Journal of Ncurochrrnisrry. 1978. Vol. 30. pp. 35-46.Pergamon Rru Printed in Great Bntain.

MOLECULAR CHARACTERIZATION OF CHOLINE ACETYLTRANSFERASE FROM BOVINE BRAIN CAUDATE NUCLEUS AND SOME IMMUNOLOGICAL PROPERTIES OF THE HIGHLY PURIFIED ENZYME D.

MAL.THE-~RENSSEN, T.

LEA, F. FONNUM and T. ESKELAND

Norwegian Defence Research Establishment, Division for Toxicology, P.O. Box 25, N-2007 Kjeller, Norway (Received 31 May 1977. Accepted 15 June 1977)

Absmct-Choline acetyltransferase from bovine brain caudate nucleus has been purified to a specific activity of 25-30pmol ACh formed per min and mg protein. Disc electrophoresis a t pH 9.5 of the purified enzyme showed two protein bands localized close to each other. We were not able to show if ChAT was linked to one or both bands. In SDS disc electrophoresis the enzyme preparation showed one major and one minor protein band with molecular weights of 69,000 and 34,000, respectively. Heterogeneity of the enzyme preparation was also demonstrated by immunodiffusion and immunoelectrophoresis. After ammonium sulphate precipitation no aggregation of the enzyme could be detected by gelfiltration on Ultrogel AC-34 whilst a high molecular weight fraction was occasionally observed by geltiltration on Sephadex G-200. The enzyme was, however, separated into two molecular forms (A and B) on CM-Sephadex chromatography. Both molecular forms had the same SiOwbut differed in heat stability and affinity for acetyl-CoA. Both forms were inactivated by an antibody preparation raised against either a purified preparation of ChAT, or A and B separately. The highly purified enzyme preparation was inactivated more than 98% by immunoprecipitation. The antibody crossreacted with ChATs from several mammalian species, but only slightly with ChAT from pigeon. The results of binding studies with affinity columns, suggest that the enzyme contains a hydrophobic lobe and a dinucleotide fold, and that a free purine rather than a free ribosyl ring of acetyl-CoA is important for the binding of the substrate to the active site. The hydrophobic lobe may be the same as the dinucleotide fold.

CHOLINE acetyltranderase (ChAT, EC 2.3.1.6), the enzyme responsible for the synthesis of acetylcholine, is present in different types of nervous tissue. The enzyme has been extensively purified from the ganglia of the squid (HUSAIN& ~ ~ U T N N E1973). R, Several attempts have beem made to purify the enzyme from brain tissue of vertebrates, but although preparations of high specific activity have been obtained, they have been heterogeneous (MALTHE-WRENSSEN et al., 1973; ROSSIER et al., 1 9 7 3 ~ ;ROSSIER, 1976a). Enzyme p r e p arations which are homogeneous on electrophoresis have been claimed by a few groups (-0 & WOLFGRAM, 1973; SINGH & MCGEER,1974~;ROSKOSKI et al., 1975), but they all have considerably lower specific activities than the heterogeneous preparations. A particular difficulty in the purification of the enzyme is its instability when highly purified and the consequent low recovery of activity ( H u m & MAUTNER,1973; ROSKOSKI et al., 1975; ROSSER, 1975; Smm & MCGFER, 1974a). There exist several reports on the formation of antibodies against ChAT from different

species (ENG et al., 1974; MALTHE-S~RENSEN, 1975; ROSIER et al., 1973b; &LISTER & OTOOLE,1974; SINGH& MCGEER,19746), but the specificities of the antibodies have been heavily criticized (ROWER,1975) as the preparations exhibited diverging properties. The aim of the present study was to purify ChAT from bovine brain extensively and to study its biochemical and immunological properties. The active site of the enzyme has been characterized further by binding to different column materials. During an early stage of this work two different forms of the enzyme were obtained by chromatography on CMSephadex of a preparation purified by ammonium sulphate precipitation. Since it has been claimed (CHAO& WOLFGRAM, 1974; BANNS,1976) that enzyme aggregation is promoted by ammonium sulphate precipitation, this has been investigated. MATERIALS AND METHODS

Chemicals [I-"CIAcetyl-CoA (59.2 mCi/mmol) was obtained Abbreoiatwns used: CM-sephadex, carboxymethyl- through New England Nuclear and acetyl-CoA was pursephadex; DEAE-cellulose, diethyl-aminoethyl-cellulose; chased from Schwartz-Mann, New York, U.S.A. Ultrogel SDS, sodium dodecyl sulphate; PEG, polyethylene glycol. AC-34 was from LKB-Produkter, Sweden. Sephadex gels 35

36

D. MAL'IHE-SC~RENSSEN, T.LEA,F. FONNUM and T.ESKEIAND

and Sepharose 4B were obtained from Pharmacia Fine Chemicals, Uppsala, Sweden, whereas sodium pthlorornercuribenzoate,polyethylene glycol 6ooo and BrCN were obtained from Koch-Light Lab. Ltd.. Bucks, U.K. 1-Ethyl3(3-dimethylaminopropyl)carbodiimide. Coomassie Brillant Blue G and R, sodium dodecyl sulphate and Blue Dextran 2000 were from Sigma. gNitrobenzoyl azide was obtained from Eastman Organic Chemicals. Acrylamide and bisacrylamide were purchased from Bio-Rad Laboratories, U.S.A. Swine immunoglobulins to rabbit IgG were purchased from Dakopatts A/S, Denmark ant Agar Noble from Difco, U.S.A. Human serum albumine, catalase. ovalbumine, malate dehydrogenase, glutamate dehydrogenase and myoglobin (sperm whale) were all purchased from CalBiochem. All other chemicals were pro analysis obtained either from BDH or Merck.

carbodiimide method outlined by CUATRECASAS (1970). Derivatized Sepharose 4B (5 ml packed gel) was added to acetyl-CoA (50 mg in 5 ml of water) and the mixture was adjusted to pH 4.8 with 0.5 N-HCI.To the suspension 2 pCi of [ l-"Clacetyl-CoA (59.2mCi/mmol) was added. l-ethyl3-(3-dimethylpropylamino) carbodiimide (1.14 mmol dissolved in 1 ml of water immediately before use) was then added to the mixture and stirred over a 5 min period while the pH was maintained at 4.8 with 0.5 N-HCI.The mixture was stirred gently at 27°C for a further 20 h, the pH being readjusted occasionally to pH 4.8 during the first hour. The gel was then washed thoroughly with 0.2 M-NaCI, the washings were free of radioactivity. The degree of coupling was measured as mentioned under (a) and 0.22pmol of acetyl-CoA was bound per ml of packed gel. According to OCarra et al. (1974) this procedure yields agaroseribosyl-acet yl-CoA.

p-Chloromercuribenzoate-Sepharose4B pChloromercuribenzoate-Sepharose4B (mercurisephar- Blue-Dextran-Sepharose 48 BrCN-activated Sepharose 48 (CUATRECASAS. 1970) was ose) was prepared as previously described (MALTHEderivatized with Blue Dextran essentially using the proSDRENSSEN, 1976) following the methods of CUATRECASAS cedure described by RYAN& VESTLING(1974). Fifty milli( 1970). litres of packed BrCN-Sepharose, prepared as described w-Aminoalkyl-Sepharose 4 8 previously, was immediately suspended in 5 O m l of wAminoalkyl-Sepharose gels were prepared as de- 0.4 m-sodium carbonate buffer pH 10.0 containing 1 g Blue Dextran. The final product was washed with 1 M-KCIuntil (1970). scribed by CWATRECASAS no Blue Dextran could be detected in the washing. Acetyl-CoA-afinity gels Columns (1 x 2cm) were prepared and equilibrated by buffer A at pH 7.2 with the proper salt concentration given (a) Immobilization of acetyl-CoA to polyacrylamide (BioGel P-200) by azolinkage. The diazoderivatives of poly- in the text. The enzyme preparation, dialysed against acrylamide beads were prepared according to INMAN 1OOOvol of the equilibration buffer for 6 h at 4°C was (1974). Polyacrylamide beads were ethylaminoalkylated loaded on the column and eluted as stated for the separate and the derivative was used for preparing arylamine experiments The flowrate was 3ml/h and fractions of (paminobenzamidoethyl) polyacrylamide. Fifty millilitre 0.5 ml were collected and tested for enzyme activity. bed volume of Bio-Gel P-200 aminoethyl derivative was suspended in 0.2 wNaCI and treated with pnitrobenzoyl Enzyme assay ChAT was determined at 3 7 T as described by FONNUM a i d e in tetrahydrofuran followed by triethylamine. The aliphatic amino groups in the product were protected by (19751). When K, of acetyl-CoA was determined, the conacetylation with acetic anhydride and the product was then centration of choline and acetyl-CoA was varied between reduced by dithionite. The arylamine derivative was 0.2 and 8 m~ and 2.5 and 100 pm respectively. 1 unit of washed thoroughly with 0.2, 2.0 and 0.2 m-NaCI in turn. enzyme activity is defined as 1 pmol ACh formed per min. Acetyl-CoA was coupled to the arylamine polyacrylamide by first preparing the diazonium form of the arylamine Purification of ChAT The enzyme was purified in part as described previously derivative. To 20mg of NaNOl in 1 ml of ice-cold water was added 2 ml of arylamine polyacrylamide (bed volume) (MALME-SC)RENSSEN.19760). The enzyme was extracted and 0.5 ml 1 N-HCI. After 8 min, 23 mg acetyl-CoA in 1 ml from acetone powder, acid precipitated to pH 4.5 and reof saturated ice-cold sodium borate was added together adjusted to pH 6.0 as described The supernatant was prewith 2 pCi of [1-'4Cjacetyl-CoA (59.2 mCi/mmol) as s u p cipitated with a saturated solution of ammonium sulphate plied from New England Nuclear. The pH of the reaction at pH 6.0. The pH was continuously adjusted to 6.0 with mixture was adjusted to pH 8.2 with I N-NaOH and the I N-NaOH during the precipitation. The enzyme solution suspension was shaken carefully for 15 h at 5"C:The gel was fractionated in several steps by slowly adding the was washed thoroughly with 0.2 M-NaCI until the washing ammonium sulphate solution to the enzyme solution and was free of radioactivity. The degree of coupling of acetyl- collecting the precipitate after centrifugation at 15,OOOg CoA to the gel was determined by measuring the amount for 30min. Fractions after 0-400/, 40-50% and 5 0 4 5 % of radioactivity per l00pl of gel suspension. A recovery saturation were prepared. Most of the enzyme activity was of 4.4% was obtained, giving 0.55 p o l acetyl-CoA coupled found in the latter fraction. The precipitate from this fracper ml bed volume of gel. According to OCARRAet al. tion was dissolved in 6Oml IOmhc-sodium phosphate(1974) this procedure yields acryhmide-8-adenine-acetyl- citrate buffer, containing 1 mM-EDTA and 7% (v/v) glycerol (buffer A) at pH 7.2. The enzyme solution was applied &A. (b) Immobilization of acetyl-CoA to Sepharose 48. The directly on a mercurisepharose column (2.5 x 3 cm) which & O'CARRA(1973) for the preparation had bem equilibrated by buffer A at pH 7.2. After applimethod of BARRY of NAD linked Sepharose was essentially followed. Scphar- cation the column was washed with a small volume (10 ml) ose 4B was activated by BrCN, aminoalkylated by dia- of the same buffer. The column was them washed with the minoethane and succinylated by succinic anhydride as de- same buffer at pH 6.0 (80 ml) before stepwise elution with scribed by CUATRACASAS (1970). Acetyl-CoA was coupled 0.25 mm and 20 mm-cysteine in b u l k A (80 ml each) at to the succinyl aminoethyl derivative by the water soluble pH 6.0. Fractions of 5ml were collected at a flowrate of

Molecular properties of ChAT from bovine brain

8ml/h. Each fraction was tested for enzyme activity and protein (Az8,,) (Fig. 1). The active fractions eluted by 20 mwcysteine, were pooled and applied directly to a column of DEAE-cellulose,DE-52 (1.5 x 30cm) which had been equilibrated by buffer A at pH 6.0. The enzyme activity was eluted with the same buffer at a flow rate of 9 d / h and the most active fractions were collected and used for PEG-6000 frao tionation. The enzyme solution was fractionated in steps of 0-15, 15-20 and 20-27% (w/v) of PEG-6000. PEG-6ooo was grounded in a mortar to a fine powder before use. The PEG-6000 was added slowly to the solution to hinder any high local concentration. The fractionation was performed at 20°C with continuous stirring. After standing for 30 min at 20°C the precipitate was removed by centrifugation at 15,000g for 30 min. The precipitate from the fraction between 20-27% (w/v) PEG-6000 was collected and dissolved in 5 ml of 22 mwsodium citrate-10 mwsodium phosphate buffer at pH 7.2 with 1 mM-EDTA and 7% (v/v) glycerol. The enzyme solution was applied directly to a CMSephadex, C-SO, column (1.5 x 3cm) previously quillbrated with buffer A a t pH 7.2. The enzyme was eluted by 0.4M-NaCl in the same buffer (Fig. 2). Fractions of 5 ml were collected a t a flowrate of 9 d/h.Protein was either measured as AZa0 or by the method of LOWRYet al. (1951). All steps in the purification were performed at 4°C unless otherwise stated. CM-Sephadex chronmtography with linear salt gradient

Enzyme after ammonium sulphate precipitation was dialysed overnight at 4°C against lo00 vol of buffer A at pH 7.2. After dialysis the enzyme solution was applied to a column of CM-Sephadex C-50 (1.5 x 20cm). The column had previously been equilibrated by buffer A at pH 7.2 and was eluted by a linear gradient of NaCl, formed by mixing 200 ml of buffer A with 200 ml of buffer A containing 0.4 M-NaCI a t pH 7.2. Fractions of 5 ml were collected at a flowrate of 9ml/h. The enzyme was eluted in two peaks called A and B (Fig. 3). Heat inactioation

Fractions A and B after CM-Sephadex chromatography were used The fractions were dialysed overnight against 1000 vol of 10 mM-sodium phosphate buffer at pH 7.4. The heat inactivation was performed at 46°C in the presence and absence of acetyl-CoA. Gel filtration of Sephadex G-2000 and Ulvogel AC-34

Enzyme fractions after ammonium sulphate precipitation, and CM-Sephadex chromatography were subjected to gelfiltration on Sephadex G-200 and Ultrogel AC-34. Both columns were equilibrated in 10 mwsodium citratephosphate buffer pH 7.2 containing 1 mM-EDTA and 0.02% sodium azide. Sodium azide had no effect on the enzyme activity. Sephadex G-200 was preswollen and deaerated before packing on the column (1.5 x 90cm) and equilibrated with buffer for 3 days. The flowrate was 9 ml/h during the packing and 7ml/h during the equilibration. Ultrogel AC-34 was stirred with the equilibration buffer and deaerated before being poured into the column (1.5 x 45cm) and equilibrated for 3 days with the same buffer as for Sephadex G-200. The flowrate was 15 ml during the packing and 12 ml/h during the equilibration. Blue Dextran 2000 and hemoglobin (rat) were used as standards for testing the exclusion volume for both columns.

37

Disc electrophoresis Disc electrophoresis was carried out essentially as described by DAVIS(1964) at pH 9.5. The current was 2.5 mA per gel and the electrophoresis time w& 1.5 h a t 5°C. The gels were either stained with Coomassie Brilliant Blue G (DIEZELet a/., 1972) or cut into 2 m m pieces and tested for ChAT activity (FONNUM,1966). When ChAT activity was to be measured the gels were polymerized with riboflavine (4 ms/lOO ml). SDS-disc electrophoresis was performed essentially as described by WEBER& &BORN (1969). The electrophoresis was performed at a constant current of 8 mA per gel. The gels were stained with Coomassie Brilliant Blue R (WEEKE, 1973). As standard proteins were used serum albumine, catalase, ovalbumine, malate dehydrogenase. glutamate dehydrogenase and myoglobin (sperm whale). Ultraconcentration of enzyme solutions

The enzyme solutions were concentrated in an Amicon Ultraconcentration cell (121111) at 4°C using a Diaflo PM 10 filter. A pressure of 1.5 kg/cm2 was used. Analytiul ulvacen tr$qatwn-Beckmann

E- M ode1 The enzyme activity peaks A and B after CM-Sephadex chromatography were run in an analytical ultracentrifuge. The enzyme peaks were loaded in a fixed partition cell and run at 60,000 rev./min at 6°C. After the run the enzyme activity in the lower compartment of the cell was determined. The S'&, value was calculated according to SCHACHMAN (1957) assuming a partial specific volume of 0.72 ml/g. Preparation of antiserum

Rabbits were given a series of three subcutaneous injections between the scapulai and two intramuscularly in the hind leg. The protein solution (0.5 mg protein) was emulsified with an equal volume of Freund's complete adjuvant. The treatment was repeated after 14 days except that Freund's incomplete adjuvant was used. After 1, 2 and 3 weeks the rabbits were bled and the Serum collected. The purified enzyme used for the immunization had a specific activity of 6.5units/mg protein. Fractions A and B after CM-Sephadex chromatography were mixed with the adjuvant for immunization and injected. Immunoprecipitation

To 25 pi of enzyme solution was added 2.5-150 yl of rabbit antiserum and 147.5-0pl of normal rabbit serum. About 0.004 units of enzyme activity was used. The specific activities of the different bovine enzyme preparations were bovine brain homogenate 0.003, ammonium sulphate prep aration 0.05. highly purified enzyme 8.4 and partially purified enzyme from other species were about 0.04 units/mg protein. After incubation for 20 min at room temperature and then at +4" for 20h, aliquots of 25p1 of the incubation mixture were transferred to a solution of 75yl of buffer (IOmg/ml of human serum albumin from Kabi. Sweden in 0.138 M-NaCI, 10 mM-sodium phosphate buffer at pH 7.4, 2 mM-EDTA and 1 mu-cysteine) and 200 yI of swine immunoglobulins to rabbit IgG in order to precipitate any antibody bound enzyme, which had not been precipitated in the first reaction. The solution was further incubated for 45 min at room temperature. After incubation the antibody-enzyme solution was tested before and after centrifugation. Any precipitate after the centrifugation was

D. MALTHE-SORENSSEN, T. LEA,F. FONNUM and T. ESKEIAND

38 TABLE1.

PURIFICATION OF CHOLINE ACETYLTRANSFERASE FROM BOVINE BRAIN CAUDATE NUCLEUS ~

Preparation Extract or acetone powder Acid precipitation Ammoniumsulphate precipitation 5 0 4 0 % Mercurisepharose chromatography DEAE-c~~u~ow chromatography Polyethylene glycol precipitation CM-sephadex chromatography

Total activity in units of ChAT

Total protein (mg)

Specific activity (units of ChAT/ min mg protein)

~~~

~~~~

Recovery

(%I

Purification

81 80.7

33,600 27,740

0.0024 0.0029

100

53.8

840

0.064

67

26

14.1

21

0.62

17

253

1.2

6.8

7.5

0.90

8

379

6.45

3.3

1.94

7.5

796

4.0

0.14

4.9

11,875

28.5

Starting material-26g of acetone powder, corresponding to 16Og wet weight of caudate nucleus, was used for the purification procedure. One unit of enzyme activity is 1 pmol ACh formed per min at 37°C. suspended in the buffer previously used and tested for lose, but omitting this step from the purification proenzyme activity. ChAT from different species were purified cedure resulted in less purification in the next step. as previously described (MALTHE-SORENSSEN & FONNUM,The chromatography on CM-Sephadex at nearly neu1972). tral pH was highly successful. Most of the proteins

applied to the column were eluted by the equilibration buffer, whereas the enzyme was retained The technique of Ouchterlony was used (OUCHTERLONY, 2). (Fig. 1949).

Douhle immunodiflusion

Properties of the purified enzyme preparation

Immunoelectrophoresis

The final preparation revealed two bands closely localized on disc electrophoresis (Fig. 3). ChAT activity was recovered on a broad band overlapping the visible bands. Increasing the amount of protein RESULTS for disc electrophoresis resulted in one diffuse band, Purification of enzyme but no additional bands appeared. ChAT from bovine brain caudate nucleus has been The relative mobility of standard polypeptides (see purified extensively to a specific activity of 28.5 Methods) on SDSdisc electrophoresis yielded a units/mg protein (Table 1). As demonstrated in Fig. straight line when plotted against the logarithm of 1 the mercurisepharose column was eluted stepwise their molecular weight (SHAPIROet al., 1967). The with 0.25 and 20 m-cysteine and 70% of the enzyme purified ChAT preparation yielded on SDSdisc elecactivity was eluted at 2 0 m . Only a small increase trophoresis one major band of molecular weight in the specific activity was achieved after DEAE-cellu- 69,000 and a smaller band of 34,000.Since the two

In principle the immunoelectrophoresis technique of WIEME(1959) was used.

Fractions

FIG. 1. Mercurisepharose chromatography of the ammonium sulphate preparation (Table I). The column (2.5 x IOcm) was eluted stepwise with cysteine as shown in the figure. (-) A,,,. (c- 4) enzyme activity. Fraction volume was 8 ml.

39

40

Pic;. 4. Double immunodiffusion and imrnunoelcctrophoresis o f CIiAT. The highly purified enzyme preparation was used after having been concentrated hy ultrafiltration. (a) Ilouble immunodiffusion of concentrated enzyme with protein concentration of 0.25 mg/ml, and specific activity of 25 unitsimg

protein. (h) lmmunoelectrophoresis of the highly purified enzyme. The protein concentration was 0.18 mg/nil and thc specific activity was the same as in (a). (c) Immunoclectrophoresis of concentrated enzyme with protein cnnccntration of 0.92 mg/ml and specific activity of 27 unitsimg protein.

Molecular properties of ChAT from bovine brain

41

I

t

€283

0

F r o c t tons

FIG.2. CM-sephadex chromatography of the PEG-preparation (Table 1). The column (1.5 x 4cm) was washed after application with buffer A pH 7.2 (see Methods) and eluted with 0.4~-NaCIin the A280, ) (A-A) ChAT activity. Fraction volume was 8 ml. same buffer. (M

bands in Fig. 3 run so close that we were unable to elute them separately in high enough concentration to run them on SDS-disc electrophoresis, we are unable to compare the two bands on disc-electrophoresis with the two bands from SDS-disc electrophoresis. In double immunodiffusion (Fig. 4a)and immunoelectrophoresis (Fig. 4b) the purified enzyme preparation yielded two and one precipitine lines respectively using a highly active antiserum preparation (see below). The result obtained by immunoelectrophoresis was dependent on the protein concentration of the sample. Only one line was revealed with protein concentrations of less than 0.2 mg/ml, whilst het-

erogeneity was observed with protein concentrations of more than 0.9 mg/ml (Fig. 4c) No heterogeneity in the molecular weight could be detected by exclusion chromatography on Ultrogel AC-34 using either the highly purified enzyme preparation (not shown) or various preparations with low specific activity (Fig. 5). Only one symmetrical peak of enzyme activity was obtained, eluted at the same position each time, just prior to hemoglobin (Fig. 5). The exclusion volume of the enzyme was independent of the amount of protein applied or the specific activity of the enzyme. Gel chromatography on Sephadex G-200 was more difficult to interpret. In most of the experiments there was no indication of heterogeneity or a high molecular weight fraction and the enzyme was eluted just prior to hemoglobin. In sucrose gradient centrifugation with high protein concentrations (>35 mdml) the enzyme peak broadened with increasing protein concentration and the middle of the activity peak moved to higher sucrose density, indicating increasing molecular weight, due either to aggregation of the enzyme or to interaction with other proteins present. Partial purification and indications of two molecular

forms -02 10--01 The ammonium sulphate preparation (Table 1) could be separated into two molecular forms (A and oe--ooe B) by CM-Sephadex chromatography using a linear 06--006 NaCl gradient (Fig. 6) as previously described -0 I (MALTHE-SQRENSSEN, 1975). Peak A, containing 70% 0 4-006 of the activity recovered, was eluted at 0.14 M-NaCI, 0 2--002 whereas peak B containing 30%, was eluted at I , 0.23 M-NaCl. On rechromatography of the separate 6 8 I0 12 14 16 18 20 peaks (A and B) on separate columns of CMFractions, 4 rnl FIG.5. Exclusion chromatography of choline acetyltrans- Sephadex, the enzyme activity was eluted as one ferase on Ultrogel AC-34. (a)0-50% saturated ammonium- single peak at the same salt concentration as orisulphate fraction, 25 mg protein/ml (b) 5 5 0 4 5 % saturated ginally. The same heterogeneity with almost the same ammonium sulphate fraction. 18 mg protein/mL The sym- distribution of enzyme activity, was also observed bols are (-0) ChAT, A-A m 4 A,,,. when the supernatant of a homogenate of caudate

D. MALIHE-%RENSSEN, T. L u ,

42

-

F.

FONNUM and T. BKELAND

A

10.13 M - N o C I

O r

T4 rn

t

3

Fractions FIG. 6.

CM-Sephadex chromatography of ammonium sulphate precipitated enzyme. The ChAT activity (A-A) was eluted by continuous salt gradient (-)Two peaks A and B were isolated from tubes as shown, dialysed against the original buffer and rechromatographed in two separate columns. The specific activities of the two peaks were for 0.7 units/mg protein for A and 0.5 units/mg protein for B.

nucleus Was Used directly for CM-Sephadex chroma- TABLE 2. PHYSICAL AND KINETIC PROPutTlES OF 'IHE DIFFERtography. These molecular forms could represent the M T MOLECULAR m m OF CHOLME A C m n w S F E m E FROM BOVINE BRAIN two molecular forms observed on disc electrophoresis. On heat inactivation at 46°C the molecular form AT K, acetyl-CoA A was less stable than B (Fig. 7a). When an equal preparation %ow oul) Peak A Peak B

5.3 f 0.2 5.3 f 0.2

8 15

SiOw,the Svedberg constant, was calculated as described in Methods. Peaks A and B refer to the molecular forms of ChAT which were separated by CM-sephadex chromatography eluted by a Linear salt gradient.

2

4

6

8

1

0

4z

-

2

4

6

8

Preincubotion time.

1

0

rnin

of the two m o k d a r forms A and B isolated by CM-Sephadex chromatography with a linear salt gradient. (a) A and B alone, and A and A and &.--A B alone, B together. (b) A and A-A B in the presence of 100 p&acetyl-&A. For details see Methods FIG. 7. Heat inactivation at 46°C

amount of enzyme activity from A and B was mixed and subjected to heat inactivation, the remaining activity corresponded almost to the arithmetic mean of A and B alone. Peak A could be partially protected against the heat inactivation by acetyl-CoA (100 p ~ ) (Fig. 7b), whereas peak B was not influenced. The apparent K, of acetyl-CoA differed for the two molecular forms, A having a higher affinity for acetylCoA than B (Table 2). The sedimentation coefficient of the two molecular forms was identical (Table 2) and both molecular forms were inactivated by immunoprecipitation to the Same extent (Fig. 8).

Aflnity column chromatography ChAT was found to bind to o-amino-alkyl sepharose with a carbon chain length of 4. Complete binding of the enzyme was achieved with a carbon chain length > 4 (Table 3). Only a small amount of the activity (less than 20%) applied to the gel could be eluted by high Salt concentration or biospecifically by acetyl-CoA. The was bound to both Blue Dextran-agarose and ribosyl-acetyl-CoA-spharose, but only slightly to 8-adenine-acetyl-CoA-polyacrylamide (Table 3). The enzyme activity could be

Molecular propertics of ChAT from bovine brain

t

I

I

20

60

40

pl

43

antiserum added

FIG.8. inactivation of ChAT from different speaes by the antibody against the bovine brain enzyme. The same amount of enzyme activity (0.004units) from the different species was used in each experiment and the enzyme activity tested after centrifugation. For details see Methods. ChAT from brains of: (m) bovine, (V) rat, (0) rat, (A) mouse, (0)guinea pig. (+) chicken and (0)pigeon. Identical precipitation patterns were observed with the following preparation of bovine brain enzyme: homogenate, ammoniumsulphate precipitation, peak A and B from CM-Sephadex chromatography and highly purified enzyme.

eluted biospecifically from these gels by acetyl-CoA at low ionic strength and to a certain extent by 0 . 4 ~ - N a C lalone. The enzyme was only slightly bound to Blue Dextran agarose if applied in 0.4 wNaCI. The highly purified enzyme (1 ml) formed a complex with Blue Dextran (0.5 mg/ml) when they were preincubated for 10min at 5". Gelfiltration of the mixture on Ultrogel AC-34 showed that they moved together in the absence and separated in the presence of acetyl-CoA (4Op~). Immunological studies

A highly active antibody preparation was obtained after immunization of rabbits with the purified TABLE3. BINDINGOF

enzyme preparation of ChAT. The antiserum inhibited ChAT from bovine brain more than 98% by immunoprecipitation (Fig. 8). The inhibition was independent of the degree of purification of the enzyme. Both fractions A and B after CM-sephadex chromatography gave rise to antibodies which inactivated ChAT. Both antibody preparations inhibited both A and B. As demonstrated in Fig. 8, the antibody inactivated ChAT from different species. The degree of inhibition of ChAT from the various species differed. Maximum inhibition at the highest dilution of the antiserum was obtained for ChAT from bovine brain (Fig. 8). O A T from pigeon brain was only slightly inhibited, whilst

CHOLINE ACENLTFWNSFERASE To DIFFERENT AFFINITY COLUMNS ~

Per cent bound enzyme activity

Per cent eluted enzyme activity Biospecific

Low ionic strength 0.1 M-NaCI

Materials w Aminoalkyl-sepharose

High ionic strength 0.4 M-NaCI

-

-CH2.CHZ.CHz.NH2

-CHZ.CH2. CH,. C H Z . NHZ -CHI. CHZ. CHI. CHI CHZ.NH2 - CH 2 . CHZ CH2 . CHI. CHZ CH 2 . NH 2 Agarose-ribosyl-acet yl-Ca A Acr ylamide-8-adenine-acet y l-CoA AgaroseBlue-Dextran 2000

High ionic strength I M-Naa

0.1 M NaCl

500 PMacety I-Co A

-

100 100

80 100 80

20 100

40

40

Low ionic strength 0.4 M-Naa

-

40 20 10

40 20 40

80 90

~

The enzyme preparation after DEAE&llulose chromatography was used for affinity chromatography on w-aminoalkylsepharose (Table 1). whereas the highly purified enzyme preparation was used for chromatography on acetyl-CoA and Blue Dextran gels. The affinity material was loaded on small columns (1 x Zcm), equilibrated with the proper salt concentration in buffer A (see Methods) and eluted with the Same buffer containing either salt or acetyl-CoA as indicated Enzyme activity corresponding to 0.17units. was used in each experiment. Per cent bound and eluted enzyme activity refers to the percentage of the total enzyme activity applied to the columns. ( - ) means not detectable or no binding.

44

D. MALIHE-SORENSSEN, T. LEA, F. FONNUM and T. ESKELAND

ChAT from mouse, cat, rat, guinea pig and chicken that a previous preparation of placenta ChAT, not brain showed more than 70% inhibition. No further claimed to be homogeneous, exhibited a specific acinhibition of ChAT was obtained using swine im- tivity of 2-3 units/mg protein (MORRIS,1966). One munoglobulins to rabbit IgG in the immunoprecipi- should not expect variations of more than 2-3-fold tation reaction. Less than 10% of the enzyme activity from the differences in assay conditions, i.e. incubaremaining after precipitation with the antibody could tion temperature, substrate concentration or salt concentration (HEBB, 1972; FONNUM, 197%). It also be traced to the precipitate after centrifugation. becomes hard to explain such large differences from inactivation of the native enzyme in their final p r e p DISCUSSION arations as has been suggested by ROSKOSKJ et al. Purification of ChAT (1975). Since ChAT was discovered by NACHMANSOHN & MACHADO (1943) in extracts from rabbit brain, many Molecular forms of ChAT ChAT from bovine brain could be isolated in two attempts have been made to purify the enzyme from different sources (PRINCE, 1967; MORRIS, 1966; different molecular forms (A and B) by gradient ion POTTERet al., 1968; HLJSAIN & MAUTNER,1973; ROS- exchange chromatography. The two forms were conSIER et a/., 1976a; MALTHE-S~~RENSSEN et a/., 1973; firmed by rechromatography of the separate fractions WENTHOLD& MAHLER,1975; CHAo & WOLFGRAM, (Fig. 6). Both molecular forms gave the same value 1973; SINGH& MCGEER, 1974a; ROSKOSKIet d., of S2,, using analytical ultracentrifugation (Table 2). 1975). The present procedure has led to the highest Fractions A and B differed in heat stability (Fig. 7) specific activity of the enzyme (25-30 units/mg pro- and affinity of acetyl-CoA (Table 2). indicating differtein) hitherto obtained from mammalian sources. Still ent molecular properties of these forms. It has not immunoelectrophoresis and disc electrophoresis indi- been possible to show if both A and B exist in the cate heterogeneity which may be due to impurities. highly purified preparation. Previously several molSDS-disc electrophoresis revealed two protein ecular forms have been demonstrated in rat and cat bands, one major component with a molecular weight brain by isoelectric focusing (MALTHE-SC~RENSSEN & 1972). In rat we have shown that the differof 69,000 and a minor component of 34,000. The FONNUM, major band presumably represents ChAT, since the ent molecular forms were not due to molecular aggremolecular weight from different species has previously gates (MALTHE-%RENSEN& FONNUM, 1972). Differbeen estimated to be within this range (BULL et al., ent molecular forms from human brain have been 1964; GL~VER & POTTER, 1971; ROSKOSKI et al., 1975; separated by cation chromatography (SINGH et a/., ROSIER, 19766), the only exception being CHAO(1975) 1975). who suggests a molecular weight of 87,000. The minor Several groups have suggested the presence of difband could represent a form of a subunit of ChAT ferent molecular forms of ChAT from various species which has been claimed to exist for G A T from due to molecular aggregates (CHAO& WOLFGRAM, & bovine brain (CHAO,1975) but most likely it r e p 1973, 1974; GLOVER& P o r n , 1971; HALJSAIN resents an impurity. Both immunodiffusion and im- MAUTNER,1973; WHITE& WLJ,1973; BANNS,1976). munoelectrophoresis showed that the purified enzyme The multiple molecular forms of O A T from bovine preparation was heterogeneous, but it is difficult to brain ( W o & WOLFGRAM,1974) and human plajudge from these results if this heterogeneity is due centa (BANNS,1976) have been claimed to be caused to different molecular forms of ChAT or to impurities by ammonium sulphate. The aggregates reported by The recovery of enzyme activity is not high for the BANNS(1976) represented only a minor part of the present procedure (Table 1). but still consistently high enzyme activity and conclusive evidence that they specific activities of ChAT were obtained. Previously, were aggregates of ChAT was not presented. ROSChAT from squid ganglia has been purified to give KOSKI et al. (19753, however, did not detect any evithree fractions of 16-60 units/mg protein ( H u m & dence for molecular aggregates in their preparations MAUTNER, 1973) and rat brain to 20 units/mg protein of human brain or human placenta ChAT. We did (RQSSIER et a/., 1976~).The latter author suggested not detect any aggregates of bovine brain ChAT after that a pure preparation from rat brain should have ammonium sulphate precipitation and gelfiltration on a final specific activity of 100 units/mg protein. Ultrogel AC-34 (Fig. 5). A high molecular weight The present preparation contains a 2Gfold higher fraction, however, was occasionally observed using specific activity than a previous preparation from Sephadex G-200 gelfiltration and also in sucrose grabovine brain claimed to be homogeneous on electro- dient centrifugation with high protein concentrations phoresis (CHAO& WOWGRAM,1973). Electrophoretic (>35 mg protein/ml) (unpublished results, MALIHEhomogeneity of ChAT purified from human brain or %RENSSEN & ESKELAND).We have previously noticed placenta has also been claimed by SINGH& MCGEER that ChAT in crude preparations from rat brain (19744 and ROSKOSKJ et al. (1975). The specific activi- synaptosomes easily interacts with other proteins & MALTHE-%RENSEN,19733. Since CHAO ties of their preparations were 0.01 and 0.04 units/mg (FONNUM protein, respectively. It is very difficult to explain & WOLFGRAM(1974) used high protein concenthese large discrepancies, particularly when we recall trations, (> 35 mg/ml) and very long columns of

Molecular properties of ChAT from bovine brain Sephadex G-200 for gelfiltration, the heterogeneity in molecular weight they observed for ChAT from bovine brain could be due to the experimental conditions. Active site of the enzyme

Recently it has been demonstrated by fractionation of ChAT from human placenta on Blue Dextran column, that the enzyme may contain a dinucleotide fold in the active site, where acetyl-CoA is supposed to fit in (ROSKOSKIet al., 1975). These results have been confirmed in the present study. ChAT was bound to immobilized Blue Dextran and was released specifically by acetyl-CoA (Table 3). In addition, Blue Dextran and ChAT moved as a complex on gelfiltration, but separated in the presence of acetyl-CoA. These results support the concept that the enzyme contains a dinucleotide fold in the active site. Stereochemically, a free adenine group rather than a free ribosyl group was important for the binding of the enzyme to immobilized acetyl-CoA (Table 3). This could imply that the adenine group of acetyl-CoA binds deep in the dinucleotide fold, whereas the r i b syl group is protruding from the fold, which is in amordance with the binding of the enzyme to immoet al., 1975). Immobilized Blue Dextran (THOMPSON bilized alkylamines (> C,) bind the enzyme almost irreversibly, suggesting that the enzyme contains a hydrophobic lobe. In agreement, both butyryl-CoA and benzoyl-CoA have previously been shown to be inhibitors of ChAT (BERMAN-REISBERG, 1957). Immobilized alkylamines and acetyl-CoA have previously been used to determine hydrophobic lobes and stereochemically important groups of complex ligands & (OCARRAet al., 1974; SHALTIEL,1974; SHALTIEL ER-EL,1973).

Immunological studies of ChAT

45

in part be explained by the fact that they raised the antibody in guinea pigs, but most likely it is due to the low specific activity of ChAT they used for immunization. The antibody raised in guinea pig crossreacts only slightly with ChAT from other species (personal communication, L. P. Chao), whereas the present antibody crossreacts easily with ChAT from various species, inactivating other mammalian enzymes more than 80%. The antibody preparation of SINGH& MCGEER(1974b)also inactivated other mammalian species, but to a much lower extent (3659%). The results indicate that a highly active antibody is raised with ChAT of high specific activity. This has also been suggested by ROSIER (1976~). Although some molecular parameters of the two forms A and B were different, the immunological properties were very similar. Both were inactivated to the same degree by the antiserum against ChAT and both molecular forms gave rise to an active antiserum, when used as an antigen. These results differ from some results reported recently by SINGHet al. (1975),describing two different molecular forms of ChAT from human brain, which were separated by phosphocellulose chromatography, where only one of the forms was inactivated by the antiserum. Acknowledgements-The skilled technical assistance of Ms H. Stuw is gratefully acknowledged The authors are also indebted to Dr. T. CHRISTENSEN, Department of Biochemis try, University of Oslo, for running the analytical ultracen-

trifuge experiments. REFERENCES BANNSH. E. (1976) J . Neurochem 26,967-971. BARRYS. & OCARRA P. (1973) Biochem. J . 135, 595-607. BERMAN-REISBERG R. (1957) Yale J . Biol. Med. 29,403-435. BULLG., FENSTEIN A. & MORRISD. (1964) Nature, Lond 201, 1326. CHAOL. P. (1975) J. Neurochem. 25, 261-266. CHAO L. P. & WOLFGRAM F. (1973) J . Neurochem. 20, 1 0 7 w 08 1. CHAOL. P. & WOLFGRAM F. (1974) J . Newochem. 23, 697-701. CUATRECASAS P. (1970) J. biol. Chem. 245, 3059-3065. DAMSB. J. (1964) Ann. N . Y Acad. Sci. 121, 404427. DIEZEL W., KOPPD~SCHL~GER G. & HOFMANNE. (1972) h l y t . Biochem 40, 617620. ENG L. F., CLYEDAC. T., CHAOL. P. & WOLFGIUMF. (1974) Nature, Lond. 250, 243-245. FONNUY F. (1966) Biochem J. 100, 479484. FONNUMF. (1975a) J . Neurochem 24, 407409. FONNUMF. (1975b) in Research Methods in Neurochemistry Vol. 111, (MARKSN. & RODNIGHT R., eds) pp. 253-275.

Several reports have described the formation of antibody against ChAT from different species (ENG et al., 1974;MALIHE-SBRENSSEN, 1975;ROSIER et al., 19736;!%LJSTER & OTOOLE, 1974;SINGH & MCGEER, 1974~) and some pictures showing the immunohistochemical localization of ChAT, using two of these preparations have been published (ENQ et al., 1974; MCGEERet al., 1974; 1975). These antibody preparations, raised against bovine brain and human brain ChAT, were claimed to be monospecific (ENG et al., 1974;SINGH& MCGEER,19746),although they showed a low activity against ChAT, precipitating a maximum of 50% of the enzyme activity. The present antibody preparation inactivates more than 98% of the ChAT activity (Fig. 8). It is not monospecific (Fig. Plenum, New York. Q,c), but is assumed to be specific since it inactivates FONNUM F. & MALTHE-S~RENSSEN D. (1973) J . Neurochem ChAT from bovine brain to the same extent, indepenm, 1351-1359. dent of the specific activity of the enzyme preparation. GLOVERV. A. S. & POTTERL. T. (1971) J . Neurochem The discrepancy between the present results and those 18, 571-580. reported by ENG et al. (1974)and SINGH& MCGEER HEBB C. (1972) Physiol. Reu. 52, 918-957. (19746) is striking. The low inactivation of ChAT HUSAIN S. S. & MAUTNERH. G. (1973) Proc. MCJI A d . sci., U.S.A. m, 3749-3753. from bovine brain observed by ENGet al. (1974)could

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D.MALlHE-%RENSSEN, T. L a . F. FONNUM and T. ESKEIAND

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