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A sensitive radiochemical assay for choline acetylase
ANALYTICAL
BIOCHEMISTRY
A Sensitive IVAN
24, go-95 (1968)
Radiochemical DIAMOND2
Department
Assay for Choline AND
EUGENE
Acetylase’
P. KENNEDY
of Biological Chemistry, Harvard Medical Boston, Massachusetts 0.9116
School,
Received July 81, 1W Several methods have been described for the measurement of choline acetylase (choline acetyltransferase, acetyl-CoA:choline O-acetyltransferase, EC 2.3.1.6). Chemical procedures are in general rather insensitive, while methods based on bioassay are time consuming. For these reasons, radiochemical methods are increasingly being used for the measurement of choline acetylase. In the method of Schuberth (1) , the conversion of labeled acetic acid to a nonvolatile form is the basis of the assay. The 1)rocedure has the advantages of simplicity and sensitivity, but is not highly specific. In the method of McCaman and Hunt (2), radioactive acetylcholine is separated as the reineckate, while the procedure of Fonnum i3) relies on the precipitation of acetylcholine by sodium tetraphenylborate. The method to be described here involves the adsorption of labeled acetylcholine (formed from radioactive acetate) on Amberlite CG-50 ion-exchange resin. Labeled acetate and acetyl-CoA pass through the column. The labeled acetylcholine is then recovered by washing with aqueous acid and counted. The procedure avoids the necessity for quantitative precipitation and washing encountered in the methods of McCaman and Hunt (2) and Fonnum (3) and is more specific than the procedure of Schuberth (1). Radioactive acetylcholine is virtually the sole labeled product measured by our procedure even when unfractionated brain homogenate is used as enzyme source. MATERIALS
AND
METHODS
Coenzyme A, acetyl-CoA, and ATP were purchased from P-L Biochemicals, eserine sulfate was obtained from Mann Research Laboratories, and acetate-3H from Nuclear-Chicago Corporation. ‘This research has been supported by grants from the National Institute for Neurological Diseases and Blindness, NB-02946, the National Institute for General Medical Sciences, GM-13952, and the Life Insurance Medical Research Fund. ‘Supported in part by a Special Fellowship 1 Fll NB li54-01 NSRB from the National Institute of Neurological Diseases and Blindness, Public Health Service. 90
ASSAY
FOR
CHOLINE
ACXTYLASE
91
A brain homogenate was prepared by homogenizing an adult guinea pig brain at 0’ in 0.01 M potassium phosphate buffer of pH 7.4 (9 ml/m fresh brain) followed by sonication in a Branson sonifier for 3 min. Preparation of acetyl-CoA synthdase: Bovine mitochondrial acetylCoA synthetase, purified through the second ammonium sulfate precipita! e step by the procedure of Campagnari and Webster (4), was kindly made available to us by Dr. A. R. Tarlov, The synthetase had retained most of its activity after storage at -20°C for over three years. The amount of acetyl-CoA synthetase needed to generate optimum amounts of labeled acetyl-CoA was determined in preliminary experiments. The enzyme was routinely diluted in 0.1 M potassium phosphate buffer of pH 7.2 containing 5 mg/ml bovine strum albumin. Assay of acetylcholine synthesis coupled to acetyE-CoA synthetase: The standard incubation mixture for the coupled assay contained the following components in a final volume of 0.2 ml: potassium phosphate (0.11 M)/tris (50 m&I) buffer of pH 7.8; MgSO, (4 mu) ; 2-mercaptoethanol (5 mM) ; eserine sulfate (0.2 mM) ; CoA-SH (0.2 mM) ; ATP (9 m&f) ; choline chloride (5 mM) ; acetate-3H (1 mM) ; acetyl-CoA synthetase (32 pg protein) ; and brain homogenate (0.4 mg protein) or other source of choline acetylase. Prior to addition of the brain homogenate the mixture was preincubated for 15 min at 37” to generate acetyl-3H-CoA; the reaction was then begun by addition of 0.04 ml brain homogenate. After incubation for 30 min at 37” the reaction was stopped by addition of 3.0 ml of 50% (v/v) ethanol containing 10 ,umoles of carrier acetylcholine chloride. The samples were centrifuged and an aliquot (2.0 ml) of the supernatant was passed over a small column containing 1.5 ml bed volume of the sodium form of Amberlite CG-50, lOO200 mesh, supported on fritted-glass filters (Pyrex 36290-3C). This eluate was discarded. The column was next washed 3 times with 3.0 ml portions of 1 mM sodium acetate and the eluate, containing acetate-3Hand acetyl-3H-CoA was discarded. The column was then washed three times with 2 ml portions of 1 N HCI. The combined fractions were collected in a graduated centrifuge tube. From preliminary experiments with authentic 14C-acetylcholine, it was determined that the pooled acid fraction contains at least 94% of the labeled acetylcholine. An aliquot (0.5 ml) was mixed with 10 ml of the counting solution described by Patterson and Green (5) and the radioactivity determined in a Packard scintillation spectrometer. An aliquot of the labeled acetate was counted under identical conditions to determine its specific activity. The results are expressed as millimicromoles of labeled acetylcholine recovered. One unit of acetylase is defined as that amount which produces one micromole of product per minute under the conditions of the assay.
92
DIAMOND
AND
KENNEDY
RESULTS
Properties of the system: The dependence of the system upon added
acetyl-CoA synthetase, choline, and eserine is shown in Table 1. The acetyl-CoA synthetase preparation was found to contain traces of choline acetylase activity. To correct for the amount of synthesis in the absence of added brain homogenate, a blank value, representing usually about 0.4 mpmole of acetylcholine, was subtracted from the experimental values shown in later figures. With crude brain homogenates, the response is linear with added amounts of enzyme up to about 0.4 mg of total protein (Fig. 1). Above
ATP,
ii iz E
4.0 .-----./
3.0-
z;; 2 s 2.0ZCE 2s l.Olz
./ / ./’ ,r
I I I 0.1 0.2 0.4 0.6 0.8 BRAIN HOMOGENATE (mg
I 1.0
PROTEIN)
FIG. 1. Assay of crude brain homogenate. The described for the standard assay under “Methods.” acetate-% was 1.4 X l(P cpm/gmole.
conditions of reaction The specific activity
are as of the
that amount, the response levels off rapidly. The reaction followed a linear rate with time for at least 30 min (Fig. 2). The over-all system was active over a rather broad pH range between 7.2 and 8.7, with an optimum about 7.8. Identification of labeled product: The fraction eluted with 1 N HCl (6 ml), derived from a typical assay such as that shown in Table 1, was neutralized with 2 N Tris free base (4.0 ml) to about pH 8.0. A 5 ml portion of the neutral solution was treated with ammonium reineckate (1.0 ml of a 2% methanolic solution), and chilled for 40 min. The crystalline acetylcholine reineckate was removed by centrifugation. Ninety-three per cent of the radioactivity was recovered as the reineckate. The reineckate was dissolved in a small volume of acetone and an
ASSAY
FOR
CHOLINE
93
ACETYLASE
aliquot spotted on Whatman #l paper. Ascending chromatography was carried out in a solvent system containing 0.2M ammonium acetate buffer of pH 4.8 (30 parts by volume) and acetone (70 parts). After drying, the paper strip was cut into sections and counted. Ninety-three
15
30 TIME
45
60
(mmutes)
Fro. 2. Time curve of reaction. The conditions are as described for the standard assay under “Methods.” The specific activity of the acetate-3H was 1.4 X l@ cpm/ pmole.
per cent of the radioactivity was recovered in a single spot with an Rf of 0.82, exactly coincident with that of authentic “C-acetylcholine carried through the entire procedure.
--
Components
TABLE 1 Required for Assay of Choline Acetylase
SY8teIll Complete Zero time control Omit ATP Omit CoA-SH Omit eserine sulfate Omit choline chloride Omit acetyl-CoA synthetase Omit brain homogenate
Acetylcholine
formed,
mpmoles
3.1 0 0 0.3 0.8 0.4 0.3 0.4
Conditions of reaction were as described for the standard assay under “Methods” except that the ATP concentration was 10 m&f. Specific activity of acetate-W was 1.4 X 10’ cpm/ctmole.
94
DIAMOND
AND
KENNEDY
DISCUSSION
Nearly all of the assays for choline acetylase described in the literature offer special advantages for one application or another. The present method was developed to meet the need for a highly sensitive assay specific enough to be used even with crude homogenates. The sensitivity of the method is limited only by the specific activity of the available acetate. With tritiated acetate of only moderately high specific activity, crude homogenates may readily be assayed in a range which is linear with added enzyme (Fig. 1). It is not easy to compare the results of this assay with that of others, since different experimental conditions are used, but it appears that the production of acetylcholine by crude homogenates tested under conditions similar to those of Figure 1 is about 25405% of the values reported by Fonnum (3) for forebrain of the guinea pig. The procedure reported here is similar in principle to the method of Reed, Goto, and Wang (6), who have measured the hydrolysis of labeled acetylcholine by an ion-exchange method. The chromatographic procedure described here for the isolation of labeled acetylcholine may easily be carried out on 20 or more samples simultaneously. Replicate assays of the standard system usually agreed TABLE 2 Replicate Assays under Varied Conditions Radioactivity remvered in acetylcholine fraction, total opm
System
Standard assay 15 min incubation
2,420 2,361 2,222
45 min incubation
omit acetyl-CoA
Control:
5,338 5,416 5,397 synthetase
recovery of added authentic
397 427 397 14C-acetylcholine (25,543 cpm)
23,845 24,302 24,056
Conditions of reaction were as described for the standard assay under “Methods.” Specific activity of acetate-aH was 1.4 X lo6 cpm/pmole. Specific activity of authentic 1”Cacetylcholine was 4 X lo6 cpm/pmole.
ASSAY
FOR
CHOLINE
ACETYLASE
95
with a standard deviation of less than 5%. The actual results of assays carried out in triplicate under standard conditions and with some variations are shown in Table 2. SUMMARY
A sensitive radiochemical assay for choline acetylase has been devised based on the formation of acetylcholine from tritium-labeled acetate. The acetylcholine is separated from other labeled components by chromatography on a short column of Amberlite CG-50 ion exchanger. The procedure is sufficiently specific and sensitive so that it can be used for the measurement of the acetylase in crude, unfractionated homogenates of brain. 1. 2. 3. 4. 5. 6.
REFERENCES SCHUBERTH, J., Acta Chem. Stand. 17, 8233 (1963). MCCAMAN, R. E., AND HUNT, J. M., J. Neurochem. 12, 253 (1965). FONNUM, F, Biochem. J. 100, 479 (1966). CAMPAQNARI, F., AND WEBSTER, L. T., JR., J. Biol. Chem. 238, 1628 (1963). PATTEBSON, M. S., AND GREENE, R. C., Anal. Chem. 37, 854 (1965). REED, D. J., GOTO, K., AND WANQ, C. H., Anal. Biochem. 16, 59 (1966).
BIOCHEMISTRY
A Sensitive IVAN
24, go-95 (1968)
Radiochemical DIAMOND2
Department
Assay for Choline AND
EUGENE
Acetylase’
P. KENNEDY
of Biological Chemistry, Harvard Medical Boston, Massachusetts 0.9116
School,
Received July 81, 1W Several methods have been described for the measurement of choline acetylase (choline acetyltransferase, acetyl-CoA:choline O-acetyltransferase, EC 2.3.1.6). Chemical procedures are in general rather insensitive, while methods based on bioassay are time consuming. For these reasons, radiochemical methods are increasingly being used for the measurement of choline acetylase. In the method of Schuberth (1) , the conversion of labeled acetic acid to a nonvolatile form is the basis of the assay. The 1)rocedure has the advantages of simplicity and sensitivity, but is not highly specific. In the method of McCaman and Hunt (2), radioactive acetylcholine is separated as the reineckate, while the procedure of Fonnum i3) relies on the precipitation of acetylcholine by sodium tetraphenylborate. The method to be described here involves the adsorption of labeled acetylcholine (formed from radioactive acetate) on Amberlite CG-50 ion-exchange resin. Labeled acetate and acetyl-CoA pass through the column. The labeled acetylcholine is then recovered by washing with aqueous acid and counted. The procedure avoids the necessity for quantitative precipitation and washing encountered in the methods of McCaman and Hunt (2) and Fonnum (3) and is more specific than the procedure of Schuberth (1). Radioactive acetylcholine is virtually the sole labeled product measured by our procedure even when unfractionated brain homogenate is used as enzyme source. MATERIALS
AND
METHODS
Coenzyme A, acetyl-CoA, and ATP were purchased from P-L Biochemicals, eserine sulfate was obtained from Mann Research Laboratories, and acetate-3H from Nuclear-Chicago Corporation. ‘This research has been supported by grants from the National Institute for Neurological Diseases and Blindness, NB-02946, the National Institute for General Medical Sciences, GM-13952, and the Life Insurance Medical Research Fund. ‘Supported in part by a Special Fellowship 1 Fll NB li54-01 NSRB from the National Institute of Neurological Diseases and Blindness, Public Health Service. 90
ASSAY
FOR
CHOLINE
ACXTYLASE
91
A brain homogenate was prepared by homogenizing an adult guinea pig brain at 0’ in 0.01 M potassium phosphate buffer of pH 7.4 (9 ml/m fresh brain) followed by sonication in a Branson sonifier for 3 min. Preparation of acetyl-CoA synthdase: Bovine mitochondrial acetylCoA synthetase, purified through the second ammonium sulfate precipita! e step by the procedure of Campagnari and Webster (4), was kindly made available to us by Dr. A. R. Tarlov, The synthetase had retained most of its activity after storage at -20°C for over three years. The amount of acetyl-CoA synthetase needed to generate optimum amounts of labeled acetyl-CoA was determined in preliminary experiments. The enzyme was routinely diluted in 0.1 M potassium phosphate buffer of pH 7.2 containing 5 mg/ml bovine strum albumin. Assay of acetylcholine synthesis coupled to acetyE-CoA synthetase: The standard incubation mixture for the coupled assay contained the following components in a final volume of 0.2 ml: potassium phosphate (0.11 M)/tris (50 m&I) buffer of pH 7.8; MgSO, (4 mu) ; 2-mercaptoethanol (5 mM) ; eserine sulfate (0.2 mM) ; CoA-SH (0.2 mM) ; ATP (9 m&f) ; choline chloride (5 mM) ; acetate-3H (1 mM) ; acetyl-CoA synthetase (32 pg protein) ; and brain homogenate (0.4 mg protein) or other source of choline acetylase. Prior to addition of the brain homogenate the mixture was preincubated for 15 min at 37” to generate acetyl-3H-CoA; the reaction was then begun by addition of 0.04 ml brain homogenate. After incubation for 30 min at 37” the reaction was stopped by addition of 3.0 ml of 50% (v/v) ethanol containing 10 ,umoles of carrier acetylcholine chloride. The samples were centrifuged and an aliquot (2.0 ml) of the supernatant was passed over a small column containing 1.5 ml bed volume of the sodium form of Amberlite CG-50, lOO200 mesh, supported on fritted-glass filters (Pyrex 36290-3C). This eluate was discarded. The column was next washed 3 times with 3.0 ml portions of 1 mM sodium acetate and the eluate, containing acetate-3Hand acetyl-3H-CoA was discarded. The column was then washed three times with 2 ml portions of 1 N HCI. The combined fractions were collected in a graduated centrifuge tube. From preliminary experiments with authentic 14C-acetylcholine, it was determined that the pooled acid fraction contains at least 94% of the labeled acetylcholine. An aliquot (0.5 ml) was mixed with 10 ml of the counting solution described by Patterson and Green (5) and the radioactivity determined in a Packard scintillation spectrometer. An aliquot of the labeled acetate was counted under identical conditions to determine its specific activity. The results are expressed as millimicromoles of labeled acetylcholine recovered. One unit of acetylase is defined as that amount which produces one micromole of product per minute under the conditions of the assay.
92
DIAMOND
AND
KENNEDY
RESULTS
Properties of the system: The dependence of the system upon added
acetyl-CoA synthetase, choline, and eserine is shown in Table 1. The acetyl-CoA synthetase preparation was found to contain traces of choline acetylase activity. To correct for the amount of synthesis in the absence of added brain homogenate, a blank value, representing usually about 0.4 mpmole of acetylcholine, was subtracted from the experimental values shown in later figures. With crude brain homogenates, the response is linear with added amounts of enzyme up to about 0.4 mg of total protein (Fig. 1). Above
ATP,
ii iz E
4.0 .-----./
3.0-
z;; 2 s 2.0ZCE 2s l.Olz
./ / ./’ ,r
I I I 0.1 0.2 0.4 0.6 0.8 BRAIN HOMOGENATE (mg
I 1.0
PROTEIN)
FIG. 1. Assay of crude brain homogenate. The described for the standard assay under “Methods.” acetate-% was 1.4 X l(P cpm/gmole.
conditions of reaction The specific activity
are as of the
that amount, the response levels off rapidly. The reaction followed a linear rate with time for at least 30 min (Fig. 2). The over-all system was active over a rather broad pH range between 7.2 and 8.7, with an optimum about 7.8. Identification of labeled product: The fraction eluted with 1 N HCl (6 ml), derived from a typical assay such as that shown in Table 1, was neutralized with 2 N Tris free base (4.0 ml) to about pH 8.0. A 5 ml portion of the neutral solution was treated with ammonium reineckate (1.0 ml of a 2% methanolic solution), and chilled for 40 min. The crystalline acetylcholine reineckate was removed by centrifugation. Ninety-three per cent of the radioactivity was recovered as the reineckate. The reineckate was dissolved in a small volume of acetone and an
ASSAY
FOR
CHOLINE
93
ACETYLASE
aliquot spotted on Whatman #l paper. Ascending chromatography was carried out in a solvent system containing 0.2M ammonium acetate buffer of pH 4.8 (30 parts by volume) and acetone (70 parts). After drying, the paper strip was cut into sections and counted. Ninety-three
15
30 TIME
45
60
(mmutes)
Fro. 2. Time curve of reaction. The conditions are as described for the standard assay under “Methods.” The specific activity of the acetate-3H was 1.4 X l@ cpm/ pmole.
per cent of the radioactivity was recovered in a single spot with an Rf of 0.82, exactly coincident with that of authentic “C-acetylcholine carried through the entire procedure.
--
Components
TABLE 1 Required for Assay of Choline Acetylase
SY8teIll Complete Zero time control Omit ATP Omit CoA-SH Omit eserine sulfate Omit choline chloride Omit acetyl-CoA synthetase Omit brain homogenate
Acetylcholine
formed,
mpmoles
3.1 0 0 0.3 0.8 0.4 0.3 0.4
Conditions of reaction were as described for the standard assay under “Methods” except that the ATP concentration was 10 m&f. Specific activity of acetate-W was 1.4 X 10’ cpm/ctmole.
94
DIAMOND
AND
KENNEDY
DISCUSSION
Nearly all of the assays for choline acetylase described in the literature offer special advantages for one application or another. The present method was developed to meet the need for a highly sensitive assay specific enough to be used even with crude homogenates. The sensitivity of the method is limited only by the specific activity of the available acetate. With tritiated acetate of only moderately high specific activity, crude homogenates may readily be assayed in a range which is linear with added enzyme (Fig. 1). It is not easy to compare the results of this assay with that of others, since different experimental conditions are used, but it appears that the production of acetylcholine by crude homogenates tested under conditions similar to those of Figure 1 is about 25405% of the values reported by Fonnum (3) for forebrain of the guinea pig. The procedure reported here is similar in principle to the method of Reed, Goto, and Wang (6), who have measured the hydrolysis of labeled acetylcholine by an ion-exchange method. The chromatographic procedure described here for the isolation of labeled acetylcholine may easily be carried out on 20 or more samples simultaneously. Replicate assays of the standard system usually agreed TABLE 2 Replicate Assays under Varied Conditions Radioactivity remvered in acetylcholine fraction, total opm
System
Standard assay 15 min incubation
2,420 2,361 2,222
45 min incubation
omit acetyl-CoA
Control:
5,338 5,416 5,397 synthetase
recovery of added authentic
397 427 397 14C-acetylcholine (25,543 cpm)
23,845 24,302 24,056
Conditions of reaction were as described for the standard assay under “Methods.” Specific activity of acetate-aH was 1.4 X lo6 cpm/pmole. Specific activity of authentic 1”Cacetylcholine was 4 X lo6 cpm/pmole.
ASSAY
FOR
CHOLINE
ACETYLASE
95
with a standard deviation of less than 5%. The actual results of assays carried out in triplicate under standard conditions and with some variations are shown in Table 2. SUMMARY
A sensitive radiochemical assay for choline acetylase has been devised based on the formation of acetylcholine from tritium-labeled acetate. The acetylcholine is separated from other labeled components by chromatography on a short column of Amberlite CG-50 ion exchanger. The procedure is sufficiently specific and sensitive so that it can be used for the measurement of the acetylase in crude, unfractionated homogenates of brain. 1. 2. 3. 4. 5. 6.
REFERENCES SCHUBERTH, J., Acta Chem. Stand. 17, 8233 (1963). MCCAMAN, R. E., AND HUNT, J. M., J. Neurochem. 12, 253 (1965). FONNUM, F, Biochem. J. 100, 479 (1966). CAMPAQNARI, F., AND WEBSTER, L. T., JR., J. Biol. Chem. 238, 1628 (1963). PATTEBSON, M. S., AND GREENE, R. C., Anal. Chem. 37, 854 (1965). REED, D. J., GOTO, K., AND WANQ, C. H., Anal. Biochem. 16, 59 (1966).