Synthesis of a naphthylvinylpyridine derivative and its use for affinity chromatography of choline acetyltransferase

ANALYTICAL

BIoCHEMISTRY

133, 120- 125 ( 1983)

Synthesis of a Naphthylvinylpyridine Derivative and its Use for Affinity Chromatography of Choline Acetyltransferase COSTANTINO COZZARI’ AND BOYD K. HARTMAN~ Department of Psychiatry and Neurobiology. Washington University School of Medicine, St. Louis, Missouri 63110 Received January 3 1, 1983 N-( lO-carboxy)decamethylene-4( I-naphthylvinyl)pyridinium chloride, a derivative of the choline acetyltransferase (CAT) inhibitor naphthylvinylpyridine (NVP) was synthesized and used as a l&and for affinity chromatography of choline acetyltransferase. The preparation of this inhibitor included the quatemization of naphthylvinylpyridine with 11Br-undecanoic acid methyl ester to obtain N-( 1O-carbomethoxy)decamethylene-4-( 1-naphthylvinyl)pyridinium bromide, followed by hydrolysis to free the carboxylic group. This inhibitor (C, r-NVP+) had a potency comparable to that of N-methyM( 1-naphthylvinyl) pyridinium iodide (Cr-NVP+) which is the most potent derivative of NVP but which lacks a functional group for conjugation to Sepharose. The C,,NVP+ was then bound through the carboxylic group to aminoalkyl Sepharose by a carbodiimide promoted condensation reaction. Interaction of CAT with the inhibitor retarded its elution from a column of Sepharose-Cr ,-NVP+ and permitted the purification of the enzyme to electrophoretic homogeneity starting from a preparation in which CAT represented about 20% of the total proteins. Conventional procedures of protein purification had previously been unsuccessful in isolating the enzyme in pure form. Inhibition studies showed that CAT could exhibit either a “high” or a “low” sensitivity to inhibition by naphthylvinylpyridine and its derivatives (I,, with C,-NVP+ = 0.57 pM or 5.2 PM). A direct relationship existed between the sensitivity of CAT to these inhibitors and the retention of the enzyme by the a8inity column. KEY WORDS: choline acetyltransferase; purification; affinity chromatography; naphthylvinylpyridine; inhibition.

Choline acetyltransferase (acetyl-CoA: choline-O-acetyltransferase, EC 2.3-l .6, CAT)3 is the enzyme which catalyzes the synthesis of acetylcholine. The difficulties encountered in the purification of this enzyme are well known (1). Even the recent advances using alhnity chromatography over Sepharose conjugates of CoA (2) or Blue Dextran (3) in conjunction

with conventional procedures have yielded only partially purified enzyme. We have achieved purification of the enzyme from bovine caudate nucleus (4,5) to a higher specific activity than previously reported. In its pure form, CAT consists of two active molecular forms with molecular weights of 72,000 and 76,000 (5). Separate injection of these two forms of CAT into rabbits produced antisera which precipitate CAT activity and meet stringent criteria of immunochemical specificity (4). The method of purification used in these studies included affinity chromatography over a new derivative of the CAT inhibitor naphthylvinylpyridine which was covalently linked to Sepharose. This chromatography step was essential to bring CAT from a stage where it represented only approximately 20% of the protein to the stage of electrophoretic

’ Present address L&oratorio di Biologia Cellulare, Via Romagnosi 18/A 00196 Rome. ’ To whom reprint requests should be addressed. 3 Abbreviations used: CAT, choline-0-acetyltransferam; BSA, bovine serum albumin; DTE, dithioerythritoh NVP, 4-( 1-naphthylvinyl)pyridine; Cr-NVP+, N-methyl-4-( Inaphthylvinyl)pyridinium iodide; &NVP+, N-(5-carboxy)pentamethylene - 4 - (1 - naphthylvinyl)pyridinium chloride; C, ,-NVP+, N-( lO-carboxy)decamethylene-4( lnaphthylvinyl)pyridinium chloride; Sepharose-C~r-NVP+, affinity column of Sepharose conjugated to C,,-NVP+; SDS, sodium dodecyl sulfate. 0003-2697183 $3.00 Copyright 0 1983 by Academic Press. Inc. All rights of reproduction in any form reserved.

120

AFFINITY

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OF

homogeneity on pH 4.3 gradient acrylamide gels and SDS gels. This paper describes the method of inhibitor synthesis, its conjugation to Sepharose4B and its use in affinity chromatography of CAT. MATERIALS

AND

METHODS

Cyanogen bromide, 3,3’-diaminodipropylamine, 1-naphthaldehyde, acetic anhydride, were purchased from Aldrich Chemical Company (Milwaukee, Wise.). 4Picoline, 6-bromohexanoyl chloride, 1 1-bromoundecanoic acid, were purchased from Eastman Organic Chemicals (Rochester, N. Y.). Sepharose 4B-200, 1-ethyl-3(3-dimethylaminopropyl)carbodiimide-HCl; choline chloride, physo&mine sulfate, Dowex 1 X 8-400, BSA, DTE, were purchased from Sigma Chemical Company (St. Louis, MO.). CI-NVPf was purchased from Calbiochem (La Jolla, Calif.). CoASAc from ICN Life Science Group (Plainview, N. Y.). CoASAc (acetyl-l-‘4C) 40-60 mCi/ mmol from New England Nuclear (Boston, Mass.). All other reagents used were analytical grade reagents. Enzyme Assay for CAT

The radiometric method of Shrier and Shuster (6) was used with the following final concentration of reagents: 0.4 mM CoASAc (acetyl-l-‘4C, 0.7 mCi/mmol), 20 mM choline chloride, 400 mrvr potassium chloride, 0.2 mM physostigmine sulfate, 1 mM EDTA, 0.1 mM DTE, 0.5 mg/ml BSA, 50 mM Tris-HCl buffer, pH 7.4, and enzyme in a total volume of 0.2 ml. Assays were incubated for 10 min at 37°C. One unit of enzyme activity (1 U) was defined as 1 pM of acetylcholine formed min-’ at 37°C. Additional details have been presented (5). Protein concentration was determined by the method of Geiger and Bessman (7) and BSA was used as the standard. Acrylamide gel electrophoresis was performed as described by Weber and Osborn (8).

CHOLINE

ACETYLTRANSFERASE

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Preparation of N-(Scarboxy)Pentamethylene-4(1-naphthylvinyifjpyridinium bromide (C&VP’)

The structural formulas for some NVP derivatives used in these experiments are shown in Fig. 1. All procedures involving NVP or its derivatives were carried out under light-protected conditions. The C6-NVP+ derivative was prepared using a modification of the method of Husain and Mautner (9). Briefly, 6-Br-hexanoic acid ethyl ester (prepared by reaction between 6-Br-hexanoyl chloride and ethyl alcohol) was refluxed with naphthylvinylpyridine in isopropyl alcohol. The N-(Qarboethoxy)pentamethylene -4( - 1 - naphthylvinyl)pyridinium bromide obtained was precipitated by the addition of hexane, hydrolyzed with 3 N hydrochloric acid, and the N-(5-carboxy)pentamethylene - 4 - ( 1 - naphthylvinyl) pyridinium chloride obtained (compound CsNVP+, Fig. 1) was purified by recrystallization from 95% ethyl alcohol. Preparation of the Inhibitor N-(lO-Carboxy)Decamethylene-4-(I-naphthylvinylk Pyridinium Chloride (C,,-NVP+) (1) Preparation of naphthylvinylpyridine (NVP). The method of Cavallito et al. (10)

was followed. A mixture of 0.4 mol of 1-naphthaldehyde, 0.4 mol of 4-picoline, and 0.6 mol of acetic anhydride were refluxed 15 h. After cooling, the reaction mixture was poured into 1 liter of 10% (w/v) sodium hydroxide. The mixture was further cooled over ice for at least 3 h and the supematant was decanted from the precipitate which was then washed several times with water. The water-washed precipitate was dissolved in 1 liter of benzene, and the hydrochloride salt was produced by the addition of concentrated hydrochloric acid (approximately 40-50 ml). After cooling for half an hour at 8-10°C the precipitate was removed by filtration and dissolved in 95% ethyl alcohol. Anhydrous diethyl ether was added to incipient precipitation and NVP hy-

122

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-

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funnel, and the water which collected at the bottom was removed. The 11-Br-undecanoic 8 acid methyl ester was collected after evaporation of the diethyl ether under vacuum, , , -CH=CH-o-CH3 C,-NVP+ yielding 21 g of product. \/ (3) Quaternization of NVP with 11-Br-undecanoic acid methyl ester. NVP (3 g) and ll, -CH=CHH-‘(-&CH~)~-COOH ClC
,

/

8

CH=CH-0

AFFINITY

CHROMATOGRAPHY

OF CHOLINE

solution of 2 M 3,31diaminodipropylamine previously titrated to pH 10 with 6 N HCI. Following reaction for 16 h at 4°C the conjugated Sepharose was exhaustively washed with large volumes of distilled water and stored at 4°C in presence of 0.02% sodium azide. Conjugation of Cl,-NVP+ to AminoalkylSepharose (Preparation of SephC,,-NVP+)

One gram of Ci ,-NVP+ was dissolved with 300 ml of conjugation solution which consisted of- N,N’-dimethylformamide:water:npentyl alcohokisopropyl alcohol in ratios of 50:50:12:5. After filtration the solution was added to 100 ml (packed volume) of aminoalkyl-sepharose previously equilibrated in conjugation solution. The suspension was stirred and 10 ml of 0.8 M 1-ethyl-3-(3dimethylaminopropyl)carbodiimide hydrochloride were slowly added. The reaction was continued for at least 24 h with continuous stirring at room temperature. The Seph-C1 i-NVP+ was then exhaustively batch washed over a 20-h period as follows: eight times with 1 liter of conjugation solution, six times with 1 liter of 20% isopropyl alcohol, eight times with 1 liter of 10% ethyl alcohol. Finally, the Seph-C, ,-NVP+ was equilibrated with 25 IIIM sodium citrate buffer, 0.2 M sodium chloride, 1 mM EDTA, 0.2 mM DTE, 2% (v/v) ethyl alcohol, pH 5.7, and packed by gravity into a 1 X 80-cm (long) column and into a I X IO-cm (short) column. These affinity columns were stable for at least a year when kept at 4°C completely protected from light.

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final concentration of ethyl alcohol. It has been shown that ethyl alcohol does not decrease enzymatic activity at these concentrations (5). CAT activity in the absence of inhibitor was considered to be 100%. Blanks consisted of the assay run in the absence of enzyme at each inhibitor concentration. The Is0 values were read from a curve of percent inhibition vs the log of inhibitor concentration. RESULTS AND DISCUSSION The Inhibition of Various CAT Preparations by Several Derivatives of NVP

The results of inhibition studies performed with three derivatives of NVP are summarized in Table 1. The Iso is the concentration of inhibitor which produces 50% inhibition of CAT activity. Somewhat surprisingly, CAT from different brain extracts could exhibit either a “high” or a “low” sensitivity to inhibition by NVP derivatives. The data in column A represents the results obtained on crude extracts from nine preparations of CAT. The results presented in column B were found for crude extracts used for four enzyme preparations. It is important to point out that no major variations of the Is0 were observed during processing of individual preparations. Thus the same IO-fold difference in sensitivity to inhibition was observed when comparing the final purified enzymes from different prepaTABLE INHIBITION

OF BOVINE

VINYLPYRIDINE ARATIONS

CAT

DERIVATIVES WITH

HIGH

I BY SEVERAL SHOWING

(A) AND

Low

NAPHTHYLENZYME

PREP-

(B) 1,, VALUES

Determination of the I,, Inhibitor

Inhibition of constant CAT activity was evaluated in duplicate at eight different inhibitor concentrations. Inhibitors were first dissolved in 60% ethyl alcohol (some compounds are poorly soluble in water) and the assays were done in the presence of 3% (v/v)

C,-NVP+

C,-NVP+ C, ,-NVP+ ‘The enzyme

A

5.2 330 19

* 2 f 180 +9

range of 1, determination preparations.

B

0.57 f 0.16 36+ 18 2.2 + 0.7 on at least

four

different

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AND

HARTMAN

20% of the proteins and exhibited a specific activity of 26 U/mg. This represented a purification of approximately 8000-fold compared to the homogenate (5). However, the enzyme preparation at that stage contained at least 10 different proteic forms, as shown by SDS gels whereas only two protein bands, with il&‘s of 72,000 and 76,000, were observed from the enzyme pools reported in Figs. 2A and B regardless of whether the enzyme preparation exhibited a high or a low sensitivity to NVP derivatives. Two protein bands were also visualized on acrylamide gradient gels (5) (7.5 to 20% acrylamide) at pH 4.3 (data not shown). In this case it was possible to demonstrate that both bands, termed CAT-A and Ajinity Chromatography of CAT over CAT-B, exhibited CAT activity (5). The idenSepharose-Cll-NVP’ tification of the enzyme was further conhrmed As expected, a direct relationship existed by demonstrating that when these bands were between the sensitivity of CAT to inhibition injected separately into rabbits both induced by C,,-NVP+ and the retention of CAT by anti-CAT antibodies (4). Three properties of the affinity column of Sepharose-C, l-NVP+ columns. Therefore, a 1 Seph-C,,-NVP+ permit the purification. The X W-cm column was used for the preparations which had an Z50between 10 and 28 pM (sim- first protein fraction which elutes from the ilar to Table 1, column A) with C, i-NVP+. If column in the void volume has no affinity for this “long” column was used for affinity chro- the inhibitor or the matrix. The second protein matography of enzyme preparations having peak which contains pure CAT is retarded by an Isa between 1.5 and 2.9 pM (similar to its affinity for the inhibitor. Although it is not clear from the chromatogram, a major portion Table 1, column B) the elution of enzyme occurred in a volume exceeding 200 ml. It of the proteins (40-60%) are separated from CAT by being retained on the column by hywas found that in these cases a 1 X lo-cm column produced an equivalent degree of res- drophobic interaction with the extensive aliphatic chain introduced to attach the inhibitor. olution with the enzyme eluting in approxiThe composition of the eluting solvent is immately 25 ml. The recovery of CAT activity portant in this regard. The concentration of from the column was excellent (approximately 70-80%) regardless of its affinity for the in- ethanol, the pH, and ionic strength were op timized for separation. The remaining proteins hibitor when the appropriate length of column was used. The enzyme which eluted from the could be eluted only by exhaustive washing affinity column had a specific activity of be- (700 ml) with the column buffer. tween 120 and 160 U/mg. Representative Since a major factor in the purification was chromatograms of both cases are shown in the hydrophobic affinity of some contaminants to the column (ionic binding to the posFigs. 2A and B. The degree of purity of the enzyme before itively charged inhibitor is unlikely since folchromathe application of affinity chromatography is lowing O-(carboxymethyl)-cellulose tography (5) all the proteins present in the important for the successful use of this method. Before affinity chromatography the preparation were slightly basic), it was important to demonstrate that the column was enzyme already constituted approximately

rations as was seen in their respective crude extracts. The relative potency of the three derivatives of NVP should be noted in Table 1. The most potent is Ci-NVP+. Adding an aliphatic chain increases the ZsO.However, it was observed that C, ,-NVPf was approximately 15-fold more potent than C6-NW+ and only slightly less potent than &NVP+. The Cl,NVP+ was, therefore, used for affinity chromatography. The fact that CAT could exhibit either a “high” or a “low” sensitivity to NVP derivatives did not produce a major problem for the use of Seph-C,,-NVP+ as an affinity column.

AFFINITY

CHROMATOGRAPHY

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125

ACETYLTRANSFERASE

to

20 30 FRACTION NUMBER

40

FIG. 2. Affinity chromatography of CAT on Sepharose-C,,-NVP+. Chromatogmphic pattern of CAT with low sensitivity to C, ,-NVP+ (Zso= 20 PM) on a 1 X 80-cm column (pattern A) and of CAT with high sensitivity to C,,-NVP+ (iso = 2.2 pM) on a 1 X IO-cm column (pattern B). In both cases, elution of the enzyme was retarded by interaction with the inhibitor. Flow rate was 2 ml/h and 1.5ml fractions were collected.

not separating two high specific activity forms of CAT from a larger quantity of low specific activity forms of the enzyme. Two facts strongly indicate this is not the case. First, the antisera prepared were capable of quantitatively precipitating CAT activity at all stages of purification (5) indicating there are no forms of CAT which are not recognized by the antisera we produced. Second, the quantity of antiserum required to titrate 10V3 units of enzyme activity was essentially the same before and after affinity chromatography (5). If low specific activity forms were removed during this step, one would predict a marked decrease in the quantity of antiserum/unit CAT activity following chromatography. ACKNOWLEDGMENTS This work was supported in part by Grants NS-123 11, NS-13672, and Research Scientist Development Award MH-7045 1 fBKH).

REFERENCES 1. Rossier, J. (1977) ht. Rev. Neurobiol. 20, 284-330. 2. Ryan, R. L., and McClure, W. 0. (1979) Biochemistry 18,5357-5365.

Hersh, L. B., Coe, B., and Casey, L. ( 1978) J. Neurochem. 30, 1077-1085. 4. Cozzari, C., and Hartman, B. K. (1980) Proc. Natl. Acad. Sci. USA 17,7453-7457. 5. Cozzari, C., and Hartman, B. K. ( 1983) J. Biol. Chem., in press. 6. Schrier, B. K., and Shuster, L. (1967) J. Neurochem. 3.

C&977-985. 7.

Geiger, P. J., and Bessman, S. P. (1972) Anal. B&hem.

8.

Weber, K., and Osbom, M. (1969) J. Biol. Chem.

49,467-473. 244,4406-4412.

Husain, S. S., and Mautner, H. G. (1973) Proc. Natl. Acad. Sci. USA 10. Cavallito, C. J., Yun, H. S., Smith, J. C., and Foldes, F. F. (1969)J. Med. Chem. 12, 134-138. 11. Cuatrecasas, P., and An&en, C. B. (197 1) in Methods in Enzymology (W. B. Jakoby, ed.), Vol. 22, pp. 345-378, Academic Press, New York. 9.