Eur. J. Biochem. 22 (1971) 563-572

Human Placental Choline Acetyltransferase

Radiometric Assay, Inhibition by Analogues of Choline and Acetylcholine, and Isotopic Exchange between Choline and Acetylcholine

David MORRIS and Darshan S. GREWAAL

Department of Physiology and Biochemistry, University of Southampton

(Received April 29/July 15, 1971)

A radiometric procedure for estimation of choline acetyltransferase using [3H]acetylphosphate, CoA and phosphate acetyltransferase as a source of [3H]acetyl-CoA is described.

Analogues of choline in which the hydroxyl group is replaced by chlorine are weak competitive inhibitors of choline acetyltransferase. The results indicate that the hydroxyl group of choline is important in the binding of this substrate.

Haloacetylcholines are potent inhibitors of choline acetyltransferase. The inhibition is short- lived and may be correlated with the short half-life of the inhibitors under the conditions used.

Isotopic exchange between [14C]choline and acetylcholine is completely dependent on the presence of CoA and indicates that a sequential rather than a “ping-pong” mechanism is operat- ing. The exchange is strongly inhibited by chloroacetylcholine.

The development of radiometric assay proce- dures [l-51 for choline acetyltransferase, which catalyses the reversible transfer of acetyl groups from acetyl-CoA to choline, has led to an increased interest in the properties of this enzyme. The discovery of potent specific inhibitors should be of great value in the elucidation of the mechanism and also in physiological studies in the nervous system. Reports of inhibition by a group of styryl-pyridine compounds [6-81, bromoacetyl-CoA [9] and by a bromoketone [lo] have appeared recently.

A kinetic study on human placental choline acetyltransferase led Schuberth [ l l ] to propose a “ping-pong” mechanism for this enzyme whereas a similar study on the rat brain enzyme [12] suggested a sequential mechanism.

The present paper describes a new and convenient radiometric assay, an investigation of the inhibition of human placental choline acetyltransferase by analogues of choline and of acetylcholine and an iso- topic exchange study designed to distinguish between the “ping-pong” and sequential mechanisms for the pla.centa1 enzyme. Brief reports [13,14] of some of this work have appeared.

MATERIALS AND METHODS

Enzymes Human placental choline acetyltransferase was

extracted from immature placentae (18-28 weeks) and partially purified (5 to 10-fold) by (NH,),SO, precipitation [15] to a specific activity of 72 nmoles acetylcholine x mi+ x mg protein-l. For isotope- exchange studies the enzyme was further purified by (NH,),SO, fractionation to a specific activity of 192 nmoles acetylcholine x m i r l x mg protein-l and was stored in 0.2 M phosphate-citrate buffer, pH 7.0 [16] a t - 18 “C.

Rabbit brain choline acetyltransferase was ex- tracted from acetone-dried powders as described by Hemsworth and Morris [17]. This enzyme was used as a standard preparation to control the incubation and assay systems.

Phosphate acetyltransferase was prepared from dried cells of Clostridium kluyverii (Worthington Biochemical Corp., Freehold, N.J., U.S.A.) as de- scribed by Nordenfelt [18].

Citrate synthase was purchased from Boehringer Mannheim GmbH (Mannheim, Germany).

Non-Standard Abbreviations. Chlorocholine, 2-chloro- ethvltrimethvlammonium : reineckate, ammonium-tetrathio- Chemiculs cyinodiammonochromate NH,Cr(NH&(SCN),. CoA (85- 95 *Ilo pure), 5,5‘-dithiobis-(2-nitrobenzo- phate acetyltransferase (EC 2.3.1.8); citrate synthase (EC ic acid), and x8 4.1.3.7). (chloride form, 200-400 mesh) were obtained from

Enzymes. Choline acetyltransferase (EC 2.3.1.6) ; phos-

564 Human Placental Choline Acetyltransferase Eur. J. Biochem.

the Sigma Chemical Co. (St. Louis, Miss., U.S.A.) and N,N-dimethyl- and N,N-diethyl-2-chloroethyl- amine-HC1 from Koch-Light Laboratories Ltd. (Bucks., U.K.). Triethylcholine chloride was a gift from Dr. C. 0. Hebb. All other standard chemicals were either of AnalaR quality or of the highest purity available.

[Me-14C]Choline chloride (54 mCi/mmole) and [,H]acetic anhydride (500 mCilmmole, both pure and as a 10.5°/, s o h in benzene) were purchased from the Radiochemical Centre (Amersham, Bucks., U.K.). Butyl-PBD scintillator was obtained from Ciba (A.R.L.) Ltd. (Duxford, Cambs., U.K.).

Chemical Preparations Acetylphosphate. Unlabelled acetylphosphate was

prepared by the method of Avison [19] as the di- lithium salt. The percentage purity on a weight basis was 800/, as determined by the method of Lipmann and Tuttle [20]. For the synthesis of [,H]acetyl- phosphate the procedure was modified as follows.

Pyridine (2.42 ml) and 0.125 M K,HPO, (10 ml) were mixed and cooled to 0 "C. [,H]Acetic anhydride (25 mCi, 10.5O/, s o h in benzene) was diluted with a similar solution (2.20 ml) of unlabelled acetic an- hydride and added to the phosphate-pyridine mix- ture over a period of 30 min a t 0 "C with rapid stirring. After a further hour a t 0 "C, 4.1 M LiOH (0.925 ml) and ethanol (90 ml) was added slowly with stirring. The product was washed a t the centrifuge with cold absolute ethanol (3 x 10 ml), Na-dried ether ( 3 ~ 10 ml) and dried in wacuo over P,O, at -10°C. The product was 80°/, pure by hydroxylamine assay [20] and had a specific radioactivity of 5.6 pCi/pmole. The latter value was determined from the radioactivity asso- ciated with the acetylcholine (estimated by bioassay) produced in the acetyl-CoA regenerating system (see below) for choline acetyltransferase estimation. For general use the acetylphosphate was diluted with unlabelled material to an appropriate specific ac- tivity.

Acetyl-CoA. Unlabelled acetyl-CoA was synthe- sized by the method of Simon and Shemin[21], assayed with citrate synthase and oxaloacetate [22] and stored under nitrogen at - 18 "C. [3H]Acetyl-CoA was prepared in a similar manner except for two modifications. First, thioglycollate was omitted from the reaction mixture, and second, the labelled acetic acid formed in the reaction was removed by repeated extraction with ether at pH 2 until the radioactivity in the ether was reduced to less than 1 of the total. The pH was then adjusted to pH 5 with 1 M KHCO, and the solution stored a t - 18 "C as before. The specific radioactivity was 2.5 pcilpmole. Before use the material was diluted appropriately with unlabelled acetyl-CoA.

Table 1.2-Chlorocholine and i ts N-ethyl analogues N,N-Dimethyl- or N,N-diethyl-2-chloroethylamine-HC1 was quaternized with the appropriate alkyl iodide as described in the Methods. The purity of the products was estimated by

volumetric analysis of their iodide content

Compound m.p. Purity

"C "lo

ClCH,CH,N(CH,), I- 210 98

ClCH,CH,N( CH,),C,H, I- 179 98

ClCH,CH,N(C,H,),CH, I- 170 96

+ + + +

CICH2CH,N(C,H,), I- 190 95

Chlorocholine and its N-Ethyl Analogues. N , N - Dimethyl- or N,N-diethyl-2-chloroethylamine-HCI (25 mmoles) was dissolved in water and neutralized with NaHCO,. The liberated free amine was extracted into ether, dried over anhydrous Na,SO, and the ether evaporated. The residue was dissolved in dry acetone (50 ml) and quaternized with the appropriate alkyl iodide (1.1 equivalents) by refluxing for 2-4 h. The product was precipitated with dry ether and recrystallized from ethanollether. The purity of the compounds on the basis of iodide analysis was 95-9S0/, (see Table 1).

Halogenated Acetylcholines. Chloro-, bromo- and iodoacetyl-choline were prepared essentially as de- scribed by Chiou and Sastry [23]. Chloroacetyltri- ethylcholine was similarly prepared and gave a single, hydroxylamine-FeC1, positive, spot on thin-layer chromatography (see below).

3-Bromoacetonyltrimethy2ammonium Bromide. This bromoketone analogue of acetylcholine was prepared from 1,3-dibromoacetone and trimethylamine as described by Persson et al. [lo].

Desulpho- CoA. CoA was desulphurized with Raney nickel and the product was isolated by DEAE-cellulose chromatography as described by Chase et al. [24].

Enzyme Assay Systems Choline Acetyltransferase. Two methods of estima-

tion were employed. In the first procedure a coupled enzyme system using phosphate acetyltransferase, CoA and acetylphosphate as an acetyl-CoA regenerat- ing system was used. The complete medium contained, in pmoles/ml of final solution: choline, 5-10; KCl, 160; CoA, 0.15 ; acetylphosphate, 7 ; cysteine-HC1 (neutralized with 1 N KOH), 23; eserine sulphate, 0.13 ; phosphate-citrate buffer pH 7.0, 40; phosphate acetyltransferase, 0.25 mg ; and choline acetyltrans- ferase, 20 pl (0.4 mg protein). The medium was pre- incubated for 10 min a t 39 "C to allow formation of acetyl-CoA before addition of choline acetyltrans-

Vol. 22, No. 4,1971 D. MoRRIs,.and D. S . GREWAAL 565

ferase. After a further period at 39 "C (usually 15 min) the reaction was stopped with either 0.5 ml of 0.3 N HC1 (prior to biological assay) or with 1 N HC10, (0.1 ml) for radiometric assay.

In the second procedure, using synthetic acetyl- CoA, the medium contained, in pmoles/ml of final solution: choline chloride, 10 ; eserine sulphate, 0.13 ; phosphate-citrate buffer pH 7.5, 40 ; acetyl-CoA, 0.40; NaCN, 40 or NaC1, 500, and choline acetyl- transferase, 2Op1 (0.4mg protein). There was no preincubation period and the incubation was stopped as described above.

In both procedures enzyme inactivated"either by heat or by HC10, was routinely incubated as a control.

Phosphate Acetyltransferase. The activity of this enzyme in the presence and absence of chloroacetyl- choline was estimated by measurement of the rate of arsenolysis of acetylphosphate [25] at 39 "C. The medium contained all the normal constituents of the acetyl-CoA regenerating system (see above) with the addition of 30mM sodium arsenate. Samples were withdrawn at various times and their acetyl- phosphate content estimated [ZO].

Assay of Acetylcholine. After non-radioactive incubations solutions were prepared for biological estimation and assayed against standard acetyl- choline chloride on the eserinized frog rectus abdo- minis muscle, using the precautions described by Feldberg [26] to control the effect of sensitizing sub- stances other than acetylcholine on the rectus muscle.

Acidified radioactive incubates were adjusted to pH 6.5, diluted to 3.5 ml with water and applied to a tightly-packed column (1 x 13 cm) of Dowex 1 X 8 in the chloride form ; the column was prepared with barely moist resin which was tamped down with a glass rod to give the required bed height. This procedure was used to avoid dilution of the radio- active acetylcholine so that its concentration in the effluent remained essentially the same as in the applied solution. 0.1 mi samples of the effluent were added to vials containing 5 ml of Butyl-PBD (8 g/1 in toluene) plus 1.72 ml of methanol and the radioactivity was estimated in a Beckman scintillation spectrometer at an efficiency of 39O/,. All radioactive solutions were estimated in this manner.

Isotope Exchnge. The incubation medium con- tained, in pmoles/ml of final solution : acetylcholine chloride, 10; NaC1,500; [l*C]choline, 0.00925 (0.5 pCi) ; eserine sulphate, 0.13 ; phosphate-citrate buffer pH 7.0, 40; CoA, 0.15, and choline acetyltransferase, 0.15 ml. Incubations a t 39 "C were begun with the addition of enzyme and stopped by the addition of 0.2 N HC10, (1 ml). Unlabelled choline chloride (10 pmoles) was added as a carrier and the incubate was allowed to stand for 1 h at 0 "C. The precipitated protein was removed by centrifugation (800 x g for 5 min), the pellet washed at the centrifuge with 0.1N HC10, and the supernatants combined. The

choline and acetylcholine were isolated as their reineckate salts and converted to the soluble chlorides as described by Hemsworth and Morris [17]. The final volume was adjusted to 0.5ml with 95O/, acetone. Samples (5-15 p1) were applied to Whatman No. 1 chromatography paper ( 1 0 0 ~ 10 cm) and sub- jected to electrophoresis (7 kV, 1 mA) for 1 h on a cooled flat bed machine (Locarte Co. Ltd., London) using 0.1 M pyridine-acetic acid buffer pH 4.0. The choline and acetylcholine were well separated (migrations of 42 cm and 36 cm, respectively) and were visualized by spraying lightly with Dragendorff's reagent [27]. The spots were cut out, snipped into vials containing the Butyl-PBDlmethanol scintillator (see above) and counted a t an efficiency of 850/,.

Thin- Layer Chromatography Cellulose thin-layer chromatography plates were

run in butanol - ethanol - acetic acid-water, (8: 2 : 1 :3, by vol.) [28] and developed either with Dragendorffs reagent or with Hestrin's reagent [29].

Kinetic Studies Initial rates of acetylcholine synthesis were ob-

tained in 15 min incubations. The effect of inhibitors was investigated both after preincubation and on simultaneous addition with substrates. K , and Ki values were derived graphically by the method of Lineweaver and Burk [30].

RESULTS INCUBATION AND ASSAY SYSTEMS

Incubation Conditions Until recently assays using the acetyl-CoA

regenerating system have given higher rates of acetyl- choline synthesis than systems employing synthetic acetyl-CoA [25]. This problem has now been solved for the choline acetyltransferase from certain species [31] by reducing the thiol content of the medium, but the placental enzyme has only given maximum rates with synthetic acetyl-CoA in the presence of NaCN [32]. Table 2 shows that rates of acetylcholine synthesis equal to that found in the acetyl-CoA re- generating medium are obtained when 0.5M NaCl is present. Bovine plasma albumin, added in the procedure of McCaman and Hunt [ 11, is not necessary under these conditions.

Radiometric Assay of Acetylcholine Table 3 shows that when acetylphosphate alone

or the products of a phosphate acetyltransferase in- cubation plus inactivated choline acetyltransferase were applied to a Dowex 1 column, radioactivity associated with acetylphosphate, acetyl-CoA or

566 Human Placental Choline Acetyltraneferase Eur. J. Biochem.

Table 2. Effect of incubation wnditwnson acetylcholine synthsis Incubations in the synthetic acetyl-CoA medium were per- formed for 15 min with the modifications described below. Rates of acetylcholine synthesis, estimated by bioassay, were compared with that given by the acetyl-CoA regenerat-

ing system. For details see Methods

Acetylcholine synthesized

nmoles/l5 min

Incubation conditions

Regenerating system 145 Synthetic acetyl-CoA system

no additions 99 + 40 mM NaCN 142 + 0.5 M NaCl 144 + 0.5 M NaCl + bovine plasma albumin (0.05O/,) 144

Table 3. Separation of [3H]acetylchZine from other radioactive in.cuba.tion products by don-exchnge chromatography

For details see Methods. Various components of the acetyl- CoA regenerating medium for choline acetyltransferase estimation were incubated for 15 min at 39 "C, subjected to Dowex-1 chromatography and the radioactivity in the column effluents determined. [3H]Acetylphosphate (7 pmoles, 1.83 x lo6 counts/min) wm present in each incubation. In two experiments with the complete system the ef5uents were rechromatographed on a second column either directly or after treatment with NaOH to hydrolyze the acetylcholine

present

Radioactivity in column effluents Conditions

Column1 Column2

counts/min - Acetylphosphate 176

Complete system with inactiv- ated choline acetyltransferase 105 -

Complete system 22 020 22 600 with NaOH treatment of first effluent 23 800 0

acetate (derived from acetylphosphate by hydrolysis) was retained on the column. On the other hand, after incubations in which acetylcholine had been formed, radioactivity was present in the column effluents and could be recovered after passage through a second column. I n addition, if the ef0uent from the fist column was treated with NaOH to hydrolyze the acetylcholine present, then all the radioactivity was subsequently retained by the second column.

Fig. 1 shows the relationship between acetyl- choline synthesized and incubation time. A linear rate was maintained over a period of 30 min with good agreement between the two assay procedures. In other experiments linear rates were obtained for up to 60 min. Table 4 illustrates the relationship between the bioassay and radiometric assays in experiments where the amount of enzyme was varied. Both sys- tems gave an approximately linear response to a

0.40 - 2 0.30 - - 2 Z i -

0.20 - .- - 0 L 0

I - c

J 0.10 -

Incubation time (rnin)

Fig. 1. Time course of acetylcholine synthesis estimated radw- metrically and by biomsay. Choline acetyltransferase WM incubated in the acetyl-CoA regenerating medium containing [aH]acetylphosphate at 39 "C (for details see Materials and

Methods). Radiometric assay (0) ; bioassay (e)

Table 4. Comparison of rad imtr i c and biological msay of acetylcholine

Different concentrations of choline acetyltransferase were incubated in the acetyl-CoA regenerating medium containing [3H]acetylphosphate for 15 min at 39 "C. The acetylcholine produced was estimated radiometrically, by direct bioaseay and by bioassay of the ion-exchange column effluent. For

details see Methods

Acetylcholine synthesized Choline

acetyltransferase xdometric Bioassay Bioassay on assay effluents

ml pmoles pmoles -ole8

0.01 0.151 0.145 0.138 0.02 0.330 0.297 0.302 0.04 0.584 0.632 0.610 0.10 1.592 1.515 1.581

10-fold range of enzyme concentration and the varia- tion between them was loo/, or less. Good agreement was found between bioassay values obtained either directly after incubation or from column effluents, thus confirming the previous finding that little or no acetylcholine was retained by the columns.

INHIBITION STUDIES

Chlorocholine and Its N-Ethyl Analogues This group of compounds were poor inhibitors of

choline acetyltransferase. It wi l l be seen from Table 6 that chlorocholine (40 &f) gave only 30°/, inhibition a t a low choline concentration (2mM). Successive substitution with ethyl groups reduced the inhibi- tion to a very low level. The competitive nature of the inhibitions is shown for some of the inhibitors in Fig2 and the approximate Ki values are given in Table 6. In order to compare the relative importance (for inhibition) of the loss of the hydroxyl group (as in chlorocholine) with that of the charged N moiety,

Vo1.22. N0.4,1971 D. MORRIS and D. S. GBEWAAL 567

Table 5. Inhibition of choline acetyltranaferase by 2-chloro- choline and its N-ethyl analogues

2-Chlorocholine, and its analogues in which the N-methyl groups had been successively replaced with N-ethyl moieties, were incubated in the acetyl-CoA regenerating medium con- taining 2 mM choline. The acetylcholine synthesized during a 15 min incubation was estimated by bioassay. For details

see Methods

Acetylcholine synthesized Inhibition Inhibitor (40 mM)

nmoles O/.

Control 204 -

+ ClCH,CH,N(CH,), 145 29

Table 6. Kc values of choline analogues The values, derived from double-reciprocal plota of initial rates of acetylcholine synthesis against choline concentra- tion obtained in the presence of a 40 mM concentration of the various inhibitors (see Fig.2), were obtained using the expression :

slope = + (1 + g) Inhibitor EX

mM

+ ClCH,CH,N(CH,), 34

+ ClCH,CH,N( CH,),C,H, 87

+ ClCH,CH,N(C,H,),CH, 250

ClCH,CH,N( C,HJ, 250

177 13

+ + C1CH,CH,N(C,H5),CH3 182 10

ClCH,CH,N(C,H,), 196 4 HOCH,CH,N(CH,), 54 +

HOCH,CH,N(C,H,), 95

1 .o 2 .o 3.0 4 .O l/[Choline](mM')

Fig.2. Double recipocul plots of inhibition of choline acetyl- transferme by choline analogues. choline acetyltransferase was incubated with the various inhibitors (40mM) for 15 min a t 39 "C in the acetyl-CoA regenerating medium (for details see Materials and Methods). The acetylcholine produced was estimated by bioassay. No inhibitor (0); diethylaminoethanol (0 ) ; 2-chloroethyldimethylethyl-ammo- nium ( A ) ; dimethylaminoethanol (A) ; 2-chloroethyltrimethyl-

ammonium (0 )

dimethyl- and diethylaminoethanol were examined as inhibitors under the same conditions; 27O/, and 401, inhibiton, respectively, was obtained under the conditions described in Table 5. The possibility that the inhibition obtained with dimethylaminoethanol was the result of its own acetylation limiting the availability of acetyl-CoA for acetylcholine synthesis was rendered very unlikely in experiments in which the acetylation of this analogue was studied. Although acetylation did occur, as shown by the

association of radioactivity with authentic dimethyl- aminoethylacetate (added as a carrier a t the end of a radioactive incubation) on thin-layer chromatog- raphy, the rate was very low; kinetic experiments gave a V of 2.7 nmoles/min per 20 pl enzyme and a Km of 20 mM. The acetyl-CoA regenerating system is capable of producing linear rates of ester synthesis of up to 46 nmoleslmin [15].

No acetylation of diethylaminoethanol could be detected under the above conditions.

Halogenated Acetylcholine Derivatives Table 7 shows that, in contrast to the halogenated

choline analogues, the halogenated acetylcholine analogues were very potent inhibitors of choline acetyltransferase. Inhibition was concentration- dependent, chloroacetylcholine being the most po- tent inhibitor. Since the acetyl-CoA regenerating incubating system was used in these experiments it was possible that the results could have been wholly or partly due to inhibition of acetyl-CoA formation. Table 8 shows that 5 mM chloroacetylcholine had no effect on phosphate acetyltransferase activity its estimated by the rate of arsenolysis of acetylphos- phate. That the observed inhibition of acetylcholine synthesis was solely due to interaction ofthe inhibitors with choline acetyltransferase was confirmed in further experiments using the synthetic [3H]acetyl- CoA system (Table 9).

The effect of chloroacetylcholine was compared with that of the alkylating agents iodoacetate, chloroacetate and iodoacetamide. Table 10 shows that none of these compounds produced a significant inhibition when preincubated at 2 mM concentra- tion with the enzyme at room temperature for

568 Human Placental Choline Acetyltransferase Eur. J. Biochem.

Table 7. Inhibition of choline acetyltransferase by halogenated derivatives of acetylcholine

Various concentration of the inhibitors were incubated with choline acetyltransferase in the acetyl-CoA regenerating medium for 15 min a t 39 "C (for details see Methods). The acetylcholine produced was assayed radiometrically and compared to that synthesized in the absence of inhibitor

Inhibitor Concentration Inhibition

@Sf Qlo

None - -

Chloroacetylcholine 20 65 40 78 62.5 92

125 89 Bromoacetylcholine 62.5 70

40 40 62.5 56

Iodoacetylcholine 20 37

Table 8. Phosphute acetyltransferase activity in the presence of chloroacetylcholine

Enzyme activity in the presence of 5 mM chloroacetylcholine was estimated by measurement of the rate of arsenolysis of acetylphosphate at 39 "C (see Methods). Samples were with- drawn a t various times and their remaining acetylphosphate content compared to that in control incubations without

inhibitor and in the presence of buffer alone

Acetvlnhosohate " - - Controls Chloroacetyl-

Incubation time

choline Buffer alone No inhibitor

min pmoles pmoles pmolea

0 0.95 0.97 0.97 5 0.95 0.60 0.60

10 0.95 0.45 0.45 15 0.92 0.35 0.35 25 0.90 0.20 0.20

Table 9. Inhibition of choline acetyltransferase by chloroacetyl- choline in the synthetic acetyl-CoA medium

Various concentrations of chloroacetylcholine were incubated with choline acetyltransferase in the synthetic [3H]acetyl-CoA medium for 15 min a t 39 "C (for details see Methods). The

acetylcholine produced was estimated radiometrically

Chloroacetylcholine Acetylchoiine synthesized Inhibition

df 0 7.8

15.6 31.2 62.5

nmoles "lo 116 0 41 65 25 78 16 85 14 88

15 min; relative to the appropriate control, 2 mM iodoacetate also failed to inhibit the enzyme when preincubated a t 4 "C for 20 h. Furthermore, alkali- treated chloroacetylcholine (30 mM) did not produce any inhibition.

Table 10. Effect of iodoacetate, chloroacetate and wdoacetami.de on choline acetyltransferase activity

Choline acetyltransferase activity was estimated in the acetyl-CoA regenerating medium after preincubation with the alkylating agents (2 mM) for 15 min a t room temperature (for details see Methods). Iodoacetate was also preincubated with the enzyme for 20 h a t 4 "C. The ACh synthesized was

estimated radiometrically

Inhibitor Preincubation conditions ~$~~~~ nmoles

None 15 rnin a t room temp. 250 Iodoacetamide 247 Iodoacetate 287 Chloroacetate 266

None 20 h a t 4°C 240 Iodoacetate 235

Table 11. Comparison of the effect of preincwbation and dialysis on inhibition by chloroacetylcholine with that given by

2-bromoacetonyltrimethylammonium (bromoketone) Choline acetyltransferase activity was estimated in the radioactive acetyl-CoA regenerating system either imme- diately following addition of inhibitor, or after preincubation at 4 "C for 1 h or overnight. For incubation details see Nethods. The effect of overnight dialysis against 0 .2M phosphate-citrate buffer pH 7.0 on the inhibition was examined. Dialysis was commenced after a 1 h preincubation with chloroacetylcholine but bromoketone was added a t the beginning of dialysis. The acetylcholine produced was

estimated radiometrically

Inhibitor Concn bation Dialysis t;E:;i Inhibition Preincu-

mM

Control Control Chloroacetyl-

choline 0.0625 0.0625 0.0625

Bromoketone 0.25 0.50 0.50 0.50 5.0 5.0

- - - overnight

- - i h -

1 h overnight

l h - overnight -

- overnight overnight -

- overnight

- -

nmoles

196 186

21 25

168 192 110

5 17 0 0

~ _ _ _ _ _ _

9 1 ~

- 4

89 87 10 2 44 97 91

100 100

The above results suggested that, in contrast to the haloacetates, the haloacetylcholine compounds might be alkylating the enzyme by virtue of their substrate-analogue properties, a possibility supported by the observation that the inhibition was not readily prevented by very high (60 mM) choline concentra- tions. Overnight dialysis, however, of inhibited en- zyme resulted in almost complete reactivation relative to the appropriate control (Table 11). This result was in marked contrast to that obtained with S-bromo- acetonyltrimethylammonium (bromoketone) where no reactivation occurred on overnight dialysis. This inhibitor required a concentration of 5mM for

V01.22, No.4,1971 D. MORRIS and D. S. GREWAAL 569

10 20 30 40 50 60 80 Incubation time (min)

Fig.3. Time course of chline acetyltransferase inhibition by chloroacetylcholine. Choline acetyltransferase activity was estimated radiometrically. No inhibitor (0); 0.04 mM chloroacetylcholine ( 0 ) ; 0.04 mM chloroacetylcholine a t time zero with further additions (40pmoles) a t 5, 10 and

15 min (A)

5 10 15 XI 25 30 Incubation time (rnin)

Fig.4. Stability of haloacetylcholines. Chloro-, bromo- and iodoacetylcholine (4 mM), respectively, were incubated in 0.04 M phosphate-citrate buffer pH 7.0, a t 39 "C. Samples were removed a t various times and their residual ester content estimated using the Hestrin [33] procedure. Chloro- acetylcholine (0) ; bromoacetylcholine (0 ) ; iodoacetylcholine

(A)

maximum inhibition and was only effective after preincubation with the enzyme for 1 h a t 0 "C.

A time course of chloroacetylcholine inhibition revealed (Fig. 3) that inhibition was maximal a t very short incubation times and that activity subse- quently returned. After 20 min the rate of acetyl- choline synthesis was almost equal to that of the 38 Eur. J. Biochem., Vol.22

I I 1 1.0 2.0 3.0 4.0 1.0 2.0 3.0 4.0

l/[Choline] (mM-')

- 2 O r ,.

1.0 2.0 3.0 4.0 l/[Choline] (mM-')

Fig.5. Double reciprocal plots of inhibition of choline acetyl- transferme by haloacetylcholines. Initial rates of acetylcholine synthesis were obtained using the radioactive acetyl-CoA regenerating system in 15 min incubations. No inhibitor (0); (A) chloroacetylcholine, 2.5 pM (o), 5.0 pM ( A ) ; (B) bromo- acetylcholine, 5.0 pM ( O ) , 10 pM ( A ) ; (C) iodoacetylcholine,

10 PM ( 0 )

control. Further additions of inhibitor a t 5, 10 and 15 min prolonged the inhibition but after about 45 min the rate again returned almost to normal.

The stability of the inhibitors in phosphate- citrate buffer, pH 7, a t 39 "C was examined using Hestrin's hydroxylamine assay [33]. The results, shown in Fig. 4, indicate that chloro- and bromoacetyl- choline were about 50°/, hydrolyzed in 15min, whereas iodoacetylcholine was considerably more stable ; acetylcholine was not hydrolyzed a t all under these conditions.

Double reciprocal plots of initial rates of acetyl- choline synthesis a t a range of choline concentrations obtained in the presence of fixed concentrations of inhibitor are shown in Fig. 5. The plots indicate either uncompetitive or non-competitive inhibition with respect to choline, depending on the inhibitor employ- ed. Average Ki values derived from a number of experiments were 1.7 pM, 5.9 pM, and 8.7 pM for chloro-, bromo- and iodoacetylcholine, respectively. In similar experiments chloroacetylcholine was also found to inhibit uncompetitively with respect to acetyl-CoA, with a Ki of 3.3 pM. The Ki values must be regarded as overestimates because of the con- tinuing hydrolysis of the inhibitors during incuba- tion.

Chloroacetyltriethylcholine gave 68 inhibition a t a concentration of 250 pM, the bulkier ethyl groups

570 Human Placental Choline Acetyltransferase Eur. J. Biochem.

Table 12. Requirements for isotopic exchange between [14C]- choline and acetylcholine

Acetylcholine (10 mM) and [14C]choline (9.25 pM, 0.5 $3) were incubated in the presence or absence of CoA and/or choline acetyltransferase for 10 min at pH 7.0 and 39 "C. The reaction was stopped by acidification with HCIO,. Unlabelled choline chloride (10 pmoles) was added, the acetylcholine and choline were isolated via their reineckate salts and after separation by high voltage paper electro- phoresis their radioactive content was estimated. For details see Materials and Methods. Recovery of radioactivity was between 90 and 100°/,,. The extent of isotopic exchange was calculated as the radioactivity in the acetylcholine expressed

as a percentage of the total

Incubation conditions Exchange

Enzyme CoA

mN "0

- 0 0.5 - 0.15 1.8 + 0 0.9 + 0.037 52 + 0.15 73 + 0.30 73

100 r

10 20 30 Time (min)

Fig.6. Time wurse of exchange between acetylcholine and [W]choline in the presence and absence of chloroacetylcholine. The extent of choline acetyltransferase-catalyzed exchange was estimated as described in Materials and Methods. No

inhibitor (0); 0.2 mM chloroacetylcholine ( 0 )

evidently interfering with enzyme inhibitor binding. A similar decrease of inhibition with incubation time was observed with this analogue.

ISOTOPE-EXCHANGE STUDIES

The requirements for isotopic exchange between acetylcholine and [14C]choline are shown in Table 12. A significant amount of exchange occurred only in the presence of both enzyme and CoA and was maximal a t a concentration of the latter of 0.15 mM. This result suggested that the exchange required the formation of acetyl-CoA and this was also indicated in a further experiment where the presence of oxalo- acetate (5 mM) and citrate synthase (20 pg) reduced exchange in a 30 rnin incubation from goo/, to 63O/,,

presumably by competing with choline acetyltrans- ferase for the available acetyl-CoA. Desulpho-CoA (0.15 or 0.3 mM) did not support exchange and reduced the level from 70°/, to 12.5O/, when present in equi- molar (0.15 mM) concentration with CoA in a 10 min incubation.

The time course of the exchange in the presence and absence of 0.2 mM chloroacetylcholine is shown in Fig. 6 ; this concentration of inhibitor gave maximal inhibition. Preincubation of the enzyme for 5 min with the inhibitor prior to a 10 min incubation in the complete medium further reduced the exchange from 2001, to 110/,. Preincubation with inhibitor plus CoA (0.15 mM), however, resulted in a 6O/, exchange which was not increased by further addition of CoA in the subsequent 10 min incubation. No exchange was found when chloroacetylcholine (10 mM) was substituted for acetylcholine.

DISCUSSION Numerous methods have been described for the

estimation of choline acetyltransferase. Colorimetric methods have serious limitations both with regard to specificity and to sensitivity, whereas methods based on biological assay are very time-consuming. Radiometric procedures may be divided into those using an acetyl-CoA regenerating system [3,5] and those employing synthetic acetyl-CoA [1, 4,7]. The former method has the advantage of giving linear rates of acetylcholine synthesis during long incuba- tions and is therefore suitable for the estimation of very small amounts of enzyme. Furthermore the synthesis of acetylcholine is less likely to be limited by acetyl-CoA hydrolase activity and by other pathways utilising acetyl-CoA present in crude enzyme extracts. Various methods have been employ- ed for the isolation and estimation of the radioactive acetylcholine. Precipitation as the reineckate [I, 21 or as the tetraphenylboron salt [3] followed by count- ing of either the solid salt or its solution in an organic solvent requires many manipulations. Recently, however, Fonnum [38] has described an improved liquid cation exchange procedure using tetraphenyl- boron. A convenient and rapid procedure is the use of ion-exchange chromatography to separate the radioactive acetylcholine from the other radio- active components (acetyl-CoA and/or acetate) with subsequent scintillation counting of the aqueous solution of acetylcholine. This procedure, using an anion exchange resin to separate the acetylcholine produced in a synthetic acetyl-CoA medium, was first described by Schrier and Schuster [4]. Diamond and Kennedy [5] sought to reap the advantages of an acetyl-CoA regenerating system coupled with ion- exchange separation of acetylcholine but used a cationic resin involving a two-step elution. These authors did not obtain linear rates of acetylcholine

Vo1.22, Wo.4, 1971 D. MORRIS and D. S. GREWAAL 571

synthesis for more than 30min nor were the rates linear over a 10-fold range of enzyme concentration using crude homogenates of guinea-pig brain.

The procedure described in this paper, employing [3H]acetylphosphate, the phosphate acetyltransferase regenerating system and isolation of the radioactive acetylcholine by anion-exchange chromatography, gives optimal yields of acetylcholine both with time (Fig. 1) and with varying amounts of enzyme (Table 4). The sensitivity of the method is only limited by the specific radioactivity of the acetylphosphate ; tri- tiated acetic anhydride of very high specific activity is readily available.

Of interest is the effect of Na+ on choline acetyl- transferase activity (Table 2). The necessity for the maintenance of thiol groups in the active enzyme has been suggested many times in the past and also recently by the observation that 10 nM 5,5’-dithiobis- (2-nitrobenzoic acid) caused 500/, inhibition of choline acetyltransferase from bovine caudate nu- cleus [34] ; complete reactivation was effected by thioglycollate. The protective effect of a high concen- tration of Na+ may be due to a conformational change in the enzyme resulting in the thiol group(s) being folded away “inside” the molecule away from oxidative influences. The lack of inhibition by halo- acetates (Table 10) suggests, however, that if thiol groups form part of the active centre, they are in- accessible to simple non-specific thiol reagents.

The weak inhibition displayed by the analogues of choline in which the hydroxyl groups had been replaced by chlorine was unexpected in view of the close similarity between the inhibitors and the normal substrate. The hydroxyl group of choline would seem to be not only necessary for esterscation but, to- gether with the quaternary nitrogen moiety, to play an important role in binding of the substrate t o the enzyme. This is borne out by the observation that dimethylaminoethanol, which lacks the quaternary nitrogen but retains the hydroxyl group, has a similar Ki to chlorocholine (Table 6). The successive substitu- tion of methyl groups in chlorocholine by ethyl groups produced a marked decrease in inhibitory potency, confirming earlier observations [ 171 that the binding site for the “cationic head” of choline cannot readily accommodate alkyl groups larger than methyl.

The haloacetyl derivatives of acetylcholine proved to be very powerful inhibitors of choline acetyltrans- ferase, the potency decreasing with increasing size of the halogen substituent (Table 7). The lack of inhibition by chloroacetate, iodoacetate and iodo- acetamide (Table 10) indicated that the halo- acetylcholines were inhibiting the enzyme by virtue of their active site directing properties and raised the possibility that specific alkylation of the enzyme might have occurred. However, alkylation had evidently not taken place since the time course SS*

of inhibition (Fig.3) and overnight dialysis (Table 11) showed that inhibition was maximal a t short incuba- tion times with subsequent return of activity. The instability of the inhibitors in phosphate buffer a t pH 7 (Fig.4) seems the most obvious explanation of the short-lived inhibition which would then be simply due to very tight binding at the choline/acetylcholine site followed by rapid hydrolysis of the inhibitor. However, none ofthe inhibitors exhibited competitive kinetics with respect to choline (Fig.5).

Bromoacetylcarnitine [35] has been shown to cause rapid inactivation of the related enzyme car- nitine acetyltransferase in the presence of CoA by a novel mechanism. The inhibitor and CoA bind to the enzyme a t the normal sites and this is followed by alkylation of the CoA by the adjacent bromo- acetyl moiety. The product, S-carboxymethyl-CoA- carnitine ester, is very tightly bound to the enzyme in a 1 : 1 stoichiometry and released with a reactivation half-time of 15 days. It is possible that inhibition of choline acetyltransferase by chloroacetylcholine in the presence of CoA has the same mechanism but that the enzyme inhibitor complex dissociates much more rapidly. In this connection it is noteworthy that carnitine, unlike choline, has a carboxyl group which could contribute to binding. An alternative cause of reactivation after the possible formation of S-carboxymethyl-CoA-choline ester could be the hydrolysis of the ester bond with subsequent rapid diffusion of S-carboxymethyl-CoA away from the enzyme. This should result in a residual inhibition of acetylcholine synthesis due to a reduction in CoA available for regeneration to acetyl-CoA. However, rates of acetylcholine synthesis (Fig. 3) approximately equal to the uninhibited rate were obtained after reactivation in an experiment where the total amount of chloroacetylcholine added was equal to that of the CoA present. More work will be required to distinguish between these various possibilities.

The “ping-pong” mechanism [36] attributed to human placental choline acetyltransferase by Schu- berth [ l l ] involves the formation of a central binary acetyl enzyme complex formed from either acetyl- choline or, in the reverse direction, from acetyl-CoA, both routes not requiring the participation of the other substrate, CoA or choline, respectively. It follows, therefore, that radioactive choline would be incorporated into acetylcholine by a reversal of this process. However the complete dependence of this exchange on the presence of CoA (Table 12), the inability of desulpho-CoA to support exchange and the inhibitory effect of oxaloacetate and citrate syn- thase do not support the “ping-pong” mechanism. The incorporation is evidently dependent on the for- mation of acetyl-CoA which can only be derived from acetylcholine by a sequential or ternary complex mechanism. The kinetics of the placental enzyme have recently been reinvestigated and the results [37]

572 D. MORRIS and D. S. GREWAAL: Human Placental Choline Acetyltransferase Em. J. Biochem.

indicate a sequential Theorell-Chance mechanism. A similar conclusion about the mechanism of calf caudate nucleus choline acetyltransferase has been published recently [7].

Inhibition of the exchange reaction by chloro- acetylcholine was increased if the latter was pre- incubated with the enzyme in the absence of acetyl- choline, suggesting common binding site for these compounds.

1.

2.

3. 4.

5.

6.

7.

8.

9.

10.

11. 12.

13,

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D. Morris University Department of Physiology and Biochemistry Southampton, SO9 5NH, Great Britain

D. S. Grewaal’s present address: Kinsmen Laboratory of Neurological Research The University of British Columbia Vancouver 8, British Columbia, Canada