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.     

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.     

Uptake, metabolism, and releasability of ethyl analogues of homocholine by rat brain

Abstract: Ethyl analogues of homocholine were synthesized and used to describe further the specificities of the processes involved in choline uptake and acetylation and acetylcholine storage and release. Monoethylhomocholine, diethylhomocholine, and triethylhomocholine decreased the transport of choline into rat brain synaptosomes. The mono- and diethyl compounds were taken up into synaptosomes with similar affinity for the transport system as choline (5.8, 8.5, and 5.5 μ M , respectively) but at a somewhat slower rate (11.3, 8.5, and 37.3 nmol/g original tissue/h, respectively); the triethyl analogue was not transported at the concentrations tested, which further defines the structural specificity of the transport system. l -Carnitine did not affect the transport of the analogues. The in situ acetylation of mono- and diethyl-homocholine by slices of rat cerebral cortex was measurable, but the in vitro acetylation by choline acetyl-transferase solubilized from rat forebrain was not. Acetylation of the diethyl analogue by slices of cerebellar cortex was <20% of that by slices of cerebral cortex. Subcellular fractionation of cerebral slices showed that acetyldiethylhomocholine localized preferentially to the cytosolic rather than vesicular stores, indicating specificity of the mechanism responsible for the incorporation of acetylated product into the vesicles. The release of acetyldiethylhomocholine and of acetylcholine was tested from sliced brain that had been incubated with the precursors. Both esters were released spontaneously but stimulation with increased K⁺ concentration enhanced the release of acetylcholine without changing the release of acetyldiethylhomocholine, suggesting that evoked transmitter release occurred from a vesicular store.     

SURFACE CHARGE OF CHOLINE ACETYLTRANSFERASE FROM DIFFERENT SPECIES

—The adsorption of partially purified choline acetyltransferase (ChAc) from cat, rat, guinea-pig and pigeon brains by the cation exchange resins, CM-Sephadex (C-50) and Amberlite CG-50 II, was studied at various pH values and ionic strengths. ChAc from cat and rat were more strongly adsorbed by cation exchangers and therefore have a stronger net positive surface charge than those from guinea pig and pigeon. Experiments showed that the difference in adsorption between these two groups of enzymes could not be explained by overloading of the resin, by competitive effect of other proteins present in the enzyme preparations or by the presence of any component suppressing the adsorption of ChAc in any of the enzyme preparations. The adsorption of ChAc by a cation exchanger is very similar to its binding to synaptosome membranes. The significance of the positive surface charge of ChAc in studies on the compartmentation of ChAc in synaptosomes is discussed.     

PURIFICATION OF RAT BRAIN CHOLINE ACETYLTRANSFERASE1

Abstract— The purification of choline acetyltransferase (ChAc) has been hampered by the increasing instability of the enzyme in the course of purification. By working with a high concentration of protein and by adding glycerol to the enzyme, the stability was increased. The purification was performed by centrifuging twice, at low and high salt concentrations, precipitation by ammonium sulphate and chromatography on carboxymethyl–Sephadex, hydroxylapatite and Sephadex G 100. The final steps were performed by using chromatography on an immunoabsorbent; this consists of agarose-coupled gammaglobulins of antisera devoid of any activity against ChAc itself and directed against other proteins still present in the purest ChAc preparation achieved by conventional biochemical techniques. The purest rat brain ChAc preparation had a specific activity of 20 μmol/min/mg of protein after a 30,000-fold purification. The enzyme was not homogeneous in polyacrylamide gel electrophoresis performed either at pH 4.5 or with sodium dodecyl sulphate. Pure ChAc from rat brain would have a specific activity of approximately 100 μmol/min/mg of protein.     

STANDARDIZATION OF A RADIOCHEMICAL ASSAY OF CHOLINE ACETYLTRANSFERASE AND A STUDY OF THE ACTIVATION OF THE ENZYME IN RABBIT BRAIN

Abstract—

• 1 The standardization of a radiochemical assay of choline acetyltransferase (acetyl-CoA: choline-O-acetyltransferase EC 2.3.1.6. [ChAc]) is described. The method depends upon the use of [l-C¹⁴]acetyl-CoA as substrate and has been modified from the procedure originally published by MCCAMAN and HUNT (1965).
• 2 The modified method gave results which were comparable with two methods depending on bioassay as the end step, which had previously given the highest recorded values for rabbit brain ChAc.
• 3 Methods of extraction and activation of the enzyme were also investigated. The results showed that ether-treated sucrose homogenates and cysteine-saline extracts of acetone-dried brain have comparable enzyme activities; both procedures give higher values than are obtained with untreated water homogenates, or sucrose homogenates treated with Nonex 501 or Triton X-100.

     

Lysophosphatidic acid,alkylglycerophosphate and alkylacetylglycerophosphate increase the neuronal nuclear acetylation of 1-acyl lysophosphatidylcholine by inhibition of lysophospholipase

Neuronal nuclei were isolated from rabbit cerebral cortex, and lipid acetylation reactions were studied because of the high nuclear concentration of acetyltransferases that generate platelet activating factor (PAF) and its acyl analogue AcylPAF. The neuronal nuclear acetylation of 1-palmitoyl lysophosphatidylcholine (lyso PC) was found to be increased more than two fold when low concentrations of lyso PC were incubated in acetylation assays in the presence of 1-palmitoyl lysophosphatidic acid (lyso PA) or 1-hexadecyl glycerophosphate (AGP). This effect was not found for a variety of other acidic and neutral 1-acyl lysoglycerophospholipids. At 4 M concentrations, AGP was the more effective in increasing rates of lyso PC acetylation, while lyso PA was more effective at 25-35 M. 1-Stearoyl, 1-alkenyl and 1-decanoyl analogues of lyso PA were all less effective than 1-palmitoyl lyso PA. Phosphatidic acid was considerably less effective than lyso PA, while the acetylated analogue of AGP, AAcGP (alkylacetylglycerophosphate), increased rates of lyso PC to maxima similar to those seen with lyso PA or AGP. In addition, AAcGP promoted these maxima at considerably lower concentrations (2-4 M). A mechanism for these effects was suggested when nuclear envelopes (NE), isolated in the presence of PMSF, showed these maximal acetylation rates at low lyso PC concentrations, and these rates were not elevated by the presence of lyso PA. PMSF is a protease inhibitor but can also inhibit lysophospholipase activity. We found a nuclear lysophospholipase that degraded lyso PC at rates more than 13 times those of nuclear lyso PC acetylation. PMSF did inhibit this nuclear lysophospholipase, as did lyso PA, AGP and AAcGP. Kinetic analyses of the effects of lyso PA, AGP and AAcGP on lyso PC lysophospholipase indicated that these three lipids acted as competitive inhibitors for the lyso PC substrate. It is possible that low rates of lyso PC acetylation seen in neuronal nuclei at low lyso PC concentrations, are caused by lyso PC loss mediated by a very strong nuclear lysophospholipase. The effects of lyso PA, AGP and AAcGP in boosting rates of lyso PC acetylation likely come from the inhibition of nuclear lysophospholipase and a preservation of lyso PC concentrations. Competing neuronal nuclear reactions for low endogenous levels of lyso PC may regulate the formation of AcylPAF, and rising lyso PA, AGP or AAcGP concentrations can increase rates of nuclear AcylPAF synthesis.     

THE SYNTHESIS OF ACETYLCHOLINE FROM ACETYL-CoA, ACETYL-DEPHOSPHO-CoA AND ACETYLPANTETHEINE PHOSPHATE BY CHOLINE ACETYLTRANSFERASE

Abstract— The synthesis of ACh by choline acetyltransferase (ChAc) has been examined using acetyl-CoA, acetyl-dephospho-CoA and acetylpantetheine phosphate. At pH 7.5 K_m values of 25.7 μ m for acetyl-CoA, 54.8 μ m for acetyl-dephospho-CoA and 382 μ m for acetylpantetheine phosphate were obtained and are similar to those at pH 6.0. This indicates that the 3-phosphate may not be required for binding the substrate to the enzyme unlike carnitine acetyltransferase.
Inhibitor constants ( K_i ) for CoA, dephospho-CoA and pantetheine phosphate were also measured and when considered with the K_m values obtained for the acetyl derivatives it is concluded that acetyl-dephospho-CoA could be a successful acetyl donor in the synthesis of ACh.
Acetyl-dephospho-CoA was found to be less satisfactory as a substrate for citrate synthase.     

Degradation of chitosans with chitinase B from Serratia marcescens. Production of chito-oligosaccharides and insight into enzyme processivity

Family 18 chitinases such as chitinase B (ChiB) from Serratia marcescens catalyze glycoside hydrolysis via a mechanism involving the N-acetyl group of the sugar bound to the -1 subsite. We have studied the degradation of the soluble heteropolymer chitosan, to obtain further insight into catalysis in ChiB and to experimentally assess the proposed processive action of this enzyme. Degradation of chitosans with varying degrees of acetylation was monitored by following the size-distribution of oligomers, and oligomers were isolated and partly sequenced using (1)H-NMR spectroscopy. Degradation of a chitosan with 65% acetylated units showed that ChiB is an exo-enzyme which degrades the polymer chains from their nonreducing ends. The degradation showed biphasic kinetics: the faster phase is dominated by cleavage on the reducing side of two acetylated units (occupying subsites -2 and -1), while the slower kinetic phase reflects cleavage on the reducing side of a deacetylated and an acetylated unit (bound to subsites -2 and -1, respectively). The enzyme did not show preferences with respect to acetylation of the sugar bound in the +1 subsite. Thus, the preference for an acetylated unit is absolute in the -1 subsite, whereas substrate specificity is less stringent in the -2 and +1 subsites. Consequently, even chitosans with low degrees of acetylation could be degraded by ChiB, permitting the production of mixtures of oligosaccharides with different size distributions and chemical composition. Initially, the degradation of the 65% acetylated chitosan almost exclusively yielded oligomers with even-numbered chain lengths. This provides experimental evidence for a processive mode of action, moving the sugar chain two residues at a time. The results show that nonproductive binding events are not necessarily followed by substrate release but rather by consecutive relocations of the sugar chain.     

CHOLINE: SELECTIVE ACCUMULATION BY CENTRAL CHOLINERGIC NEURONS

Abstract— Most of the cholinergic input to the hippocampus was destroyed by placement of lesions in the medial septal area. In animals with such lesions we found that hippocampal ChAc activity was reduced by 85–90% and endogenous acetylcholine levels were reduced by more than 80 %. When hippocampal synaptosomes from animals with lesions were incubated with [³H]choline at concentrations of 7.5 nm, 1 μm and 10 μm there was approximately a 60 % reduction in the uptake of [³H]choline, suggesting that cholinergic nerve endings were mainly responsible for [³H]choline uptake. At 0.1 mm concentrations of [³H]choline, there was only a 25 % reduction of choline uptake, suggesting that at higher concentrations of choline there was more nonspecific uptake. The uptake of radiolabelled tryptophan, glutamate and GABA were only slightly or not at all affected by the lesions. There was a significant reduction of uptake of radiolabelled serotonin and norepinephrine, since known monoaminergic tracts were disrupted. Choline uptake was reduced only in brain regions in which cholinergic input was interrupted (i.e. the cerebral cortex and hippocampus) and remained unchanged in other regions (i.e. the cerebellum and striatum). The time course of the reduction in choline uptake was similar to that of the reductions in ChAc activity and endogenous ACh levels; there was no decrease at 1 day, a significant decrease at 2 days, and the maximal decrease at 4 days postlesion. There was a close correlation among choline uptake, ChAc activity and ACh levels in the four brain regions examined (i.e. the striatum, cerebral cortex, hippocampus and cerebellum). Our results suggest that when hippocampal synaptosomes (and perhaps synaptosomes from other brain areas as well) are incubated in the presence of choline, at concentrations of 10 μm m or lower, then cholinergic nerve endings are responsible for the bulk of the choline accumulated by the tissue.     

Acetylation of N-terminal valine of glycine N-methyltransferase affects enzyme inhibition by folate

Native liver glycine N-methyltransferase (GNMT) is N-acetylated while the recombinant enzyme is not. We show here that acetylation of the N-terminal valine affects several kinetic parameters of the enzyme. Glycine N-methyltransferase is a regulatory enzyme mediating the availability of methyl groups by virtue of being inhibited by folate. N-acetylation does not affect the overall structure of the protein and does not affect basal enzyme activity of GNMT. Binding of both the mono- and pentaglutamate forms of 5-methyltetrahydrofolate is the same for the acetylated and non-acetylated forms of the enzyme, however the pentaglutamate form is bound more tightly than the monoglutamate form in both cases. Although binding of the folates is similar for the acetylated and non-acetylated forms of the enzyme, inhibition of enzyme activity differs significantly. The native, N-acetylated form of the enzyme shows 50% inhibition at 1.3 microM concentration of the pentaglutamate while the recombinant non-acetylated form shows 50% inhibition at 590 microM. In addition, the binding of folate results in cooperativity of the substrate S-adenosylmethionine (AdoMet), with a Hill coefficient of 1.5 for 5-methyltetrahydrofolate pentaglutamate.     

System-wide studies of N-lysine acetylation in Rhodopseudomonas palustris reveal substrate specificity of protein acetyltransferases

N-lysine acetylation is a posttranslational modification that has been well studied in eukaryotes and is likely widespread in prokaryotes as well. The central metabolic enzyme acetyl-CoA synthetase is regulated in both bacteria and eukaryotes by acetylation of a conserved lysine residue in the active site. In the purple photosynthetic α-proteobacterium Rhodopseudomonas palustris, two protein acetyltransferases (RpPat and the newly identified RpKatA) and two deacetylases (RpLdaA and RpSrtN) regulate the activities of AMP-forming acyl-CoA synthetases. In this work, we used LC/MS/MS to identify other proteins regulated by the N-lysine acetylation/deacetylation system of this bacterium. Of the 24 putative acetylated proteins identified, 14 were identified more often in a strain lacking both deacetylases. Nine of these proteins were members of the AMP-forming acyl-CoA synthetase family. RpPat acetylated all nine of the acyl-CoA synthetases identified by this work, and RpLdaA deacetylated eight of them. In all cases, acetylation occurred at the conserved lysine residue in the active site, and acetylation decreased activity of the enzymes by >70%. Our results show that many different AMP-forming acyl-CoA synthetases are regulated by N-lysine acetylation. Five non-acyl-CoA synthetases were identified as possibly acetylated, including glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and Rpa1177, a putative 4-oxalocrotonate tautomerase. Neither RpPat nor RpKatA acetylated either of these proteins in vitro. It has been reported that Salmonella enterica Pat (SePat) can acetylate a number of metabolic enzymes, including GAPDH, but we were unable to confirm this claim, suggesting that the substrate range of SePat is not as broad as suggested previously.     

Direct determination of acetyl-enzyme intermediate in the acetylcholinesterase-catalyzed hydrolysis of acetylcholine and acetylthiocholine

Acetylcholinesterase from Electrophorus electricus was acetylated during the hydrolysis of [3H]acetylcholine and [3H]acetylthiocholine. The steady state levels of [3H]acetyl-enzyme were measured at different pH and different concentrations of substrate. The maximum acetylation fraction [S)----infinity) at pH 7.0 in 0.5 M salt was 0.65 with acetylcholine as substrate and 0.57 with acetylthiocholine as substrate. Acetylation is faster than deacetylation. The fraction of acetyl-enzyme was not affected by pH which indicates that acetylation and deacetylation are equally affected by changes in pH. This results supports the concept that acetylation and deacetylation involve similar mechanisms.     

Sirtuin 3 (SIRT3) Protein Regulates Long-chain Acyl-CoA Dehydrogenase by Deacetylating Conserved Lysines Near the Active Site

Long-chain acyl-CoA dehydrogenase (LCAD) is a key mitochondrial fatty acid oxidation enzyme. We previously demonstrated increased LCAD lysine acetylation in SIRT3 knockout mice concomitant with reduced LCAD activity and reduced fatty acid oxidation. To study the effects of acetylation on LCAD and determine sirtuin 3 (SIRT3) target sites, we chemically acetylated recombinant LCAD. Acetylation impeded substrate binding and reduced catalytic efficiency. Deacetylation with recombinant SIRT3 partially restored activity. Residues Lys-318 and Lys-322 were identified as SIRT3-targeted lysines. Arginine substitutions at Lys-318 and Lys-322 prevented the acetylation-induced activity loss. Lys-318 and Lys-322 flank residues Arg-317 and Phe-320, which are conserved among all acyl-CoA dehydrogenases and coordinate the enzyme-bound FAD cofactor in the active site. We propose that acetylation at Lys-318/Lys-322 causes a conformational change which reduces hydride transfer from substrate to FAD. Medium-chain acyl-CoA dehydrogenase and acyl-CoA dehydrogenase 9, two related enzymes with lysines at positions equivalent to Lys-318/Lys-322, were also efficiently deacetylated by SIRT3 following chemical acetylation. These results suggest that acetylation/deacetylation at Lys-318/Lys-322 is a mode of regulating fatty acid oxidation. The same mechanism may regulate other acyl-CoA dehydrogenases.     

Active site-directed and allosteric effectors of regulatory enzymes: the activation of aspartate transcarbamylase by substrate and transition state analogues

An extension of the two-state theory of Monod, Wyman &; Changeux (1965) has been used to explain quantitatively the effects of substrate and transition state analogues on the kinetic and physcco-chemical properties of aspartate transcarbamylase. The derived equations accurately predict the observed activation at low concentrations followed by inhibition at higher concentrations of such analogues. They also predict the binding, kinetic and physical behaviour of the enzyme in the presence of both active site-directed effectors, such as succinate, and allosteric effectors, such as cytidine triphosphate, both of which the enzyme is subjected to physiologically. The equations are applicable to other enzymes provided that the system behaves as though there is only one substrate and that binding equations can be converted to kinetic equations by replacing a dissociation constant by a microscopic Michaelis constant.     

Multiple Forms of Choline Acetyltransferase from Rat Brain

WE have found that several different forms of choline acetyl-transferase (ChAc) from rat brain can be separated by isoelectric focusing. Such heterogeneity of ChAc is of particular interest in the context of its ultrastructural localization. Subcellular fractionation^1–4 and histochemistry⁵ have shown that the enzyme in rat, in conditions of low ionic strength and pH, adhered to several different membranous structures.     

Neurochemical Characterization of an Antimony-Choline Analog in Rat Cortical Synaptosomes

Abstract: An analog of choline, in which nitrogen was replaced by antimony, was neurochemically characterized in rat cortical synaptosomes. It was found to be a substrate for several cholinergic enzymes, transported by a Na⁺-dependent, hemicholinium-3-sensitive process, acetylated, and subsequently released by depolarization in a calcium-dependent manner. Sb-choline also competed with choline for Na⁺-dependent uptake and for acetylation by [¹⁴C]acetyl-CoA in synaptosomes. These results suggest that Sb-choline and its acetylated product should be useful substrates for the x-ray microanalytical localization of cholinergic pools in intact nerve terminals.     

SEPARATION OF APPARENT MULTIPLE FORMS OF HUMAN BRAIN CHOLINE ACETYLTRANSFERASE BY ISOELECTRIC FOCUSING

Abstract— Choline acetyltransferase (acetyl-CoA: choline O -acetyl transferase; EC 2.3.1.6; ChAc) purified from human brain (basal ganglia) and sciatic nerve were separated into apparent multiple enzyme forms by the method of isoelectric focusing (pH gradient 3-10) on acrylamide gel. A preparative separation of enzyme forms of human brain was accomplished by the column method, by using a sucrose gradient. When each separated form was re-electrofocused, only a portion of the ChAc activity was observed in its original pH region while more than one-half of the recovered activity for each fraction appeared at pH 7.8-8. Gel filtration and kinetic studies of separated forms indicated that the more acidic forms might be aggregates, while more basic forms might be configurational isomers. Human ChAc of sciatic nerve did not exhibit acidic forms on electrofocusing, but otherwise yielded an electrofocusing profile similar to that of human brain. ChAc of rabbit brain and sciatic nerve each exhibited only a single form at pH 7.1 ± 0.2. Although ChAc differs among species, the enzyme of brain and sciatic nerve of the same species cannot be clearly distinguished by electrofocusing.     

Purification and characterization of sheep platelet cyclo-oxygenase. Acetylation by aspirin prevents haemin binding to the enzyme.

Arachidonate cyclo-oxygenase (prostaglandin synthetase; prostaglandin endoperoxide synthetase; EC 1.14.99.1) was purified from sheep platelets. The purification procedure involved hydrophobic column chromatography using either Ibuprofen-Sepharose, phenyl-Sepharose or arachidic acid-Sepharose as the first step followed by metal-chelate Sepharose and haemin-Sepharose affinity chromatography. The purified enzyme (Mr approximately 65,000) was homogeneous as observed by SDS/polyacrylamide-gel electrophoresis and silver staining. The enzyme was a glycoprotein with mannose as the neutral sugar. Haemin or haemoglobin was essential for activity. The purified enzyme could bind haemin exhibiting a characteristic absorption maximum at 410 nm. The enzyme after metal-chelate column chromatography could undergo acetylation by [acetyl-3H]aspirin. The labelled acetylated enzyme could not bind to haemin-Sepharose, presumably due to acetylation of a serine residue involved in the binding to haemin. The acetylated enzyme also failed to show its characteristic absorption maximum at 410 nm when allowed to bind haemin.     

Kinetic Data on the Inhibition of High-affinity Choline Transport into Rat Forebrain Synaptosomes by Choline-like Compounds and Nitrogen Mustard Analogues

Abstract: A series of choline analogues and nitrogen mustard derivatives were evaluated as inhibitors of high-affinity transport of choline in rat forebrain synaptosomes. When synaptosomes were preincubated for 10 min with choline mustard aziridinium ion, monoethylcholine and monoethylcholine mustard aziridinium ion, the agents appeared to be equipotent as inhibitors of high-affinity uptake (K_i=2.63, 3.15 and 2.72 μm , respectively). Acetylcholine mustard aziridinium ion was less potent than these compounds (K_i= 27.8 μm ), but it was more potent than ethoxycholine and ethoxycholine mustard aziridinium ion (K_i= 500 and 403 μm ) as a blocker of choline transport. From study with these compounds it was concluded that the high-affinity choline transport mechanism shows specificity for hydroxylated compounds over those in which the same hydroxyl has been acetylated (10-fold) and that the carbonyl oxygen of the acetylated analogues is important, as its removal (to form the ethylether derivative) decreased affinity another 20-fold. The presence of an aziridinium ring on the quaternary nitrogen in place of two methyl groups did not affect the blocking of transport at 10 min of inhibitor preincubation and replacement of a methyl group on the nitrogen by an ethyl group did not alter affinity for the high-affinity carrier. The aziridinium ring on the nitrogen of the mustard analogues was important, however, in determining the extent of reversibility of the binding of these agents to the carrier protein. Choline transport was not restored by washing synaptosomes that were incubated with choline mustard aziridinium ion or monoethylcholine mustard aziridinium ion, but was readily obtained in washed synaptosomes preincubated with monoethylcholine, hemicholinium-3, or pyrrolcholine. The results indicate that the mustard analogues may be potent alkylators of the high-affinity choline carrier and thus, useful agents in monitoring acetylcholine turnover in systems where the carrier is blocked.