Neurochemical Research (2) 281-291 (1977)

STUDIES ON CHOLINE A C E T Y L T R A N S F E R A S E ISOLATED FROM H U M A N BRAIN VIJENDRA K. SINGH AND PATRICK L. MCGEER Division of Neurological Sciences Department of Psychiatry University of British Columbia Vancouver, British Columbia V6T l W5 Canada

Accepted December 29, 1976

The purified choline acetyltransferase from human striatal tissue wag found to have a K,,~ value of 8 /zM for acetyl-coenzyme A and 250/zM for choline. The predominant enzyme component has a molecular weight of about 67,000 daltons, measured by molecular filtration through Sephadex G-100. In a sucrose-density gradient, the enzyme cosedimented with bovine serum albumin with an estimated S-value of 4.5. The enzyme activity was enhanced 2- to 3-fold by KCI, NaCI, (NH4)~SO4, and chelating agents like EDTA or EGTA. Cupric sulfate (0.1 raM) inhibited the enzyme activity almost completely. This inhibition was circumvented by increasing concentrations of enzyme protein, dithiothreitol, and EDTA, but not by the substrates, histidine, or imidazole.

INTRODUCTION The e n z y m e choline acetyltransferase (CAT) (acetyl-coenzyme A, choline-acetyltransferase, E.C. 2.3.1.6.) is responsible for the synthesis of neurotransmitter acetylcholine and is, therefore, a very important enz y m e in the nervous system. N u m e r o u s studies of the distribution, properties, and the reaction mechanism of this enzyme, in crude or partially purified preparations, have been described (l-5). Recently the e n z y m e has been purified to electrophoretic h o m o g e n e i t y from bovine brain (6), squid head ganglia (7), and human brain striatal nuclei (8). Furthermore, antibodies have been prepared against purified C A T which appeared to be highly specific as determined by immunochemical (8-11) as well as immunocytohistochemical tools (10,12,13). 281 (~) 1977 Plenum Publishing Corp., 227 West 17th Street, New York, N.Y. 10011. To promote freer access to published material in the spirit of the 1976 Copyright Law, Plenum sells reprint articles from all its journals. This availability underlines the fact that no part of this publication may be reproduced, stored in a retrieval system, or transmitted, in any form or by any means, electronic, mechanical, photocopying, microfilming, recording, or otherwise, without written permission of the publisher. Shipment is prompt; rate per article is $7.50.

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There has been some difficulty in obtaining electrophoretically pure enzyme from rat brain (14) and, thus, the purity of this enzyme protein and of the antibody to it as reported for other mammalian sources (8,10,11) has been doubted (14). Although such skepticism exists, it should be noted that the methodology of purifying mammalian CAT as developed by us (8) has been successfully reproduced (15). In this paper we wish to describe some of the important properties of an antigenic form of choline acetyltransferase which has been isolated previously by us (8,9).

EXPERIMENTAL PROCEDURE Preparation of Purified Enzyme The striatal nuclei (caudate and putamen) of human brains, obtained from accident victims 2-8 h postmortem, were used to prepare the enzyme. The enzyme was extracted by homogenization of the tissue in 50 mM potassium phosphate buffer, pH 7.4, containing 2 mM EDTA, followed by centrifugation at 34,800g for 1 h. The solubilized enzyme was first fractionated on a hydroxyapatite column (8), and finally on a phosphocellulose column (9). From the phosphocellulose column, most of the CAT activity was eluted with 0.15 M potassium phosphate buffer, pH 6.8, containing 0.5 mM dithiothreitol and 10% glycerol. The purity of this enzyme fraction was judged by SDS-polyacrylamide gel electrophoresis and immunochemical means, and was found to be a single polypeptide (9). The enzyme fraction was always dialyzed against PDG-buffer (10 mM potassium phosphate, pH 6.8, -0.5 mM dithiothreitol-10% glycerol) prior to its use in the present investigation. Enzyme Assay. The method of McGeer et al. (16), with minor modification of the reaction mixture, was used for the CAT assays. The final incubation mixture contained 50 mM potassium phosphate, pH 7.4, 10 mM choline chloride, 20 tzg of bovine serum albumin, 0.1 mM eserine sulfate, 10 mM EDTA, [14Clacetyl CoA (21,000 counts per rain/ assay of a specific activity of 4.5 x 108 disint, per min/t~mole), and enzyme fraction in a final volume of 0.1 ml. As specified in some experiments, EDTA was omitted from the assay mixture, The incubation was done at 37 ~ for 30 rain, and the reaction was terminated by the addition of 1 ml of 0.025 N perchloric acid containing 0.011 N acetic acid. To this, 1 ml of 0.5 M Tris-acetate, pH 7.0, was added, and the contents were poured on top of Amerlite CG-50 columns (approximately 5 x 40 mm in size) preequilibrated with 3 ml of 0.5 M Tris-acetate, pH 7:0. The reaction product [14C]acetylcholine was adsorbed on the resin, and, after washing of the column with about 30 ml of distilled water, was eluted with 3 ml of 4 N acetic acid directly into the counting vials, Ten milliliters Bray's scintillant was dispensed in each vial, and the radioactivity was counted with about 80% efficiency using a Nuclear Chicago liquid scintillation counter. Sephadex G-IO0 column chromatography. The swollen Sephadex G-100 gel, preequilibrated with 50 mM potassium phosphate buffer, pH 6.8, containing 0.2 mM dithiothreitol and 10% glycerol, was packed to yield a column of size 1.5 x 85 cm. The sample, containing about 80-100/zg protein in 0.5 ml, was applied on top of this column, allowed to adsorb, and, after about 50 ml of the equilibrating buffer had percolated through the column, about 130 fractions of l-l.5-ml volume each were collected. An aliquot (20 /~l) from each fraction was assayed for CAT activity. The recovery of enzyme activity in the

STUDIES ON CHOLINE ACETYLTRANSFERASE

283

column fractions was about 95-110% in three experiments whereas the protein, owing to its very low content, was not measured. The molecular-weight determination was canied out according to the method of Andrews (17). The calibration of the column was performed with nonenzymic protein markers such as y-globulin, albumin, ovalbumin, myoglobin, and cytochrome C. The source of these reference proteins was Schwartz/Mann Research Laboratories, New York. Sucrose-Density-Gradient Centrifugation. The continuous 5-20% sucrose gradients were prepared in cellulose nitrate tubes (5-ml capacity) by the procedure of Martin and Ames (18). The sucrose solutions were buffered with t0 mM potassium phosphate, pH 7.4, containing 0.2 mM dithiothreitol. After 20 rain of precooling at 4~ 0.2 ml (containing about 70 /.~g protein) of purified human brain CAT was carefully layered on top of the sucrose gradient. The tubes were then loaded into a swinging-bucket rotor #SW39 and centrifuged at a rotor speed of 37,000 rpm for 16 h at 4~ in a Beckmann Spinco Model L ultracentrifuge. After centrifugation, fractions of 12 drops each were collected by piercing the bottom of the tube with a hypodermic needle. Each fraction was then assayed for CAT activity. The yield of enzyme activity was about 90-95%. The standard protein markers such as cytochrome C (S = 2), bovine serum albumin (S = 4.5), and glucose oxidase (S = 8) were separately subjected to similar density-gradient centrifugation and analyzed in the fractions by measuring absorbance at 280 nm.

RESULTS

AND DISCUSSION

General T h e p u r i f i e d e n z y m e r e q u i r e d c h o l i n e a n d a c e t y l - C o A as s u b s t r a t e s for activity. Carnitine could not be substituted for choline, suggesting t h e l a c k o f c a r n i t i n e a c e t y l t r a n s f e r a s e in t h e e n z y m e p r e p a r a t i o n . T h e p r e s e n c e o f e s e r i n e s u l f a t e in t h e r e a c t i o n m i x t u r e h a d no e f f e c t o n t h e enzyme activity. This shows that the enzyme preparation was free of acetylcholinesterase. The removal of bovine serum albumin from the assay mixture lowered the CAT activity by 50-60%. The presence of d i t h i o t h r e i t o l ( 0 . 2 - 0 . 5 r a M ) a n d 10% (v/v) g l y c e r o l w a s a b s o l u t e l y essential for the maintenance of enzyme activity during the purification procedures. There was a direct proportionality between enzyme activity a n d p r o t e i n c o n c e n t r a t i o n . T h e r e a c t i o n w a s l i n e a r u p to 30 m i n , t h e n showed a gradual decline. The enzyme was more or less inactive below p H 5 a n d e x h i b i t e d a s h a r p p H o p t i m u m a r o u n d 7.9. P r e i n c u b a t i o n o f t h e e n z y m e f r a c t i o n at 60~ f o r 3 rain c a u s e d c o m p l e t e l o s s o f a c t i v i t y d u e to h e a t d e n a t u r a t i o n . T h e M i c h a e l i s - M e n t e n c o n s t a n t (Kin) v a l u e s f o r m a m m a l i a n c h o l i n e a c e t y l t r a n s f e r a s e , in e i t h e r c r u d e e x t r a c t s o r p a r t i a l l y p u r i f i e d f r a c t i o n s , h a v e b e e n f o u n d to b e o f t h e o r d e r 0 . 3 - 1 . 2 m M f o r c h o l i n e a n d 6 - 2 0 / x M for acetyl-CoA (2-4,19). Within the concentration range of the substrates (0-2 mM for choline and 0-100/xM for acetyl-CoA) studied for

284

SINGH AND McGEER

this purified CAT from human brain, the Lineweaver-Burk plots were linear. The extrapolated Km value was 250/xM for choline and 8/zM for acetyl-CoA.

Molecular Weight Based on gel-filtration chromatography, molecular weights of 105 or higher have been reported for CAT from rat brain (20), squid head ganglia (7), bovine brain (21), and human placenta (22). The high values have been attributed to aggregation caused by the ammonium sulfate used in the fractionation procedure (21). Since we have routinely utilized ammonium sulfate in our procedure for purifying CAT from human striatal tissue (8,9), we might expect similar results. As shown in Figure 1, it is clear that the predominant peak of enzyme activity (peak II) indicated a molecular weight of about 67,000. There was a small peak (peak I) of a high-molecular-weight (about 125,000 daltons) form of the enzyme, presumably due to aggregation. Further evidence for a molecular weight of 67,000 daltons for the most abundant species of CAT was obtained when purified enzyme was centrifuged through a linear gradient

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285

STUDIES ON CHOLINE ACETYLTRANSFERASE

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of 5-20% sucrose density; the peak of CAT activity sedimented in the same fraction as bovine serum albumin does (Figure 2). Since this reference protein has a sedimentation coefficient (S) of 4.5 and a molecular weight of approximately 67,000, human brain CAT may be expected to have a similar S value and molecular weight. This centrifugal analysis did not reveal any aggregated form of CAT. These observations lend support both to our previous measurements of 67,000 + 2,000 daltons as the molecular weight of purified human brain CAT using SDS-polyacrylamide gel electrophoresis (9) and to those reporting similar values in the literature (2,22-25).

Effects of Salts and Chelating Agents Three different salts [KCI, NaCI, and (NH4)~SO4] stimulated the purified CAT from human brain (Figure 3). KC1 was the most effective and at the optimum concentration of 0.3 M elicited about 2-fold stimulation in enzyme activity. The optimum concentration (0.3 M) of these three salts for human brain enzyme parallels closely that found for rabbit brain enzyme (26) and rat brain enzyme (27). However, the order

286

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of effectiveness appeared to be different, since rabbit brain enzyme is activated more effectively by NaC1 than by KC1 (26). The enzyme from bovine brain, on the other hand, has been variously reported as being unaffected by KC1 up to 0.3 M (2) or as requiring 0.45-0.6 M NaC1 or KC1 to cause maximum enhancement (about 2-3 times that of control) of enzyme activity (21). The mechanism of salt activation of CAT is not understood; however, it appears to be a general feature of this enzyme from various sources. EDTA is commonly used in many CAT assay mixtures and has been shown to have a stabilizing effect on CAT activity in both bacterial and bovine brain extracts (20). In contrast, other workers did not find any effect of 0.1 mM EDTA on bovine brain enzyme (2). In our experiments with purified human brain CAT, we found that chelating substances like EDTA or EGTA (at 0.2-0.4 mM concentrations) increased the enzyme activity 2- to 3-fold over the control. However, there was no absolute requirement for each of these chelating agents for the detection of CAT activity. Our preliminary evidence indicates that enzyme might aggregate in the presence of EDTA (28).

Effect of Metallic Cations Cupric sulfate at 0.1 mM concentration almost completely suppressed (95% inhibition) the CAT activity (Figure 4). At this concentration, other

287

STUDIES ON CHOLINE ACETYLTRANSFERASE

cations (MnCI~, F e S Q , and ZnSO4) had no effect. However, zinc sulfate at 5-fold higher concentration (0.5 mM) elicited about 45% inhibition. The inhibition of enzyme activity by cupric sulfate was circumvented in vitro by increasing concentrations of enzyme protein, dithiothreithol, or EDTA (Figure 5), but not by the substrates (acetyl-CoA or choline), by histidine, or by imidazole (Figure 6). The inhibition of purified CAT by cupric sulfate, as reported here, was similar to that found with other mammalian brain enzyme preparations (21,27,29). Moreover, the counteracting action of EDTA and dithiothreitol is expected because of the metal-chelating properties of the former and the thiol groups of the latter. The finding that the inhibition was also circumvented by increasing concentrations of enzyme protein, but not by additional substrate indicates that the Cu ++ probably complexes with the enzyme. The reversal of Cu++-mediated inhibition of bovine brain enzyme by histidine and the consideration that Cu ++ at low concentrations preferentially interacts with imidazole have led to the proposal that imidazole moities have a catalytic role in this enzyme (29). However,

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288

SINGH AND McGEER

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cupric ions also have a strong affinity for free sulfhydryl groups of proteins and, thus, may bind to CAT through a thiol group. Moreover, histidine or imidazole, as observed in this study, had no protective action against Cu § inhibition of human brain enzyme. These observations, however, must be interpreted with great caution because of the lack of specificity in interactions of Cu +§ with active groups of proteins. Several kinetic studies suggest a Theorell-Chance mechanism of choline acetyltransferase reaction (3,19,29,30). The inhibition experiments indicate the involvement of a sulfhydryl group (5,15,27) or a

STUDIES ON CHOLINE ACETYLTRANSFERASE

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