Arylamine N-acetyltransferase from chicken liver. I. Monoclonal antibodies, immunoaffinity purification, and amino acid sequences

Five monoclonal antibodies against arylamine acetyltransferase (EC 2.3.1.5) from the chicken liver were established by immunizing a mouse with a partially purified enzyme preparation. None of the antibodies cross-reacted with arylamine N-acetyltransferase from the livers of cow, rabbit, and rat, nor with arylalkylamine N-acetyltransferase from the chicken pineal gland, indicating a high specificity of the antibodies. By using the antibodies, two immunoaffinity purification procedures were elaborated: A partially purified enzyme preparation was incubated with the monoclonal antibody, and the resulting enzyme-IgG complex was separated by a protein A-Sepharose column. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a single protein band with a molecular mass of 34 kDa in addition to the heavy and light chains of IgG. Secondly, an immunoaffinity column was prepared by immobilizing a monoclonal antibody to Sepharose 4B. After a partially purified enzyme preparation was absorbed on the column, N-acetyltransferase activity was eluted with 1 M NaCl and 1 M urea. The eluted sample contained a single 34-kDa protein. The purified enzyme preferred arylamines to arylalkylamines as substrates, indicating that it was arylamine N-acetyltransferase. The purified protein was subjected to digestion by lysylendopeptidase and separated by high performance liquid chromatography. Partial amino acid sequences of three peptides were determined by a gas-phase sequence analyzer.     

Effect of phenylpyruvate on pyruvate dehydrogenase activity in rat brain mitochondria

1. The effects of phenylpyruvate, a metabolite produced in phenylketonuria, on the pyruvate dehydrogenase-complex activity were investigated in rat brain mitochondria. 2. Pyruvate dehydrogenase activity was measured by two methods, one measuring the release of (14)CO(2) from [1-(14)C]pyruvate and the other measuring the acetyl-CoA formed by means of the coupling enzyme, pigeon liver arylamine acetyltransferase (EC 2.3.1.5). In neither case was there significant inhibition of the pyruvate dehydrogenase complex by phenylpyruvate at concentrations below 2mm. 3. However, phenylpyruvate acted as a classical competitive inhibitor of the coupling enzyme arylamine acetyltransferase, with a K(i) of 100mum. 4. It was concluded that the inhibition of pyruvate dehydrogenase by phenylpyruvate is unlikely to be a primary enzyme defect in phenylketonuria.     

From Arylamine N-Acetyltransferase to Folate-Dependent Acetyl CoA Hydrolase: Impact of Folic Acid on the Activity of (HUMAN)NAT1 and Its Homologue (MOUSE)NAT2

Acetyl Coenzyme A-dependent N-, O- and N,O-acetylation of aromatic amines and hydrazines by arylamine N-acetyltransferases is well characterised. Here, we describe experiments demonstrating that human arylamine N-acetyltransferase Type 1 and its murine homologue (Type 2) can also catalyse the direct hydrolysis of acetyl Coenzyme A in the presence of folate. This folate-dependent activity is exclusive to these two isoforms; no acetyl Coenzyme A hydrolysis was found when murine arylamine N-acetyltransferase Type 1 or recombinant bacterial arylamine N-acetyltransferases were incubated with folate. Proton nuclear magnetic resonance spectroscopy allowed chemical modifications occurring during the catalytic reaction to be analysed in real time, revealing that the disappearance of acetyl CH ₃ from acetyl Coenzyme A occurred concomitantly with the appearance of a CH ₃ peak corresponding to that of free acetate and suggesting that folate is not acetylated during the reaction. We propose that folate is a cofactor for this reaction and suggest it as an endogenous function of this widespread enzyme. Furthermore, in silico docking of folate within the active site of human arylamine N-acetyltransferase Type 1 suggests that folate may bind at the enzyme’s active site, and facilitate acetyl Coenzyme A hydrolysis. The evidence presented in this paper adds to our growing understanding of the endogenous roles of human arylamine N-acetyltransferase Type 1 and its mouse homologue and expands the catalytic repertoire of these enzymes, demonstrating that they are by no means just xenobiotic metabolising enzymes but probably also play an important role in cellular metabolism. These data, together with the characterisation of a naphthoquinone inhibitor of folate-dependent acetyl Coenzyme A hydrolysis by human arylamine N-acetyltransferase Type 1/murine arylamine N-acetyltransferase Type 2, open up a range of future avenues of exploration, both for elucidating the developmental role of these enzymes and for improving chemotherapeutic approaches to pathological conditions including estrogen receptor-positive breast cancer.     

Purification and partial characterization of chicken liver acetyl coenzyme A: arylamine N-acetyltransferase

Arylamine acetyltransferase (EC 2.3.1.5) was purified 120-fold from chicken liver. The enzyme showed a rise in activity from pH 6.5 to 7.7 followed by a constant activity to about pH 8.6. The relative molecular weight of the enzyme was about 34,000. The apparent Km for acetyl-CoA was 13 microM with 4-nitroaniline as acetyl-acceptor. CoA was a noncompetitive inhibitor relative to acetyl-CoA with apparent Ki value of 110 microM. With 4-methylaniline as substrate, arylamine acetyltransferase activity in pigeon liver was about 8 times greater than in chicken liver, and about 40 times greater than in rabbit.     

Immobilization of acetyl-CoA:arylamine N-acetyltransferase and the preparation of an enzyme reactor for the synthesis of N-[11C]acetylserotonin

The enzyme arylamine acetyltransferase (acetyl-CoA:arylamine N-acetyltransferase, EC 2.3.1.5) from pigeon liver is immobilized onto differently derivatized controlled pore glass beads. Different silanes, spacer arms and reactive end-groups were tested, and immobilized enzyme stability tests were performed. From these experiments, the method of choice was selected: immobilization on controlled pore glass beads (24 nm pore size, 75-125 microns particle size) derivatized with gamma-aminopropyl and glutaraldehyde as the reactive end group. The kinetic properties of an enzyme reactor were investigated and optimized. The goal was to obtain a rapid high-yield conversion of 0.5-1 mumol acetyl-CoA to N-acetylserotonin, so that the reactor is useful for the 11C-labelling of N-acetylserotonin. Using an enzyme reactor (9.8 x 0.5 cm i.d.) containing 4.6 U active arylamine acetyltransferase immobilized onto 930 mg carrier, a 70% conversion of acetyl-CoA was obtained within 4 min.     

Effect of phenylpyruvate on enzymes involved in fatty acid synthesis in rat brain

1. The effects of phenylpyruvate, a metabolite produced in phenylketonuria, on the pyruvate dehydrogenase-complex activity were investigated in rat brain mitochondria. 2. Pyruvate dehydrogenase activity was measured by two methods, one measuring the release of ¹⁴CO₂ from [1-¹⁴C]pyruvate and the other measuring the acetyl-CoA formed by means of the coupling enzyme, pigeon liver arylamine acetyltransferase (EC 2.3.1.5). In neither case was there significant inhibition of the pyruvate dehydrogenase complex by phenylpyruvate at concentrations below 2mm. 3. However, phenylpyruvate acted as a classical competitive inhibitor of the coupling enzyme arylamine acetyltransferase, with a K_i of 100μm. 4. It was concluded that the inhibition of pyruvate dehydrogenase by phenylpyruvate is unlikely to be a primary enzyme defect in phenylketonuria.     

Regulation of the diurnal cycle in activity of serotonin acetyltransferase in the chick pineal gland.

When chick pineal glands were explanted into organ culture at midlight phase of a diurnal cycle of illumination and incubated in the dark, they developed marked increases in serotonin acetyltransferase (acetyl coA:arylamine N-acetyltransferase; EC 2.3.1.5) activity. Either this increase in activity was inhibited or its onset was retarded in glands incubated under constant illumination. Supplements of theophylline, isobutylmethylxanthine, quinidine, and compound Ro 20-1724 (4-(3-butoxyl-4-methoxybenzyl)-2-imidazolidinone) elicited very marked increases in serotonin acetyltransferase activity in glands cultured in the dark. Levels of activity attained after 6 h in culture approached or exceeded the maximum levels attained at middark phase of the diurnal cycle in vivo. Effects of theophylline and compound Ro 20-1724 were additive. Supplements of dibutryl cAMP had little or no effect upon levels of serotonin acetyltransferase activity when tested alone or in combination with theophylline but further enhanced the increase in the level of enzyme activity elicited by Ro 20-1724. Adenosine and cAMP had little or no effect upon levels of serotonin acetyltransferase activity. It is concluded that levels of serotonin acetyltransferase activity in the chick pineal gland are regulated by a repressive, negative-control mechanism, which probably involves a membranous adenosine receptor.     

N-hydroxyarylamine O-acetyltransferase in hamster liver: identity with arylhydroxamic acid N,O-acetyltransferase and arylamine N-acetyltransferase

N-Hydroxyarylamine O-acetyltransferase, arylhydroxamic acid N,O-acetyltransferase, and arylamine N-acetyltransferase in hamster liver cytosol were co-purified almost to electrophoretical homogeneity by ion exchange chromatography on DEAE-cellulose, gel filtration on Cellulofine GCL-2000-sf and high-performance KB-hydroxyapatite chromatography. The molecular weight of the acetyltransferase was estimated to be 33,000 by gel filtration and SDS-polyacrylamide gel electrophoresis. The three acetyltransferase activities were inhibited by iodoacetamide, pentachlorophenol, and 1-nitro-2-naphthol. Furthermore, 2-aminofluorene, a substrate for arylamine N-acetyltransferase, inhibited the reactions of N-hydroxyarylamine O-acetyl transfer and arylhydroxamic acid N,O-acetyl transfer. These results suggest that the same enzyme catalyzes the three types of acetyl transfer reactions. The acetyltransferase could activate N-hydroxyarylamines, such as 2-hydroxyamino-6-methyldipyrido[1,2-alpha:3',2'-d]imidazole, 3-hydroxyamino-1-methyl-5H-pyrido[4,3-b]indole, and N-hydroxy-2-aminofluorene, to the corresponding N-acetoxyarylamines, which are capable of binding to nucleic acid. Polyguanylic acid was most efficiently modified by the N-acetoxyarylamines formed by the acetyltransferase.     

Spectrophotometric measurement of pyruvate dehydrogenase complex activity in cultured human fibroblasts

A spectrophotometric assay for the pyruvate dehydrogenase complex (PDHC) has been adapted for use with cultured human firbroblasts. It is a coupled enzyme assay utilizing pigeon liver arylamine acetyltransferase to measure the acetyl-CoA produced by PDHC. Activity is proportional to fibroblasts protein and to tine and depends completely on added pyruvate, CoA and NAD. In extracts in which PDHC had been activated (dephosphorylated) by the method of Sheu et al. (Sheu, R.K.-F., Hu, C.C. and Utter, M.F. (1981) J. Clin. Invest. 67, 1463–1471), activities in control cell lines are 5–50 fold higher than in earlier reports. Low activity has been demonstrated in a line previously eported to be PDHC-deficient.     

Regulation and possible role of serotonin N-acetyltransferase in the retina

The activity of retinal serotonin N-acetyltransferase (NAT) (arylamine acetyltransferase, EC 2.3.1.5), the penultimate enzyme in melatonin biosynthesis, exhibits properties of a circadian rhythm comparable to that seen in the pineal gland. Using an eye cup preparation we have found that circadian properties persist in vitro, which indicates that an endogenous circadian oscillator controlling NAT is present in the eye. Nighttime increases in NAT activity are suppressed by light, protein synthesis inhibitors, and catecholamines. In light, NAT activity is induced by conditions expected to increase intracellular levels of cyclic AMP (cAMP). This suggests that catecholamines and cAMP are normally involved in the regulation of NAT. Circadian indoleamine metabolism may play a role in the control of rhythmic photoreceptor metabolism as evidenced by the observation that melatonin and related compounds are potent activators of disk shedding.     

2-aminoanthracene as an analytical tool with the acetylation reaction catalyzed by arylamine N-acetyltransferase.

The polynuclear aromatic amine, 2-aminoanthracene, was found to be acetylated with high efficiency in the presence of acetyl-CoA by pigeon liver arylamine N-acetyltransferase (EC 2.3.1.5). As a consequence of acetylation the fluorescence properties of the compound dramatically change and the reaction time course can be easily followed fluorometrically at the emission wavelength of 425 nm upon excitation at 360 nm. When 2-aminoanthracene is employed with pigeon arylamine N-acetyltransferase, as the ultimate acceptor of the acetyl group in coupled fluorometric assays, it is possible to measure enzymatic activities, such as pyruvate dehydrogenase or carnitine acetyltransferase, in continuous assays rapidly and with high sensitivity or to determine with as much sensitivity important metabolites such as acetylcarnitine or acetyl-CoA.     

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.     

Biosynthesis of platelet-activating factor (PAF-acether). V. Enhancement of acetyltransferase activity in murine peritoneal cells by calcium ionophore A23187

The activity of the acetyltransferase capable of transferring the acetyl moiety of acetyl-CoA onto 2-lyso PAF-acether (1-alkyl-sn-glycero-3-phosphocholine) to form PAF-acether was compared in ionophore A23187-stimulated and in non-stimulated rat peritoneal cells. Stimulation resulted in a doubling of the acetyltransferase activity within 30 s. This effect was abolished in the presence of EDTA (1 mM) or EGTA (1 mM) and restored by addition of Ca2+ (10 mM). The specificity of acetyltransferase measured in ionophore-stimulated as well as in untreated cells is the same. In both situations we observed the same Km values for acetyl-CoA, whereas the Vmax values were different. The wide similarities of the two enzyme preparations lead us to conclude that stimulation by the ionophore involves an increase in the number of enzyme molecules rather than a change in the kinetic parameters of the acetyltransferase.     

Probing the Role of Cysteine Residues in Glucosamine-1-Phosphate Acetyltransferase Activity of the Bifunctional GlmU Protein from Escherichia coli: Site-Directed Mutagenesis and Characterization of the Mutant Enzymes

The glucosamine-1-phosphate acetyltransferase activity but not the uridyltransferase activity of the bifunctional GlmU enzyme from Escherichia coli was lost when GlmU was stored in the absence of β-mercaptoethanol or incubated with thiol-specific reagents. The enzyme was protected from inactivation in the presence of its substrate acetyl coenzyme A (acetyl-CoA), suggesting the presence of an essential cysteine residue in or near the active site of the acetyltransferase domain. To ascertain the role of cysteines in the structure and function of the enzyme, site-directed mutagenesis was performed to change each of the four cysteines to alanine, and plasmids were constructed for high-level overproduction and one-step purification of histidine-tagged proteins. Whereas the kinetic parameters of the bifunctional enzyme appeared unaffected by the C296A and C385A mutations, 1,350- and 8-fold decreases of acetyltransferase activity resulted from the C307A and C324A mutations, respectively. The K_m values for acetyl-CoA and GlcN-1-P of mutant proteins were not modified, suggesting that none of the cysteines was involved in substrate binding. The uridyltransferase activities of wild-type and mutant GlmU proteins were similar. From these studies, the two cysteines Cys307 and Cys324 appeared important for acetyltransferase activity and seemed to be located in or near the active site.     

Genetic polymorphism and cancer susceptibility: evidence concerning acetyltransferases and cancer of the urinary bladder

Acetyltransferase enzymes expressed in hepatic and extrahepatic tissues are products of an acetyltransferase gene locus. Acetylation capacity is regulated by simple autosomal Mendelian inheritance of two codominant alleles at this locus. Human slow acetylators are predisposed to bladder cancer from arylamine chemicals. The role of the bladder in arylamine metabolism and of bladder acetyltransferases in the etiology of bladder cancer is not fully understood, but the acetylator genotype-dependent expression of arylamine N-acetyltransferase and N-hydroxyarylamine O-acetyltransferase in bladder cytosol may contribute towards the genetic predisposition of human slow acetylators to arylamine-induced bladder cancer.     

Phosphorylation of 69-kDa choline acetyltransferase at threonine 456 in response to amyloid-beta peptide 1-42

Choline acetyltransferase synthesizes acetylcholine in cholinergic neurons. In the brain, these neurons are especially vulnerable to effects of beta-amyloid (A beta) peptides. Choline acetyltransferase is a substrate for several protein kinases. In the present study, we demonstrate that short term exposure of IMR32 neuroblastoma cells expressing human choline acetyltransferase to A beta-(1-42) changes phosphorylation of the enzyme, resulting in increased activity and alterations in its interaction with other cellular proteins. Using mass spectrometry, we identified threonine 456 as a new phosphorylation site in choline acetyltransferase from A beta-(1-42)-treated cells and in purified recombinant ChAT phosphorylated in vitro by calcium/calmodulin-dependent protein kinase II (CaM kinase II). Whereas phosphorylation of choline acetyltransferase by protein kinase C alone caused a 2-fold increase in enzyme activity, phosphorylation by CaM kinase II alone did not alter enzyme activity. A 3-fold increase in choline acetyltransferase activity was found with coordinate phosphorylation of threonine 456 by CaM kinase II and phosphorylation of serine 440 by protein kinase C. This phosphorylation combination was observed in choline acetyltransferase from A beta-(1-42)-treated cells. Treatment of cells with A beta-(1-42) resulted in two phases of activation of choline acetyltransferase, the first within 30 min and associated with phosphorylation by protein kinase C and the second by 10 h and associated with phosphorylation by both CaM kinase II and protein kinase C. We also show that choline acetyltransferase from A beta-(1-42)-treated cells co-immunoprecipitates with valosin-containing protein, and mutation of threonine 456 to alanine abolished the A beta-(1-42)-induced effects. These studies demonstrate that A beta-(1-42) can acutely regulate the function of choline acetyltransferase, thus potentially altering cholinergic neurotransmission.     

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.     

Assay of the pyruvate dehydrogenase complex by coupling with recombinant chicken liver arylamine N-acetyltransferase

The activity of the pyruvate dehydrogenase complex has long been determined in some laboratories by coupling the production of acetyl-coenzyme A (acetyl-CoA) to the acetylation of 4-aminoazobenzene-4'-sulfonic acid by arylamine N-acetyltransferase. The assay has some advantages, but its use has been limited by the need for large amounts of arylamine N-acetyltransferase. Here we report production of recombinant chicken liver arylamine N-acetyltransferase and optimization of its use in miniaturized assays for the pyruvate dehydrogenase complex and its kinase.     

Inhibitors of Escherichia coli serine acetyltransferase block proliferation of Entamoeba histolytica trophozoites

The protozoan parasite Entamoeba histolytica is the etiologic agent of amebiasis, a major global public health problem, particularly in developing countries. There is an effective anti-amoebic drug available, however its long term use produces undesirable side effects. As E. histolytica is a micro-aerophilic organism, it is sensitive to high levels of oxygen and the enzymes that are involved in protecting against oxygen-stress are crucial for its survival. Therefore serine acetyltransferase, an enzyme involved in cysteine biosynthesis, was used as a target for identifying potential inhibitors. Virtual screening with Escherichia coli serine acetyltransferase was carried out against the National Cancer Institute chemical database utilizing molecular docking tools such as GOLD and FlexX. The initial analysis yielded 11 molecules of which three compounds were procured and tested for biological activity. The results showed that these compounds partially block activity of the E. coli enzyme and the growth of E. histolytica trophozoites but not mammalian cells.     

The effects of substrates on the optical rotatory dispersion of carnitine acetyltransferase

1. The optical rotatory dispersion of carnitine acetyltransferase is altered in the presence of l-carnitine or acetyl-l-carnitine. These changes, which include an increase in the reduced mean residue rotation at 233nm. ([M'](233)), suggest that substrate binding causes the enzyme to unfold. 2. CoA and acetyl-CoA have no immediate effect on [M'](233) and CoA has no effect on the change in this parameter induced by l-carnitine. 3. The change in [M'](233) was used as a measure of the degree of saturation of the enzyme with carnitine substrates. Dissociation constants for the enzyme complexes with l-carnitine, d-carnitine and acetyl-l-carnitine were determined in this way. 4. Prolonged incubation of carnitine acetyltransferase in the presence of CoA leads to a small increase in the value of [M'](233) accompanied by irreversible inhibition of the enzyme. 5. Optical-rotatory-dispersion studies of two specifically inhibited enzyme forms are reported.