The choline acetyltransferase of human placenta

1. Various methods for the extraction of choline acetyltransferase (acetyl-CoA-choline O-acetyltransferase, EC 2.3.1.6) from immature human placenta (18-28 weeks of gestation) are described. 2. The crude enzyme was found to be stable at -18 degrees and +4 degrees under a variety of conditions. 3. Purification methods, including ammonium sulphate fractionation, gel filtration on various grades of Sephadex and DEAE-Sephadex fractionation, have yielded a preparation of high specific activity.     

Neurons with choline acetyltransferase immunoreactivity and mRNA are present in the human cerebral cortex

 We examined the cerebral cortex of five autopsied individuals without neurological and psychiatric diseases by immunohistochemistry using an anti-human recombinant choline acetyltransferase (ChAT) polyclonal antibody and in situ hybridization with ³⁵S-labeled human ChAT riboprobes. The immunohistochemistry detected positive neurons which were medium-sized or large pyramidal neurons located predominantly in layers III and V. The density of such neurons was higher in the motor and secondary sensory areas than in other cortical areas; the immunoreactive neurons in layer V were more densely distributed in the motor area and those in layer III were distributed in the secondary sensory areas. Positively stained, non-pyramidal neurons were observed in the superficial layer of the cingulate gyrus and parahippocampus. No immunoreactive neurons were found in the primary sensory areas. The in situ hybridization detected some neurons with signals for ChAT mRNA in the cerebral cortex, most of which were distributed in layer V of the motor area and in layer III of the secondary visual area. These results indicate that the human cerebral cortex contains cholinergic neurons and displays regional and laminal variations in their distribution. Accepted: 17 November 1998     

Evidence for the extreme overestimation of choline acetyltransferase in human sperm, human seminal plasma and rat heart: a case of mistaking carnitine acetyltransferase for choline acetyltransferase

Detection of choline acetyltransferase (ChAc) in a number of non-neuronal tissues has been extremely overestimated. There are two major types of errors encountered. Type 1 error occurs when endogenous substrates (e.g. L-carnitine) are acetylated by acetyltransferase enzymes (e.g. carnitine acetyltransferase ( CarAc ) ) yielding an acetylated product mistaken for acetylcholine (AcCh). In the past, human sperm and human seminal plasma putative ChAc activity has been extremely overestimated due to Type 1 error. This study demonstrates (1) an endogenous acetyltransferase and substrate activity in human sperm and human seminal plasma forming an acetylated product that is not AcCh but probably acetylcarnitine ( AcCar ); (2) that the addition of 5 mM choline substrate does not significantly increase acetyltransferase activity; (3) that boiled seminal plasma contains an endogenous acetyltransferase substrate which is not choline, but probably L-carnitine. Type 2 error occurs when endogenous carnitine acetyltransferase synthesizes true AcCh, resulting in mistaken evidence for ChAc. This is demonstrated by the fact that the choline substrate Km-value for the neuronal or true ChAc from mouse brain is 0.73 +/- 0.06 mM while the Km-value of choline substrate for purified CarAc from pigeon breast muscle is 108 +/- 4 mM. Type 2 error has occurred for the estimation of putative ChAc in rat heart. The rat heart ChAc was measured in previous studies utilizing a concentration of 30 mM choline substrate. While saturation of neuronal ChAc is observed at 2-5 mM choline, saturation of the rat heart CarAc enzyme is not reached until over 800 mM. Purified CarAc significantly synthesizes AcCh at 30 mM choline. Thus, putative ChAc has been greatly overestimated in the scientific literature for mammalian sperm, human seminal plasma and rat heart.     

Two methods for large-scale purification of recombinant human choline acetyltransferase

Choline acetyltransferase (ChAT) catalyzes the transfer of an acetyl group from acetyl-CoA to choline to produce the neurotransmitter acetylcholine (ACh). We have produced large quantities of pure human ChAT using two different bacterial expression systems. In the first, ChAT is fused to a chitin-binding domain via a self-cleavable linker allowing the release of ChAT without the use of proteases. In the second, ChAT is fused to a hexahistidine (His6) tag at the N-terminus with a linker incorporating a TEV protease cleavage site. In both cases, pure ChAT was produced that has a final specific activity of approximately 50 micromol ACh/min/mg and is suitable for structural characterization. Analysis of purified ChAT by Western blots and mass spectrometry revealed that the C-terminal 15 amino acids were slowly removed by endogenous proteolytic activity, to produce a stable 615 residue protein. Furthermore, we show that purified recombinant human ChAT is highly prone to oxidation, leading to the formation of covalent dimers and/or a loss of catalytic activity. Kinetic parameters of our purified proteins were obtained and, when compared to previously published constants for human placental ChAT, we found that recombinant human ChAT displays lower values for Michaelis and inhibition constants for ACh, which may be due to the complete absence of post-translational modifications.     

Differential assay for choline acetyltransferase

A rapid and sensitive radiochemical assay for choline acetyltransferase (EC 2.3.1.6) is reported. The assay allows for the fact that during incubation of [¹⁴C]acetyl-CoA and choline with a cell homogenate, at least one product is formed besides [¹⁴C]acetylcholine, which passes an anion exchange column. In contrast to [¹⁴C]acetylcholine, this major contaminant ([¹⁴C]acetylcarnitine) is not hydrolyzed apparently by Electrophorus acetylcholinesterase. Therefore, two types of assays are performed, the one in the presence of an acetylcholinesterase inhibitor, the other in the presence of acetylcholinesterase from Electrophorus. After passing the reaction mixtures over anion exchange columns, the radioactivities of the effluents are determined. Their difference is proportional to the choline acetyltransferase activity.     

The kinetic properties of human placental choline acetyltransferase

1. Michaelis constants for human placental choline acetyltransferase were shown to be dependent on the concentration of the second substrate present. The primary plots indicate a sequential rather than a Ping Pong mechanism and are of the same type with 300mm- and 500mm-sodium chloride. 2. Similar results have been obtained with rabbit brain choline acetyltransferase. 3. Product inhibition of the forward reaction has been studied. CoA inhibits competitively with respect to acetyl-CoA and non-competitively with respect to choline. Acetylcholine inhibits competitively with respect to choline and non-competitively with respect to acetyl-CoA. No inhibition is given by acetylcholine when the enzyme is saturated with choline. 4. It is concluded that human placental choline acetyltransferase has an ordered mechanism of the Theorell-Chance type.     

Localization of choline acetyltransferase in human brain stem neurons

Berth's method was used to study the cytochemical activity of choline acetyltransferase in truncus cerebri neurons of 6-8 lunar month-old human fetuses. Three types on neurons were diagnosed in the nuclei of the truncus cerebri with regard to cholinacetyltransferase localization: (1) cholinergic cholinoceptive neurons; (2) cholinergic non-cholinoceptive neurons; (3) non-cholinergic cholinoceptive neurons. The distribution of the neurons in 27 nuclei of the truncus cerebri is described.     

Spectrophotometric assay for choline acetyltransferase

A rapid and simple spectrophotometric assay for choline acetyltransferase is described. The method employs 4,4′-dithiodipyridine to measure the coenzyme A produced by the enzymic reaction. The conditions of the assay are described. The results are compared with those obtained by the radiochemical assay of the enzyme.     

Studies on choline acetyltransferase isolated from human brain

The purified choline acetyltransferase from human striatal tissue was found to have aK _m value of 8 μM for acetyl-coenzyme A and 250 μM for choline. The predominant enzyme component has a molecular weight of about 67,000 daltons, measured by molecular filtration through Sephadex G-100. In a sucrose-density gradient, the enzyme cosedimented with bovine serum albumin with an estimatedS-value of 4.5. The enzyme activity was enhanced 2- to 3-fold by KCl, NaCl, (NH₄)₂SO₄, and chelating agents like EDTA or EGTA. Cupric sulfate (0.1 mM) inhibited the enzyme activity almost completely. This inhibition was circumvented by increasing concentrations of enzyme protein, dithiothreitol, and EDTA, but not by the substrates, histidine, or imidazole.     

Expression of cholinesterase gene(s) in human brain tissues: translational evidence for multiple mRNA species

To resolve the origin(s) of the molecular heterogeneity of human nervous system cholinesterases (ChEs), we used Xenopus oocytes, which produce biologically active ChE when microinjected with unfractionated brain mRNA. The RNA was prepared from primary gliomas, meningiomas and embryonic brain, each of which expresses ChE activity with distinct substrate specificities and molecular forms. Sucrose gradient fractionation of DMSO-denatured mRNA from these sources revealed three size classes of ChE-inducing mRNAs, sedimenting at approximately 32S, 20S and 9S. The amounts of these different classes of ChE-inducing mRNAs varied between the three tissue sources examined. To distinguish between ChEs produced in oocytes and having different substrate specificities, their activity was determined in the presence of selective inhibitors. Both 'true' (acetylcholine hydrolase, EC 3.1.1.7) and 'pseudo' (acylcholine acylhydrolase, EC 3.1.1.8) multimeric cholinesterase activities were found in the mRNA-injected oocytes. Moreover, human brain mRNAs inducing 'true' and 'pseudo' ChE activities had different size distribution, indicating that different mRNAs might be translated into various types of ChEs. These findings imply that the heterogeneity of ChEs in the human nervous system is not limited to the post-translational level, but extends to the level of mRNA.     

Substrate binding and catalytic mechanism of human choline acetyltransferase

Choline acetyltransferase (ChAT) catalyzes the synthesis of the neurotransmitter acetylcholine from choline and acetyl-CoA, and its presence is a defining feature of cholinergic neurons. We report the structure of human ChAT to a resolution of 2.2 A along with structures for binary complexes of ChAT with choline, CoA, and a nonhydrolyzable acetyl-CoA analogue, S-(2-oxopropyl)-CoA. The ChAT-choline complex shows which features of choline are important for binding and explains how modifications of the choline trimethylammonium group can be tolerated by the enzyme. A detailed model of the ternary Michaelis complex fully supports the direct transfer of the acetyl group from acetyl-CoA to choline through a mechanism similar to that seen in the serine hydrolases for the formation of an acyl-enzyme intermediate. Domain movements accompany CoA binding, and a surface loop, which is disordered in the unliganded enzyme, becomes localized and binds directly to the phosphates of CoA, stabilizing the complex. Interactions between this surface loop and CoA may function to lower the KM for CoA and could be important for phosphorylation-dependent regulation of ChAT activity.     

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.     

Antibody production to choline acetyltransferase purified from human brain

Choline acetyltransferase (CAT) was isolated from human caudate and putamen. The enzyme was highly purified by a series of steps involving fractionation by protamine sulfate and ammonium sulfate followed by chromatography on DEAE-Sephadex, hydroxyapatite and carboxymethyl cellulose columns. The isolated CAT gave a single protein band on polyacrylamide gel electrophoresis at pH 8.3 which corresponded with CAT activity. A single band was also obtained at pH 6.8. Rabbit antiserum was prepared to the purified homogeneous CAT from carboxymethyl cellulose columns. It exhibited a single sharp precipitin band on double diffusion tests on Ouchterlony I.D. plates when tested against the partially purified hydroxyapatite enzyme. On preincubation, the antiserum inhibited CAT activity to 50–60% of control independently of the concentration of enzymatic protein. Normal rabbit serum neither produced a precipitin band on double diffusion tests nor inhibited the CAT activity on incubation. The anti-CAT rabbit antibody thus appeared to be specific.