An Evaluation of Irreversible Inhibition of Synaptosomal High-Affinity Choline Transport by Choline Mustard Aziridinium Ion

Abstract: Choline mustard aziridinium is a potent, irreversible and selective blocker of sodium-dependent, high-affinity transport of choline into rat forebrain synaptosomes; it was found to be 30 times less potent against low-affinity transport of choline. The IC₅₀ value for high-affinity transport was 0.94 μM, compared to 29 μM for low-affinity uptake. The inhibitory action of choline mustard aziridinium ion on high-affinity transport of choline was graded with respect to time; a 12-fold increase in potency was obtained by increasing the inhibitor preincubation times from 1 to 30 min. Low concentrations of choline mustard aziridinium ion could produce significant blockade of choline carriers providing the exposure time was prolonged. The characteristics of the blockade of synaptosomal high-affinity choline transport by choline mustard aziridinium ion also changed depending upon preincubation time. The kinetics of inhibition of high-affinity choline transport by choline mustard aziridinium ion showed apparent competitive inhibition initially, followed by noncompetitive characteristics at longer preincubations with inhibitor. The rate of irreversible inhibition of carriers by this nitrogen mustard analogue would appear to be rapid; the rate constant was determined to be 5 × 10^?2 s^?1for micromolar concentrations of inhibitor. This action may preclude the transport of the mustard analogue into the nerve terminal, although initially some reversible binding with the carrier may result in the translocation of some choline mustard aziridinium ion into the presynaptic ending. The progressive alkylation of high-affinity carriers by the analogue could indicate the presence of excess carrier sites in the presynaptic membrane, or subpopulations of carriers in an inactive state in equilibrium with active carriers. A model is described for the inhibitory action of choline mustard aziridinium ion on synaptosomal high-affinity choline carriers.     

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.     

Inhibition of choline transport into human erythrocytes by choline mustard aziridinium ion.

Acetylcholine mustard aziridinium ion inhibited the transport of [3H]choline into human erythrocytes. Treatment of the erythrocytes with 1 X 10(-4) M tetraethylpyrophosphate prevented the inhibition of [3H]choline transport by acetylcholine mustard aziridinium ion. Hydrolyzed acetylcholine mustard aziridinium ion inhibited choline transport both in the presence and absence of 1 X 10(-4) M tetraethylpyrophosphate. The product of hydrolysis was equipotent with acetylcholine mustard in its ability to inhibit choline transport; incubation of this product with sodium thiosulfate prevented inhibition of choline transport thereby indicating the presence of an aziridinium ion. The hydrolysis product is likely to be choline mustard aziridinium ion. Results on the efflux of [3H]choline from erythrocytes in the presence of the proposed choline mustard aziridinium ion showed that the mustard moiety was transported into the red cells on the choline carrier. The rate of efflux of [3H]choline produced by choline mustard aziridinium ion was 55% of that produced by the same concentration of choline. It is concluded that acetylcholinesterase (EC 3.1.1.7) of red cells rapidly hydrolyzes acetylcholine mustard aziridinium ion to acetate and choline mustard aziridinium and the latter compound can act as a potent inhibitor of choline transport. This finding would indicate that the hemicholinium-like toxicity of acetylcholine mustard in the mouse is due to the formation of choline mustard aziridinium ion.     

Choline mustard: an irreversible ligand for use in studies of choline transport mechanisms at the cholinergic nerve terminal

It has been shown in our laboratory that choline mustard aziridinium ion is a potent and irreversible inhibitor of choline transport into rat brain synaptosomes; this compound showed selectivity for the sodium-dependent, high affinity carrier in that it was 30 times more potent as an inhibitor when compared with the effect on sodium-independent, low affinity choline uptake. In the present study, this mustard analogue did not inhibit synaptosomal uptake of 5-hydroxytryptamine, noradrenaline, or gamma-aminobutyric acid, thereby confirming further the specificity of this compound for the choline carrier. Studies of the effect of depolarization of the nerve terminals on the inactivation of choline carriers by choline mustard were performed. It was determined that alkylation of the carrier was significantly increased in nerve endings previously depolarized. The enhancing effect of depolarization on choline transport velocity and on the alkylation of choline carriers by choline mustard was dependent upon the presence of sodium in the external medium. Possible mechanisms for the enhanced inactivation of choline carriers by choline mustard aziridinium ion are proposed, and kinetic interactions of choline mustard with the high affinity choline carrier and with choline acetyltransferase are reviewed and discussed.     

Studies on the Alkylation of Choline Acetyltransferase by Choline Mustard Aziridinium Ion

Although a potent irreversible inhibitor of high-affinity choline transport in rat brain synaptosomes, choline mustard aziridinium ion (ChM Az) appeared to be a relatively weak inhibitor of choline acetyltransferase (ChAT) in rat brain homogenates, and evidence for irreversible binding of this compound to the enzyme had not been established. Accordingly, the irreversible inactivation of partially purified rat brain ChAT by ChM Az was studied. This compound is a rather weak inhibitor of the enzyme, with 50% inhibition of ChAT activity achieved following 30 min incubation at 37 degrees C with 0.6 mM ChM Az. This result indicates that although ChM Az has affinity for many nucleophiles there was little diluting effect of the inhibitor in the crude brain homogenate which could be attributed to such reactions (50% inhibition caused by 1.8 mM ChM Az following 10 min incubation). Although the initial binding of ChM Az to ChAT may be of a competitive nature, irreversible bond formation resulted. The time-dependent alkylation reaction conformed to pseudo-first-order kinetics with an observed forward rate constant (kobs) of 0.173 min-1; the half-time (t 1/2) for irreversible binding was about 4 min. The irreversible inactivation of ChAT by ChM Az would appear to be slower than the alkylation of high-affinity choline carriers in synaptosomes by this compound, and the relatively weak inhibitory action of ChM Az against either partially purified ChAT or ChAT activity in crude rat brain homogenates is in striking contrast to previous evidence that ChAT in intact synaptosomes was inhibited irreversibly by lower concentrations of the inhibitor.     

AF64A: An Active Site Directed Irreversible Inhibitor of Choline Acetyltransferase

Ethylcholine mustard aziridinium ion (AF64A, MEChMAz) has been proposed as a cholinergic neuron-specific neurotoxin. We report that in further studies on its mechanism of action incubation of the cholinergic neuroblastoma X glioma cell line, NG-108-15, with 100 microM AF64A resulted in a rapid decrease in cellular choline acetyltransferase (ChAT) activity which preceded cytotoxicity. Thus, a 60-85% decrease in ChAT activity was measured within 5 h of AF64A exposure, whereas cell lysis (measured as the release of the cytosolic enzyme lactate dehydrogenase into the medium) did not become apparent until 18 h of AF64A exposure. This led us to examine the effects of AF64A on partially purified ChAT. We report a concentration- and time-dependent inhibition of partially purified ChAT by AF64A that could not be reversed by dialysis but could be prevented by coincubation of the enzyme and AF64A with choline but not with acetyl-coenzyme A. We present kinetic evidence that choline and AF64A compete for the same site on the enzyme. In addition, thiosulfate, which inactivates the aziridinium ion, eliminated AF64A's capacity to inhibit the enzyme. AF64A also irreversibly inhibited partially purified choline kinase and acetylcholinesterase but not lactate dehydrogenase, alcohol dehydrogenase, carboxypeptidase A, or chymotrypsinogen, enzymes that do not use choline as a substrate or product. Thus, the data suggest that AF64A acts as an irreversible active site directed inhibitor of ChAT and possibly other enzymes recognizing choline.     

Presynaptic effect of the aziridinium ion of acetylcholine mustard (methyl-2-acetoxyethyl-2'-chloroethylamine) on the phrenic nerve--rat diaphragm preparation.

The rat diaphragm has been used to investigate the neuromuscular blocking action of acetylcholine mustard which yields a potent nicotinic agonist, an aziridinium ion, in aqueous medium. Evidence was obtained that the acetylcholine mustard aziridinium ion impaired neuromuscular activity when the phrenic nerve was stimulated and that the ion did not directly inhibit muscle contraction. Impairment of neuromuscular activity was characterized by a latent period and depended both on the concentration of aziridinium ion and the frequency of stimulation of the phrenic nerve. Elevated concentrations of Ca-2+ and choline changed the response of the rat diaphragm to the aziridinium ion, the former increasing the rate of development of neuromuscular block and the latter protecting against neuromuscular block. These results indicated that the aziridinium ion may act either at the site of choline uptake or have an effect on acetylcholine synthesis in the nerve ending and that impairment of neuromuscular transmission in the rat diaphragm involved the availability of acetylcholine. Similar results were obtained with acetylcholine mustard aziridinium ion subjected to alkaline hydrolysis. This substance is thought to be choline mustard aziridinium ion. Although difficult to prove with the rat diaphragm it is possible that acetylcholinesterase of this preparation could hydrolyze acetylcholine mustard aziridinium ion at the neurotransmitter site and the resultant choline mustard aziridinium ion would interfere with the uptake of choline and eventually prevent neuromuscular transmission. This hemicholinium-like hypothesis for the mechanism of action of choline mustard aziridinium ion is compatible with reported date for toxicity of acetylcholine mustard aziridinium ion in the mouse.     

Regulation of Rat Brain Synaptosomal [3H]Hemicholinium-3 Binding and [3H]Choline Transport Sites Following Exposure to Choline Mustard Aziridinium Ion

Abstract: Choline uptake by cholinergic nerve terminals is increased by depolarization; the literature suggests that this results from either the appearance of occult transporters or the increased activity of existing ones. The present experiments attempt to clarify the mechanism by which choline transport is regulated by testing if the preexposure of synaptosomes to choline mustard aziridinium ion prevents the stimulation-induced appearance of hemicholinium-3 binding sites and/or choline transport activity. Choline mustard inhibited irreversibly most of the “ground-state” (basal) high-affinity choline transport but only 50% of “ground-state” hemicholinium-3 binding sites. Exposure of both striatal and hippocampal synaptosomes to the mustard, before stimulation, inhibited K⁺-stimulated increases in choline transport and of [³H]hemicholinium-3 binding. We conclude that the mechanism by which choline transport is regulated involves the increased activity of a pool of transport sites that are occluded to hemicholinium-3 but are available to choline mustard aziridinium ion, and presumably to choline, before stimulation. However, the concentration of mustard needed to inhibit the stimulation-induced increase of [³H]hemicholinium-3 binding and choline transport was lower for striatal synaptosomes than for hippocampal synaptosomes. In the absence of extracellular Ca²⁺ or presence of high Mg²⁺ levels, the choline mustard did not prevent the appearance of extra striatal hemicholinium-3 binding sites. Also, high Mg²⁺ levels removed the ability of the mustard to inhibit K⁺-stimulated increases of either [³H]hemicholinium-3 binding or choline transport by hippocampal synaptosomes. In contrast, the preexposure of hippocampal synaptosomes to the mustard in the presence of a calcium ionophore (A23187) reduced the concentration of inhibitor needed to prevent the activation of [³H]hemicholinium-3 binding and choline uptake. Thus, we conclude that the ability of the choline mustard to alkylate the pool of choline transporters that are activated by stimulation appears dependent on the entry of extracellular Ca²⁺.     

Specificity of Ethylcholine Mustard Aziridinium as an Irreversible Inhibitor of Choline Transport in Cholinergic and Noncholinergic Tissue

The sensitivity of choline transport to inhibition by ethylcholine mustard aziridinium (ECMA) was studied in several tissues. Choline transport was found to be inhibited irreversibly by ECMA in guinea pig and rat synaptosomes but not inhibited in erythrocytes or kidney slices. If this finding can be extended to other tissues ECMA sensitivity may provide a simple criterion for identifying the choline carrier associated with cholinergic tissue.     

Synaptosomal "Membrane-Bound" Choline Acetyltransferase Is Most Sensitive to Inhibition by Choline Mustard

The objectives of the present study were to validate the presence of cytoplasmic and membrane-associated pools of choline acetyltransferase (ChAT) in rat brain synaptosomes, and to evaluate inhibition of these different forms of the enzyme by the nitrogen mustard analogue of choline, choline mustard aziridinium ion (ChM Az). The relative distribution of ChAT and lactate dehydrogenase (LDH) was followed in subfractions of synaptosomes to establish whether ChAT activity associated with salt-washed presynaptic membranes represents membrane-bound protein rather than cytosolic enzyme trapped within undisrupted synaptosomes or revesiculated membrane fragments. The percentage of total synaptosomal ChAT activity (14%) recovered in the final membrane pellet always exceeded that of LDH (6%), lending support to the hypothesis that much of the ChAT associated with the membranes was a membrane bound form of the enzyme. Incubation of purified synaptosomes with ChM Az led to irreversible inhibition of ChAT activity; this loss of enzyme activity could not be accounted for by lysis of nerve terminals during incubation in the presence of the mustard analogue. Subfractionation of the ChM Az-treated nerve terminals revealed that the membrane-bound form of ChAT was inhibited to the greatest extent, followed by the ionically membrane-associated enzyme, with the activity of the water-solubilized enzyme not differing significantly from control. Preparation of the synaptosomal ChAT subfractions from untreated nerve terminals prior to incubation with varying concentrations of ChM Az or naphthylvinylpyridine revealed that under these conditions water-solubilized, ionically membrane-associated, and detergent-solubilized membrane-bound pools of ChAT were not differentially inhibited by either compound.(ABSTRACT TRUNCATED AT 250 WORDS)     

Selective Presynaptic Cholinergic Neurotoxicity Following Intrahippocampal AF64A Injection in Rats

Compound AF64A, ethylcholine mustard aziridinium ion (0.4-8 nmol) was stereotaxically administered into rat dorsal hippocampus, and neurochemical changes were determined 5 days later. AF64A treatment, over an almost 10-fold dose range, resulted in a significant (up to 70%) decline in choline acetyltransferase activity. In the same tissue samples, Na+-dependent choline transport activity was also lowered, with most decreases ranging between 10 and 50% of controls; however, there was no significant correlation (r = 0.39) between these two parameters. Acetylcholinesterase activity was not affected by AF64A treatment when assayed by either histochemical or enzymatic methods. AF64A reduced acetylcholine levels by 43%, but did not alter norepinephrine content or serotonin uptake. These results demonstrate that AF64A can induce a specific, long-term reduction of cholinergic presynaptic biochemical markers in rat hippocampus. Thus, AF64A can serve as a useful new tool to study the cholinergic system and as an important agent to help develop animal models representing disorders of central cholinergic hypofunction.     

Affinity Labelling and Identification of the High-Affinity Choline Carrier from Synaptic Membranes of Torpedo Electromotor Nerve Terminals with [3H]Choline Mustard

The physiological mechanisms regulating activity of the sodium-dependent, high-affinity choline transporter and the molecular events in the translocation process remain unclear; the protein has not been purified or characterized biochemically. In the present study, [3H]choline mustard aziridinium ion [( 3H]ChM Az), a nitrogen mustard analogue of choline, bound irreversibly to presynaptic plasma membranes from Torpedo electric organ in a hemicholinium-sensitive, and sodium-, time-, and temperature-dependent manner. Specific binding of this ligand was greatest when it was incubated with membranes in the presence of sodium at 30 degrees C. Separation of the 3H-labelled membrane proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that most of the radiolabel was associated with a polypeptide of apparent molecular mass of approximately 42,000 daltons; labelling of this species was abolished in membranes incubated with ligand in the presence of HC-3. Two other 3H-labelled polypeptides were detected, with apparent molecular masses of approximately 58,000 and 90,000 daltons; radiolabelling of the former was also HC-3 sensitive. [3H]ChM Az may be a useful affinity ligand in the purification of the choline carrier from cholinergic neurons.     

High-affinity choline uptake in regions of rat brain and the effect of antidepressants.

The effect of tricyclic antidepressants, chlorpromazine, and some monoamine oxidase inhibitors on the accumulation of [14C]choline by crude synaptosomal (P2) fraction from different regions of rat brain (cortex, striatum, and hippocampus) was investigated. Analysis of choline uptake kinetics resulted in high- and low-affinity components with different Michaelis constants. All tricyclic antidepressants tested inhibited in a dose-dependent manner the high-affinity choline uptake in the three regions, amitriptyline being the most potent. The IC50 values correlated significantly with the relative potencies of imipramine congeners in binding to muscarinic receptors in the brain. Neither tranylcypromine nor pargyline in concentrations up to 0.1 mM had any effect on choline transport. Concentrations of tricyclic antidepressants effective in inhibiting the uptake of choline failed to influence significantly the activity of choline acetyltransferase in brain regions examined. The results suggest that the effect of imipramine congeners on high-affinity choline uptake may be reflected in the anticholinergic properties of these compounds.     

Ethylcholine mustard aziridinium ion lesions of the rat cortex result in retrograde degeneration of basal forebrain cholinergic neurons: Implications for animal models of neurodegenerative disease

Multiple injections of 2 nmols of cyclised ethylcholine mustard aziridinium ion (ECMA), a putative cholinergic neurotoxin, were made (unilaterally) into the cortical terminal field of cholinergic neurons projecting from the nucleus basalis of Meynert (NBM) in the rat basal forebrain. After 30 days, choline acetyltransferase enzymatic activity, a marker for cholinergic function, was significantly lowered in both ipsilateral cortex and NBM, and cholinergic cell bodies in the latter reduced in cross-sectional area, a spectrum of effects characteristic of retrograde degeneration of this pathway. These results are discussed in the context of neurodegenerative diseases affecting cholinergic function.     

Differential Effects of Nerve Growth Factor on Expression of Choline Acetyltransferase and Sodium-Coupled Choline Transport in Basal Forebrain Cholinergic Neurons in Culture

Abstract: Nerve growth factor (NGF) treatment of primary cultures of embryonic day 17 rat basal forebrain differentially altered activity of choline acetyltransferase (ChAT) and high-affinity choline transport; ChAT specific activity was increased by threefold in neurons grown in the presence of NGF for between 4 and 8 days, whereas high-affinity choline transport activity was not changed relative to control. Dose-response studies revealed that enhancement of neuronal ChAT activity occurred at low concentrations of NGF with an EC₅₀ of 7 ng/ml, with no enhancement of high-affinity choline transport observed at NGF concentrations up to 100 ng/ml. In addition, synthesis of acetylcholine (ACh) and ACh content in neurons grown in the presence of NGF for up to 6 days was increased significantly compared with controls. These results suggest that regulation of ACh synthesis in primary cultures of basal forebrain neurons is not limited by provision of choline by the high-affinity choline transport system and that increased ChAT activity in the presence of NGF without a concomitant increase in high-affinity choline transport is sufficient to increase ACh synthesis. This further suggests that intracellular pools of choline, which do not normally serve as substrate for ACh synthesis, may be made available for ACh synthesis in the presence of NGF.     

Kinetics of irreversible inhibition of choline transport in synaptosomes by ethylcholine mustard aziridinium

Summary Ethylcholine mustard aziridinium (ECMA) inhibits choline transport in synaptosomes at a half-maximal concentration of about 20 m. The rate of inhibition falls off rapidly after 10 min and the concentration dependency reaches a plateau at about 100 m. The inhibition is not removed by washing the synaptosomes, and choline and hemicholinium-3 protect the carrier against attack by the mustard. Choline efflux, particularly that stimulated by choline in the medium (transactivation) is also inhibited by the aziridinium compound. Similarly choline influx activated by preloaded internal choline is inhibited by ECMA. The mustard can enter the synaptosomes in an active form but most of the carrier is alkylated when facing the outside. Prior depolarization of the synaptosomes causes an increase in the rate of inhibition by ECMA which is proportionally about the same as the increase in choline influx also caused by depolarization. At low ECMA concentrations the rate of inhibition is that of a first-order reaction with the carrier but at high ECMA concentrations the translocation of the carrier to the outward-facing conformation controls the rate of inhibition. Using a model of choline transport with some simplifying assumptions it is possible to estimate the amount of carrier; cholinergic synaptosomes carry about six times the concentration of carrier found in noncholinergic ones. In noncholinergic synaptosomes the carrier faces predominately out, the reverse in cholinergic ones. The rate constant of carrier translocation is increased by combination with choline some six- to sevenfold to about 3.5 min^–1. The rate constant of ECMA attack on the carrier is about 440m ^–1 sec^–1.     

Differentiation Effects of Ciliary Neurotrophic Factor on Human Neuroblastoma Cells

Abstract: In the human neuroblastoma cell line LA-N-2, recombinant rat ciliary neurotrophic factor (CNTF) induced neurite growth and cholinergic differentiation that were both half-maximally saturated at <100 p M of the neurokine, but was not required for cell survival in serum-free conditions over a 13-day period. CNTF markedly stimulated choline acetyltransferase activity and acetylcholine synthesis, whereas high-affinity choline transport was only slightly enhanced and acetylcholinesterase activity was unchanged. Leukemia inhibitory factor had effects identical to CNTF on neurite growth and choline acetyltransferase activity, but interleukin 6 had no effect. Radioiodinated CNTF binding and affinity cross-linking studies were consistent with tripartite receptor activation as a mediator of the observed biological effects.     

Reconstitution of carrier-mediated choline transport in proteoliposomes prepared from presynaptic membranes oftorpedo electric organ,and its internal and external ionic requirements

Summary Proteoliposomes made by a butanol-sonication technique from electric organ presynaptic membranes showed choline transport activity. In contrast to intact nerve terminals, the uptake of choline was dissociated from its conversion to acetylcholine in this preparation. The kinetics of choline uptake by proteoliposomes was best described by two Michaelis-Menten components. At a low concentration of choline, uptake was inhibited by hemicholinium-3 and required external Na⁺ and, thus, closely resembled high-affinity choline uptake by intact cholinergic nerve terminals. Choline transport could be driven by the Na⁺ gradient and by the transmembrane potential (inside negative) but did not directly require ATP. External Cl^–, but not a Cl^– gradient, was needed for choline transport activity. It is suggested that internal K⁺ plays a role in the retention of choline inside the proteoliposome. Proteoliposomes should prove a useful tool for both biochemical and functional studies of the highaffinity choline carrier.Abbreviations ACh acetylcholine - HC-3 hemicholinium-3 - ChAT choline acetyltransferase     

Effect of ethyl choline mustard on choline dehydrogenase and other enzymes of choline metabolism

The effect of ethyl choline mustard (ECMA), and effective irreversible inhibitor of choline transport, was investigated on the enzymes of choline metabolism. ECMA at concentrations of 50 microM hardly affected choline acetyltransferase and caused only a 20% inhibition of choline kinase at a concentration of 1 mM. However, the mustard was an extremely effective inhibitor of choline dehydrogenase, producing 50% inhibition at concentrations of 6 microM. The inhibition was prevented by incubation in the presence of choline or by prior reaction of the mustard with thiosulphate. Separation of the components of the ECMA solution on TLC suggested that only the compound with an aziridine ring was an effective inhibitor of choline dehydrogenase. The inhibition was resistant to the washing out of excess unreacted mustard. The rate constant of inhibition was 395 M-1 X S-1. By the use of [3H]ECMA a single polypeptide in the enzyme preparation having a MW of 67,000 was labelled. The labelling was thiosulphate-sensitive and prevented by incubation with choline. It is concluded that ECMA is an irreversible inhibitor of choline dehydrogenase. It is at least as effective an inhibitor of choline dehydrogenase as of the choline transport system, although it does not appreciably inhibit choline acetyltransferase or choline kinase in the micromolar range.