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.     

Protein kinase C isoforms differentially phosphorylate human choline acetyltransferase regulating its catalytic activity

Choline acetyltransferase (ChAT) synthesizes acetylcholine in cholinergic neurons; regulation of its activity or response to physiological stimuli is poorly understood. We show that ChAT is differentially phosphorylated by protein kinase C (PKC) isoforms on four serines (Ser-440, Ser-346, Ser-347, and Ser-476) and one threonine (Thr-255). This phosphorylation is hierarchical, with phosphorylation at Ser-476 required for phosphorylation at other serines. Phosphorylation at some, but not all, sites regulates basal catalysis and activation. Ser-476 with Ser-440 and Ser-346/347 maintains basal ChAT activity. Ser-440 is targeted by Arg-442 for phosphorylation by PKC. Arg-442 is mutated spontaneously (R442H) in congenital myasthenic syndrome, rendering ChAT inactive and causing neuromuscular failure. This mutation eliminates phosphorylation of Ser-440, and Arg-442, not phosphorylation of Ser-440, appears primarily responsible for ChAT activity, with Ser-440 phosphorylation modulating catalysis. Finally, basal ChAT phosphorylation in neurons is mediated predominantly by PKC at Ser-476, with PKC activation increasing phosphorylation at Ser-440 and enhancing ChAT activity.     

Substrate binding and catalytic mechanism in ascorbate peroxidase: evidence for two ascorbate binding sites

The catalytic mechanism of recombinant soybean cytosolic ascorbate peroxidase (rsAPX) and a derivative of rsAPX in which a cysteine residue (Cys32) located close to the substrate (L-ascorbic acid) binding site has been modified to preclude binding of ascorbate [Mandelman, D., Jamal, J., and Poulos, T. L. (1998) Biochemistry 37, 17610-17617] has been examined using pre-steady-state and steady-state kinetic techniques. Formation (k1 = 3.3 +/- 0.1 x 10(7) M(-1) s(-1)) of Compound I and reduction (k(2) = 5.2 +/- 0.3 x 10(6) M(-1) s(-1)) of Compound I by substrate are fast. Wavelength maxima for Compound I of rsAPX (lambda(max) (nm) = 409, 530, 569, 655) are consistent with a porphyrin pi-cation radical. Reduction of Compound II by L-ascorbate is rate-limiting: at low substrate concentration (0-500 microM), kinetic traces were monophasic but above approximately 500 microM were biphasic. Observed rate constants for the fast phase overlaid with observed rate constants extracted from the (monophasic) dependence observed below 500 microM and showed saturation kinetics; rate constants for the slow phase were linearly dependent on substrate concentration (k(3-slow)) = 3.1 +/- 0.1 x 10(3) M(-1) s(-1)). Kinetic transients for reduction of Compound II by L-ascorbic acid for Cys32-modified rsAPX are monophasic at all substrate concentrations, and the second-order rate constant (k(3) = 0.9 +/- 0.1 x 10(3) M(-1) s(-1)) is similar to that obtained from the slow phase of Compound II reduction for unmodified rsAPX. Steady-state oxidation of L-ascorbate by rsAPX showed a sigmoidal dependence on substrate concentration and data were satisfactorily rationalized using the Hill equation; oxidation of L-ascorbic acid by Cys32-modified rsAPX showed no evidence of sigmoidal behavior. The data are consistent with the presence of two kinetically competent binding sites for ascorbate in APX.     

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.     

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.     

Purification and immunochemical properties of choline acetyltransferase from human brain

Choline acetyltransferase (CAT) was purified to homogeneity from 363 g of human neostriatum by means of ammonium sulfate and protamine sulfate fractionation, followed by chromatography on DEAE-Sephadex, hydroxyapatite, phosphocellulose, and agarose-hexane-Co A columns. The final product migrated as a single component on 7.5% gels with or without SDS. It had a molecular weight of 66,000 daltons and a specific activity of 7.3 mol acetylcholine formed per milligram protein per minute. Antibodies prepared in rabbits gave single precipitin lines against this protein on Ouchterlony immunodiffusion and immunoelectrophoresis plates. The CAT-anti-CAT IgG complex migrated as a single band on gel electrophoresis, establishing the monospecificity of the antibodies. Strong cross-reactivity to the IgG was obtained with CAT from rat, rabbit, and guinea pig, but only weak reactivity with chicken. Fab fragments were prepared from the rabbit IgG and were used to stain CAT-containing neurons in the spinal cord and nerve endings at the neuromuscular junction using the PAP technique.     

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.     

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.     

Membrane-bound choline acetyltransferase from human brain: Purification and properties

Choline acetyltransferase (ChAT; EC 2.3.1.6) was separated from human caudate/putamen into three fractions by successive extractions into apotassium phosphate buffer, a high salt (NaCl) buffer and a buffer containing 0.6% Triton X-100. The Triton-X-solubilized fraction is the membrane-bound ChAT (mChAT) and represents about 40% of the total ChAT. After centrifugation, mChAT was precipitated by ammonium sulfate at 35–65% saturation. The crude enzyme preparation was fractionated in turn on a DEAE-Sepharose, a hydroxylapatite and a phosphocellulose columns. Finally, mChAT was applied to a CoA-Sepharose column equilibrated with buffer containing 100 mM choline chloride and was specifically eluted with buffer containing acetyl-CoA. The presence of both substrates greatly stabilized the enzyme and ChAT was recovered almost quantitatively. The final preparation of mChAT has a specific activity of 37.2 mol of acetylcholine synthesized per min-mg protein. The purified mChAT has a pH optimum of 8.3. It migrated as two bands on SDS-PAGE with molecular weights of 67,000 and 62,000 daltons, respectively. Immunoblot autoradiography showed that an antiserum prepared previously against soluble ChAT also cross-reacted with both bands of mChAT, indicating that both forms of this enzyme are related. Furthermore, as previously reported for soluble ChAT, Fab-Sepharose chromatography could be used for the purification of mChAT and this preparation also resolved into two bands of 10% SDS gel.Special Issue dedicated to Prof. Eduardo De Robertis.     

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.     

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.     

Carrier-mediated inhibition of choline acetyltransferase

Incubation of rat forebrain synaptosomes with choline mustard aziridinium ion in a sodium-rich medium caused a time-dependent inhibition of the high-affinity transport of choline, as well as a significant decrease in intrasynaptosomal choline acetyltransferase activity. In the absence of added sodium choline uptake by a sodium-independent mechanism was also blocked in a time-dependent manner but intrasynaptosomal choline acetyl-transferase activity was unaltered. Neither monoethylcholine nor hemicholinium-3 changed intrasynaptosomal choline acetyl-transferase activity but competitively inhibited the transport of choline. The results indicate that there may be a fraction of choline acetyltransferase that is closely associated with the sodium-dependent high-affinity choline transport system and that this fraction can be irreversibly inhibited by choline mustard aziridinium ion, perhaps indirectly mediated by alkylation of the carrier.     

The phosphorylation of choline acetyltransferase

Human placental Choline Acetyltransferase (ChAT) has been shown to be phosphorylated in vitro by kinases present in rat brain. Phosphorylation occurs at a single site with the exclusive phosphoamino acid being serine. ChAT phosphorylation was shown to be calcium, and not cyclic nucleotide, dependent and was inhibited by inhibitors of calcium/calmodulin protein kinases including anti-calmodulin anti-sera. ChAT phosphorylation was stimulated by calmodulin (9 fold) and, to a lesser extent, by phosphatidylserine (4 fold). These results indicate the involvement of a calcium/calmodulin and possibly also a calcium/phosopholipid kinase. This finding was confirmed by demonstrating ChAT phosphorylation using both purified multifunctional calcium/calmodulin protein kinase (CaMK) and calcium/phospholipid protein kinase C (PKC) from rat brain. A stoichiometric incorporation of 0.9 mol phosphate/mol ChAT was achieved by CaMK. Phosphorylated ChAT could be isolated from freshly prepared rat brain synaptosomes. The results obtained with this model system support the hypothesis that in vivo a fraction of ChAT exists phosphorylated.     

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.     

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.     

Expression of multiple mRNA species for choline acetyltransferase in human T-lymphocytes

Acetylcholine (ACh) is synthesized by choline acetyltransferase (ChAT) in cholinergic neurons. However, both ACh and mRNA for ChAT are expressed in mononuclear leukocytes and various human leukemic T-cell lines. Multiple ChAT mRNA species (R-, N0-, N1-, N2-, and M-types) having an identical coding region and different 5'-noncoding regions have been discovered in human brain and spinal cord. These mRNAs are transcribed by a combination of use of different promoter regions and alternative splicing. However, which types of ChAT mRNA species are expressed in T-lymphocytes remains to be elucidated. In the present study, we used two human leukemic T-cell lines, CCRF-CEM (CEM) and MOLT-3, which express the same ChAT mRNA as that in the nervous system. Major mRNA species in CEM were N2- and M-type, and to a lesser extent N1-type, while MOLT-3 expressed only N2-type. Neither CEM nor MOLT-3 expressed R-type mRNA. We previously found a lack of mRNA expression encoding vesicular acetylcholine transporter (VAChT) in CEM and MOLT-3, which mediates ACh transport to synaptic vesicles in cholinergic neurons. These findings suggest that the mechanisms regulating ChAT mRNA expression in T-lymphocytes differ from those in cholinergic neurons.