may provide a powerful avenue for exploring the role of discrete substances within the CNS.     

High-Level Synthesis and Fate of Acetylcholine in Baculovirus-Infected Cells: Characterization and Purification of Recombinant Rat Choline Acetyltransferase

Rat choline acetyltransferase (ChAT) has been expressed at a high level in Spodoptera frugiperda Sf9 cells using a baculovirus expression system. A cDNA containing the coding sequence for ChAT was inserted into the transfer vector pAcYM1 to yield the recombinant vector pAcYM1/ChAT. Sf9 cells were then coinfected with pAcYM1/ChAT and the wild-type Autographa californica virus. One recombinant virus particle, containing the cDNA for ChAT, was selected that expressed a protein of 68.5 kDa. Forty hours after infection of cells with the recombinant virus, the specific activity of ChAT in the cytosol was 190 nmol of acetylcholine/min/mg of protein, accounting for approximately 24% of the cell cytosolic proteins as being ChAT. The apparent Km values of the enzyme for choline and acetyl-CoA were 299 and 221 microM, respectively, whereas the respective Vmax values were 10.6 and 11.4 mumol of acetylcholine/min/mg of protein. In addition, analysis of the protein revealed that ChAT is phosphorylated in Sf9 cells. About 0.5 mg of ChAT was obtained from a one-step purification procedure starting with 10(8) infected Sf9 cells. Addition of choline to the incubation medium led to accumulation of high amounts of acetylcholine in the cytosol of the infected cells. The neurotransmitter was not released by Sf9 cells in response to membrane depolarization or on ionophore-mediated calcium entry. Some acetylcholine, which most likely originated from cell death inherent to viral infection, accumulated in the culture medium. The infected insect cells, which synthesize and store neurotransmitter, provide a new and convenient model for analyzing synaptic transmission at the molecular level.