Human Choline Acetyltransferase Research Articles

Distribution of choline acetyltransferase immunopositive structures in the rat brainstem

The distribution of neurons, fibers and terminal fields in rat brainstem displaying positive immunoreactivity to a polyclonal antiserum to human placental choline acetyltransferase (ChAT) is described. The antiserum was used at the high dilution of 1:10,000 and was coupled with a sensitive detection system using the nickel ammonium sulfate intensification method. In addition to previously described ChAT immunopositive groups of large cells in the cranial motor nuclei, and the parabrachial and reticular complexes, many small or medium size, weakly immunopositive neurons were identified. Some of these appeared in structures in the region of the fourth ventricle, including the area postrema. Others were in structures associated with the superior olivary complex, including the lateral superior olive, and the medioventral, lateroventral and superior periolivary nuclei. Scattered, weakly positive cells were seen in numerous other structures, including the ventral tegmental area of Tsai, central gray, superior colliculus, spinal nucleus of nerve 5, dorsal cochlear nucleus and non-motor regions of the spinal cord. The prominent ascending fiber tract of the laterodorsal tegmental pathway was traceable from the parabrachial area to the subgeniculate region of the thalamus. Prominent terminal fields were seen in a number of brainstem structures, including the superior colliculus, pontine nuclei, anterior pretectal nucleus, interpeduncular nucleus and spinal nucleus of nerve 5. The association of small ChAT positive cells and terminal fields with many sensory structures suggests a significant cholinergic participation in the physiology of sensory function.

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/phospholipid 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.

Studies on detergent released choline acetyltransferase from membrane fractions of rat and human brain

The relationship between soluble and membrane choline acetyltransferase (ChAT) was studied. Differential solubilization of rat and human brain yielded ChAT in the soluble and membrane fractions. The addition of 1% Triton X-100 to membrane fractions resulted in a release of ChAT. A comparable release of lactate dehydrogenase was also observed. The Triton released ChAT and soluble ChAT from rat and human brain were efficiently purified by immuno-affinity chromatography. A single molecular weight of 68,000 was observed for both forms of rat and human brain ChAT. Epitope maps produced from both forms of human brain ChAT were identical. It is concluded that Triton released ChAT is identical to soluble ChAT and simply represents occluded soluble ChAT.

Immunohistochemical staining of cholinergic neurons in the human brain using a polyclonal antibody to human choline acetyltransferase

Antibodies against human placental choline acetyltransferase (ChAT) were used to immunohistochemically stain cholinergic neurons in the neostriatum and nucleus basalis of Meynert in human brain. Cells in both regions were intensely stained as were nerve fibers. Comparable cells were stained in these same brain regions in the rat. This anti-human ChAT antibody will enable the further detailed characterization of cholinergic neurons in the human brain in both health and disease.

Multiple isoelectric and molecular weight variants of choline acetyltransferase. Artifact or real?

The existence of isoelectric variants of bovine brain and human placental choline acetyltransferase was confirmed by chromatofocusing. The identification of molecular weight variants (bovine brain, Mr = 68,000 and 63,000; human placental, Mr = 66,000 and 64,000) was also demonstrated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by "Western blotting" and immunochemical visualization of choline acetyltransferase with monoclonal antibodies. No correlation between the isoelectric variants and molecular weight variants could be observed; however, in the case of the bovine brain enzyme the more alkaline isoelectric variant was enriched in the higher molecular weight form of the enzyme. Treatment of the bovine brain enzyme with Staphylococcus aureus V8 protease resulted in the conversion of the Mr = 68,000 form of the enzyme to the Mr = 63,000 form. During this conversion no change in the enzyme activity was observed demonstrating that the Mr = 63,000 form of the enzyme and probably also the Mr = 68,000 form of the enzyme are active. Preparation of the bovine brain enzyme in the presence of proteolytic enzyme inhibitors yielded a new higher molecular weight form of the enzyme, Mr = 73,000, which was enzymatically active. The Mr = 73,000 form of the enzyme exhibited a single isoelectric form when analyzed by chromatofocusing. These results suggest that the Mr = 73,000 form of the enzyme represents the native form, and that other molecular weight and isoelectric variants may arise by proteolysis.

Anti-human choline acetyltransferase fragments antigen binding (FAB)-sepharose chromatography for enzyme purification.

Fragments Antigen Binding (FAB)-Sepharose immunoaffinity chromatography was successfully applied to the purification of human choline acetyltransferase. The mild elution conditions made it possible to obtain the enzyme in its full catalytic state. The method should be generally applicable to enzyme purification.

Immunohistochemical staining of the human forebrain with monoclonal antibody to human choline acetyltransferase

Monoclonal antibody to human choline acetyltransferase (ChAT) was successfully produced from a mouse hybridoma cell line. The antibody was found to be of the IgM molecular species. By using this monoclonal antibody, immunohistochemical staining for ChAT was obtained on human brain sections. Only large sized cells were stained in the putamen and the substantia innominata. The specificity of the staining was comparable to that with polyclonal rabbit antibody to human ChAT produced by standard immunization procedures. No staining was observed when mouse monoclonal antibodies prepared against other human or bacterial antigens, or when normal mouse IgM, was employed.

Immunochemical studies of bovine and human choline-O-acetyltransferase using monoclonal antibodies.

Immunochemical properties of bovine and human choline acetyltransferase (ChAT, EC 2.3.1.6, acetyl-CoA:choline-O-acetyltransferase) were studied using six monoclonal antibodies (AB1, AB5, AB6, AB7, AB8, and AB9) reactive with the enzyme. All antibodies except AB1 bound specifically to two proteins of 68,000 and 70,000 MW on "Western" blots of sodium dodecyl sulfate-polyacrylamide gels containing human or bovine ChAT. The enzyme was specifically absorbed to immobilized antibody and could not be eluted by low pH and/or high salt concentrations although the enzyme retained activity on the immunoabsorbent. Pure bovine enzyme consisting of the same two proteins as seen in the Western blotting studies was eluted from immobilized AB1 in the presence of sodium dodecyl sulfate. Although active enzyme could not be eluted from immobilized antibodies by standard conditions, various combinations of free and immobilized antibodies were effective in competing off bound enzyme. Free antibody AB1 quantitatively eluted the active enzyme from immobilized AB1. The different capacities of the antibodies to elute enzyme from various immunoabsorbents reflect interesting properties of both the enzyme and the antibodies.

Cross-species and intraspecies reactivities of monoclonal antibodies against choline acetyltransferase

We have previously reported the first successful production of monoclonal antibodies against the cholinergic neuronal marker, choline acetyltransferase (ChAT). We now report some inter- and intraspecies cross-reactivity studies of the monoclonal antibody AB1. This antibody reacted most strongly with bovine ChAT and weakly with sheep and human enzyme. Similar amounts of binding were observed with the two forms of bovine ChAT separated by ion exchange chromatography. The antibody also reacted equally well with human ChAT derived from either brain or placenta.

Anti-choline acetyltransferase fragments antigen binding (Fab) for immunohistochemistry

The preparation of fragments antigen binding (Fab) from serum of rabbits immunized against human brain choline acetyltransferase (CAT) is described. Ant-CAT Fab fragments were less potent than anti-CAT immunoglobulin (IgG) in inhibiting mammalian CAT activity and did not precipitate CAT activity. They proved, however, far superior to anti-CAT IgG for the immunohistochemical staining of CAT in mammalian neurons.

Choline acetyltransferase-containing neurons in rodent brain demonstrated by immunohistochemistry.

Choline acetyltransferase was demonstrated in neuronal structures of the rodent central nervous system by immunohistochemistry through the application of Fab fragments obtained from monospecific antiserums to human choline acetyltransferase. The specificity of the antiserum for the enzyme was confirmed by the staining of both the ventral horn motor neurons in the rat spinal cord and the neuromuscular junction of the guinea pig diaphragm. Enzyme-containing cell bodies were observed in frontal sections of rat and guinea pig brain in the neostriatum, accumbens, nucleus of the diagonal band, medial septum, and olfactory tubercle. Positively staining fibers and probable nerve terminals were also found in the olfactory tubercle field and other areas of the basal forebrain. The results provide information on the distribution of the cholinergic systems in the rostral forebrain of the rodent.

Effect of sodium chloride on changing the rate-limiting step in the human placental choline acetyltransferase reaction.

The kinetic constants, Km and Vmax, for the choline acetyltransferase reaction were determined for choline and eight choline analogs under conditions of high (0.3 M) and low (approximately 0.01 M) sodium chloride. At high sodium chloride, the maximal velocities of the different substrates varied over 27-fold, while at low sodium chloride, less than a 5-fold variation was observed. Dead-end inhibition studies using acetylaminocholine as the inhibitor showed that under conditions of high sodium chloride, inhibition changes from noncompetitive to competitive as the reactivity of the substrate decreases. Under conditions of low sodium chloride, acetylaminocholine inhibition is nonlinear and noncompetitive with respect to all substrates tested. These results suggest that increased ionic strength increases the rate of coenzyme A dissociation from the enzyme. The rate-determining step of the reaction can be ternary complex interconversion, coenzyme A release, or both, depending on the ionic strength and the substrate employed.

Human placental choline acetyltransferase: Nature and molecular aspects of the inhibition by iodo- and bromoacetylcholines

Human placental choline acetyltransferase (ChA) was assayed by the formation of acetyl[ 14C]choline (ACh) from choline and acetyl[ 14C]coenzyme A (ACoA). Among the four halogenoacetylcholines, iodo- and bromoacetylcholines were potent inhibitors (I 50: 2 × 10 −6 M) of ChA. They were studied in the following experiments: (1) variation of initial velocity as a function of choline concentration (5 × 10 −5 to 2 × 10 −4 M, ACoA 10 −5 M) at four levels of iodo- and bromoacetylcholines (5 × 10 −7 to 2.5 × 10 −6 M); (2) variation of initial velocity as a function of ACoA concentration (6 × 10 −6 to 2.5 × 10 −5 M, choline 10 −4 M) at four levels of iodo- and bromoacetylcholines; (3) dialysis of the inhibited ChA; and (4) capacities of iodo- and bromoacetates to inhibit ChA. In (I) and (2), primary plots were noncompetitive and secondary plots of slopes and intercepts were linear. In (3), only 70–80 per cent of the inhibition was reversible. In (4), high levels of halogeno-acetates (10 −2 M) inhibited ChA by 20–30 per cent. These investigations support the formation of ternary intermediates (CoA ChA · IACh or CoA · ChA · BrACh) during the reaction of placental ChA. The quaternary ammonium head and the positive carboxyl-carbon do seem to contribute to the ChA inhibition by IACh and BrACh. The irreversible component of the inhibition (20–30 per cent) with XACh or iodo- and bromoacetates can be explained by possible (1) formation of R-carboxymethylated ChA (where R = -O-, -S-or = N-) which is less active than the original ChA, (2) formation of S-carboxymethylated CoA which is slowly dialyzed free from the enzyme surface, or (3) conversion of the part of the enzyme to the — N—carboxymethylated form which is inactive.

Relationships between Chemical Structure and Inhibition of Human Placental Choline Acetyltransferase by Keto Analogs of Acetylcholine

Seven keto analogs of acetylcholine were synthesized and evaluated as inhihitors of human placental choline acetyltransferase. Their potencies for inhibition of horse serum cholinesterase and stimulation of cholinergic receptors in the longitudinal ileal muscle of the guinea pig were investigated. The most potent and selective inhibitor of choline acetyltransferase was (2-benzoylethyl)trimethylammonium chloride with an I50 of 3 × 10–6M. It exhibited considerably low activities at muscarinic and nicotinic receptors and cholinesterases. Its high potency for inhibiting choline acetyltransferase was attributed to: (a) its cationic terminal, a site for an electron acceptor interaction; (b) an aryl moiety for hydrophobic and electron donor contributions; and (c) a positive charge on the carbon atom adjacent to the benzene ring due to the presence of the carbonyl group, which interacts with the nucleophilic residue on the enzyme.

Effect of salts on the physical and kinetic properties of human placental choline acetyltransferase.

Abstract— The effects of salt on the properties of human placental choline acetyltransferase have been examined. Increases in enzyme activity, thermal denaturation and susceptibility to proteolysis can be related to increases in ionic strength, rather than to specific salt effects. Increased ionic strength increases the maximal velocity (Km) of the reaction, with no change in the kinetic parameter Vmax/Km (choline). The pH‐Km profile, measured over the range of 6.5–8.0, indicates the requirement of a dissociated acidic residue whose pKa is below 7.5 at high ionic strength, and a protonated residue whose pKa is above 7.5 at low ionic strength. It is proposed that the conformation of the enzyme is different at high ionic strength and at low ionic strength, and that these different conformational states of the enzyme result in different rate‐determining steps of the reaction.

Re-evaluation of the kinetic mechanism of the choline acetyltransferase reaction.

Initial velocity patterns for human placental choline acetyltransferase show a series of converging lines for both the forward and reverse reaction; with Kacetyl coenzyme A = 11.9 micron, Kcholine = 0.41 mM, KCOA = 8.8 micron, and Kacetylcholine = 1.3 mM. The relative rates of acetylcholine synthesis (Vf) to acetylcholine breakdown (Vr) is 4.5. Product inhibition by acetylcholine is competitive with respect to choline and noncompetitive with respect to acetyl-CoA, while product inhibition by choline is competitive with respect to acetylcholine and noncompetitive with respect to coenzyme A. Chlorocholine, diethylaminoethanol, and acetylaminocholine were used as dead-end inhibitors and shown to inhibit competitively with respect to acetylcholine, and noncompetitively with respect to choline, acetyl-CoA, and CoA. At high choline concentrations, uncompetitive substrate inhibition is observed, and inhibition by acetylaminocholine changes from noncompetitive to competitive. Comparing the reactivity of dimethylaminoethanol to choline, and acetyldimethylaminoethanol to acetylcholine, the maximal velocities obtained with these analogues was approximately 25% of the natural substrates. These data are not consistent with the previously proposed ordered Theorell-Chance reaction mechanism, and have been interpreted in terms of a random binding mechanism.

Human brain and placental choline acetyltransferase: purification and properties.

Choline acetyltransferase (EC 2.3.1.6) catalyzes the biosynthesis of acetylcholine according to the following chemical equation: acetyl-CoA + choline in equilibrium to acetylcholine + CoA. In addition to nervous tissue, primate placenta is the only other animal source which contains appreciable acetylcholine and its biosynthetic enzyme. Human brain caudate nucleus and human placental choline acetyltransferase were purified to electrophoretic homogeneity using ion-exchange and blue dextran-Sepharose affinity chromatography. The molecular weights determined by Sephadex G-150 gel filtration and sodium dodecyl sulfate gel electrophoresis are 67000 plus or minus 3000. N-Ethylmaleimide, p-chloromercuribenzoate, and dithiobis(2-nitrobenzoic acid) inhibit the enzyme. Dithiothreitol reverses the inhibition produced by the latter two reagents. The pKa of the group associated with N-ethylmaleimide inhibition is 8.6 plus or minus 0.3. A chemically competent acetyl-thioenzyme is isolable by Sephadex gel filtration. The enzymes from the brain and placenta are thus far physically and biochemically indistinguishable.

Cross-immunity of antibodies to human choline acetyltransferase in various vertebrate species

Cross-immunity of antibodies to human choline acetyltransferase in various vertebrate species

Kinetic mechanisms of human placental choline acetyltransferase

Choline acetyltransferase (ChA) from human placenta was assayed by the formation of [acetyl- 14C]choline (ACh) from choline (Ch) and [acetyl- 14C]CoA (ACoA). The bisubstrate kinetics of this enzyme (0.15 nmoles ACh min mg protein; ACoA, 10 −5 M; Ch, 5 × 10 −4; pH 7.4, 37°) were studied by the following experiments: (1) variation of the initial velocity as a function of Ch concentration (6 × 10 −5 to 2.5 × 10 −4 M) at fixed ACoA concentrations (6 × 10 −6 to 2.5 × 10 −5 M); (2) variation of the initial velocity as a function of ACoA concentration (6 × 10 −6 to 2.5 × 10 −5 M) at fixed Ch concentrations (6 × 10 −5 to 2.5 × 10 −4 M); (3) enzyme inhibition by ACh (10 −2 M) and CoA (2.5 × 10 −5, 10 −4 M) with Ch as the variable substrate and ACoA as the fixed substrate; and (4) enzyme inhibition by ACh and CoA with ACoA as the variable substrate and Ch as the fixed substrate. In experiments (1) and (2), double reciprocal plots intersecting in the third quadrant were obtained. In experiment (3), ACh is a competitive inhibitor and CoA is a noncompetitive inhibitor. In experiment (4), CoA is a competitive inhibitor and ACh a noncompetitive inhibitor. These results suggest that the steady state concentrations of ternary complexes (ACoA. ChA. Ch, CoA. ChA. ACh) are very low. CoA was about 100 times stronger as a product inhibitor than ACh. Therefore, an ordered Theorell-Chance mechanism was tentatively postulated with ACoA as the leading substrate and CoA as the obligatory product. A ping-pong mechanism has been proposed for the human placental ChA in the published literature which was based upon the parallel double reciprocal plots in experiments (1) and (2) at higher substrate concentrations for ACoA and choline than those used by us. At these high substrate concentrations, the experimental points fall within the region of mixed first-order and zero-order kinetics instead of first-order kinetics on the initial velocity-substrate concentration curves. Therefore, the intersecting double reciprocal plots in experiments (1) and (2) were altered into plots which appeared approximately parallel. At high substrate concentrations of ACoA, a significant degree of substrate inhibition and product inhibition due to CoA was observed.

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.