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A radiometric method for the determination of choline acetylase activity based on thin-layer chromatography
ANALYTICAL
BIOCHEMISTRY
74, 354-358 (1976)
A Radiometric Method for the Determination Choline Acetylase Activity Based on Thin-Layer Chromatography1
of
ZORAN GRUBIC, TOMAZ KIAUTA, AND MIRO BRZIN Institute of Pathophysiology, 61105 Ljubljnna,
University of Zjubljana, Yugoslavia
Received September 18, 1975; accepted April 15, 1976 A rapid thin-layer chromatographic method on Gelman I.T.L.C. plates for the determination of choline acetylase activity has veloped. The mobile phase consists of sodium tetraphenylboron in acetonitrile. The Rt values for acetyl coenzyme A and acetate 0.1, while that for acetylcholine is about 0.9. The method compares with other radiometric methods as far as recovery, separation, and are concerned.
type SA been dedissolved are about favorably simplicity
Choline acetylase (acetyl-CoA: choline O-acetyltransferase, EC 2.1.3.6) (ChAc) catalyzes the synthesis of acetylcholine (ACh) from choline and acetyl coenzyme A (acetyl-CoA). Because of the important role of this enzyme in the function of the nervous system, many methods have been devised to measure its activity. Radioactively labeled substrates afforded the development of sensitive and accurate assay methods based upon the isolation of synthesized labeled ACh from the reaction mixture on an anion-exchange column (l), by precipitation of ACh (2-4), by liquid cation extraction (5-Q, and by electrophoresis (9). After very favorable experiences with the thin-layer chromatographic (tic) method for the assay of acetylcholinesterase activity (10,l l), an attempt was made in our laboratory to devise a tic procedure for the separation of labeled acetyl-CoA and acetate from ACh needed for ChAc activity measurements, using sodium tetraphenylboron in ethyl butyl ketone (5) or in acetonitrile (6) as the mobile phase. Since the separation of ACh with acetonitrile as the mobile phase was better, only the results obtained with this mobile phase are described. MATERIALS Chemicals.
[1-14C]acetate
AND METHODS
[ lJ4C]Acetylcholine chloride (sp act 11.8 mCi/mmol), (sp act 58 mCi/mmol), and [1-14C]acetyl coenzyme
1 This work was supported in part by the Slovene Research Community and in part by National Institutes of Health Grant No. 02-008-1, Z-ZF-6. Copyright 0 1976 by Academic Press, Inc. All rights of reproduction in any form reserved.
354
TLC RADIOMETRIC
METHOD
FOR ChAc
355
A(sp act 58 mCi/mmol) were obtained from the Radiochemical Centre, Amersham, and stored at -30°C. They were not additionally purified. Acetyl-CoA, L&-salt, and AChJ (Serva), choline iodide and physostigmine sulphate (BDH), sodium tetraphenylboron (Kemika, Zagreb), and acetonitrile (Schuchardt) were used. The tic strips (I.T.L.C. type SA, 5 x 20 cm) were purchased from Gelman Instrument Co. The buffer solution used throughout the experiments was 50 mM sodium phosphate buffer, pH 6.8, containing 138 mM NaCl, 4 mM KCl, and 3 mM MgSO,. Radioactivity was measured by liquid scintillation spectrometry (Isocap/300, Nuclear Chicago), using a modified Bray’s liquid scintillation mixture containing 5 g of diphenyloxasole (PPO), 0.5 g of p-bis(2-5-phenyloxasolyl) benzene (POPOP), and 80 g of naphthalene/litre of solvent consisting of equal volumes of toluene, p-dioxane and ethyleneglycol monomethyl ether. Chromatographic separation of ACh from acetyl-CoA and acetate. The tic strips (1.5 x 12 cm) were preprepared by applying 20-~1; aliquots of saturated cold sodium acetate solution 2 cm from the end of the strips in order to prevent the upward movement of [ I-‘*Cl acetate. The interval between the application of cold acetate and the beginning of chromatography may vary from minutes to days without affecting the results in any way. Aliquots (10 ~1) of the buffer solution containing a mixture of [ l-14C]ACh, [1-14C]acetate, and [l-14C]acetyl-CoA were taken by Pedersen constriction micropipets and spotted 2 cm from the end of the strips. Six strips at a time were developed in a 400-ml beaker containing 15 ml of mobile phase (130 mg of sodium tetraphenylboron/ml of acetonitrile). In order to achieve saturation, the lower two thirds of the beaker had been lined with a filter-paper roll and wetted with the solvent, and the beaker was covered with parafilm at least 30 min before chromatography. Then the strips were placed into the beaker, covered, and allowed to develop ascendingly for 9 cm, which took lo-15 min. The chromatograms were removed with tweezers and cut into l-cm pieces which were dropped into counting vials containing 10 ml of the liquid scintillation mixture. In order to determine ACh recovery, six solutions of [l-14C]ACh at concentrations ranging from 5 to 50 PM were prepared, and lo-p1 aliquots were spotted on strips and chromatographed. The portion between 4.5 cm from the start to just beyond the solvent front was cut from the strip and dropped into a counting vial prefilled with 20 ml of the cocktail solution. The radioactivity of the vials containing parts of the strips was compared with the control radioactivity of the vials containing 10 ~1 of the respective ACh solution.
356
GRUBIC, KIAUTA
acetyl
AND BRZIN
CoA
+ acetate
23L56789 0
cm
FIG. 1. Histogram of a typical chromatogram spot on chromatogram; F, front.
F of a reaction mixture.
0, origin
of
Assay procedure. The enzyme preparation: Male Wistar strain albino rats, weighing from 150 to 250 g, were anesthetized with ether and bled to death; their brains were removed and placed in the icecold buffer solution (340 mg of brain tissue/ml). The tissue was homogenized for 3 min in a Sorvall Omnimixer at about 0°C and a speed of 10 scale units. The homogenate was centrifuged in a Sorvall RCZB centrifuge at 13,OOOg and at 0-4°C for 1 hr. The supernatant, which served as the enzyme stock preparation, was removed and stored in the vapor phase of a liquid nitrogen freezer. In order to obtain five concentrations of the enzyme preparation, 5, 10, 15, 20, and 25 ~1 of the enzyme stock preparation were brought up to 50 ~1 with the buffer solution. Physostigmine sulphate (0.4 mM) was included in all the samples. The mixtures were prepared in small glass tubes (length, 4 cm; inner diameter, 4 mm), mixed on a vortex mixer, and preincubated for 1 hr at about 0°C in order to ensure inhibition of cholinesterases present in the enzyme preparation. After preincubation, 10 ~1 of the substrate solution were added to 10 ~1 of each of the preincubation mixtures, mixed on a vortex mixer, and incubated for 20 min at 38°C. In blanks, the buffer solution was substituted for the enzyme solution. The final concentration of [1-14C]acetyl-CoA (sp act 3.0 pCi/pmol) was 0.5 mM; of choline iodide, 30 mM; and of physostigmine sulphate, 0.2 mM. After incubation, the tubes with the samples were cooled in melting ice in order to stop the enzyme reaction. Aliquots (10 ~1) of the samples were
TLC RADIOMETRIC
0
5
10 ~1 of
METHOD
20
15 enzyme
stock
357
FOR ChAc
25
preporatlon
FIG. 2. The synthesis of [I-W]ACh in the presence of increasing concentrations of ChAc. The abscissa represents the volumes of the enzyme stock preparation in 50 ~1 of the preincubation mixtures. Incubation time was 20 min. [I-W]Acetyl-CoA and choline iodide concentrations were 0.5 mM and 30 mM, respectively. Open circles are means of three parallel runs.
spotted on tic strips and chromatographed immediately thereafter. No spot drying was necessary. All measurements were done in triplicate. RESULTS
When the mixture of [l-14C]ACh, [1-14C]acetate, and [1-14C]acetylCoA was spotted and developed, [l-‘*C]ACh separated totally from the other two compounds. The same separation was obtained after enzyme synthesis of [l-14C]ACh (Fig. 1). The R, values at 21°C determined after chromatographing each compound alone, were about 0.1 for acetyl-CoA and acetate, and about 0.9 for ACh. The recovery of [ l-14C]ACh was found to be quantitative (99 to 100%). Synthesis of [l-14C]ACh by ChAc was calculated by dividing the radioactivity present as [1-14C]ACh by the radioactivity of [ l-*4C]acetylCoA added to the chromatogram. There was a linear relationship between percent synthesis of ACh and the concentration of the enzyme preparation present (Fig. 2). Reproducibility was 2 to 4% (SD). Identical results were obtained when this experiment was repeated with Fonnum’s method (6). The blanks amounted to 0.80 2 0.08% (mean of eight parallel runs + SD) and were probably due to labeled impurities in the commercial isotope preparations. DISCUSSION
The presented tic method for the assay of ChAc activity makes use of sodium tetraphenylboron dissolved in acetonitrile as the mobile
358
GRUB&,
KIAUTA
AND BRZIN
phase. This mixture was originally introduced by Fonnum (6) for the liquid cation extraction method for ChAc. The recovery of the reaction product is quantitative and its separation from other labeled compounds of the incubation medium total, The sensitivity of the assay is limited only by the specific activity of the labeled substrate. Reproducibility is good. The method is rapid: Following the basic incubation step, 40 samples can be conveniently analyzed in 40 mitt, using seven beakers for tic. In this method, the enzyme reaction is stopped by cooling the samples to 0°C which according to Big1 (8) is a satisfactory way of stopping the reaction after incubation. The method is also potentially adaptable for the differential assay of ChAc (12), by which other possible acetylated products of the enzyme reaction would be excluded. In conclusion, we feel that the method compares favorably with and might in some instances be a useful alternative of other radiometric methods for ChAc assay. ACKNOWLEDGMENTS The authors wish to thank Dr. L. Kosta of the Department of Analytical Chemistry, University of Ljubljana, for his gift of sodium tetraphenylboron. The skilled technical assistance of Mr. Vasilij Loboda, MrsDarka Ka&Zupan, and Mrs. Sonja TrPek is gratefully acknowledged.
REFERENCES 1. 2. 3. 4.
Schrier, B. K., and Schuster, L. (1967) J. Neurochem. 14, 977-985. McCaman, R. E., and Hunt, J. M. (1%5) J. Neurochem. 12, 253-259. Fonnum, F. (1966) Biochem. J. 100,479-484. Goldberg, A. M., Kaita, A. A., and McCaman, R. E. (1%9) J. Neurochem. 16, 823-824.
5. 6. 7. 8. 9.
Fonnum, F. (1969) Biochem. J. 115, 465-472. Fonnum, F. (1975) J. Neurochem. 24,407-409. Glover, V., and Green, D. P. L. (1972) J. Neurochem. 19, 2465-2466. Bigl, V. (1975)Acta Biol. Med. Germ. 34, 1437-1440. Potter, L. T., Glover, V. A. S., and Saelens, J. K. (1%8) J. Biol. 243, 3864-3870. 10. Lewis, M. K., and Eldefrawi, M. E. (1974)Anal. Biochem. 57, 588-592. Il. Grub& Z., Sketelj, J., and Brzin, M. (1975) Anal. Biochem. 69, 306-309. 12. Hamprecht, B., and Amano, T. (1974) Anal. Biochem. 57, 162-172.
Chem.
BIOCHEMISTRY
74, 354-358 (1976)
A Radiometric Method for the Determination Choline Acetylase Activity Based on Thin-Layer Chromatography1
of
ZORAN GRUBIC, TOMAZ KIAUTA, AND MIRO BRZIN Institute of Pathophysiology, 61105 Ljubljnna,
University of Zjubljana, Yugoslavia
Received September 18, 1975; accepted April 15, 1976 A rapid thin-layer chromatographic method on Gelman I.T.L.C. plates for the determination of choline acetylase activity has veloped. The mobile phase consists of sodium tetraphenylboron in acetonitrile. The Rt values for acetyl coenzyme A and acetate 0.1, while that for acetylcholine is about 0.9. The method compares with other radiometric methods as far as recovery, separation, and are concerned.
type SA been dedissolved are about favorably simplicity
Choline acetylase (acetyl-CoA: choline O-acetyltransferase, EC 2.1.3.6) (ChAc) catalyzes the synthesis of acetylcholine (ACh) from choline and acetyl coenzyme A (acetyl-CoA). Because of the important role of this enzyme in the function of the nervous system, many methods have been devised to measure its activity. Radioactively labeled substrates afforded the development of sensitive and accurate assay methods based upon the isolation of synthesized labeled ACh from the reaction mixture on an anion-exchange column (l), by precipitation of ACh (2-4), by liquid cation extraction (5-Q, and by electrophoresis (9). After very favorable experiences with the thin-layer chromatographic (tic) method for the assay of acetylcholinesterase activity (10,l l), an attempt was made in our laboratory to devise a tic procedure for the separation of labeled acetyl-CoA and acetate from ACh needed for ChAc activity measurements, using sodium tetraphenylboron in ethyl butyl ketone (5) or in acetonitrile (6) as the mobile phase. Since the separation of ACh with acetonitrile as the mobile phase was better, only the results obtained with this mobile phase are described. MATERIALS Chemicals.
[1-14C]acetate
AND METHODS
[ lJ4C]Acetylcholine chloride (sp act 11.8 mCi/mmol), (sp act 58 mCi/mmol), and [1-14C]acetyl coenzyme
1 This work was supported in part by the Slovene Research Community and in part by National Institutes of Health Grant No. 02-008-1, Z-ZF-6. Copyright 0 1976 by Academic Press, Inc. All rights of reproduction in any form reserved.
354
TLC RADIOMETRIC
METHOD
FOR ChAc
355
A(sp act 58 mCi/mmol) were obtained from the Radiochemical Centre, Amersham, and stored at -30°C. They were not additionally purified. Acetyl-CoA, L&-salt, and AChJ (Serva), choline iodide and physostigmine sulphate (BDH), sodium tetraphenylboron (Kemika, Zagreb), and acetonitrile (Schuchardt) were used. The tic strips (I.T.L.C. type SA, 5 x 20 cm) were purchased from Gelman Instrument Co. The buffer solution used throughout the experiments was 50 mM sodium phosphate buffer, pH 6.8, containing 138 mM NaCl, 4 mM KCl, and 3 mM MgSO,. Radioactivity was measured by liquid scintillation spectrometry (Isocap/300, Nuclear Chicago), using a modified Bray’s liquid scintillation mixture containing 5 g of diphenyloxasole (PPO), 0.5 g of p-bis(2-5-phenyloxasolyl) benzene (POPOP), and 80 g of naphthalene/litre of solvent consisting of equal volumes of toluene, p-dioxane and ethyleneglycol monomethyl ether. Chromatographic separation of ACh from acetyl-CoA and acetate. The tic strips (1.5 x 12 cm) were preprepared by applying 20-~1; aliquots of saturated cold sodium acetate solution 2 cm from the end of the strips in order to prevent the upward movement of [ I-‘*Cl acetate. The interval between the application of cold acetate and the beginning of chromatography may vary from minutes to days without affecting the results in any way. Aliquots (10 ~1) of the buffer solution containing a mixture of [ l-14C]ACh, [1-14C]acetate, and [l-14C]acetyl-CoA were taken by Pedersen constriction micropipets and spotted 2 cm from the end of the strips. Six strips at a time were developed in a 400-ml beaker containing 15 ml of mobile phase (130 mg of sodium tetraphenylboron/ml of acetonitrile). In order to achieve saturation, the lower two thirds of the beaker had been lined with a filter-paper roll and wetted with the solvent, and the beaker was covered with parafilm at least 30 min before chromatography. Then the strips were placed into the beaker, covered, and allowed to develop ascendingly for 9 cm, which took lo-15 min. The chromatograms were removed with tweezers and cut into l-cm pieces which were dropped into counting vials containing 10 ml of the liquid scintillation mixture. In order to determine ACh recovery, six solutions of [l-14C]ACh at concentrations ranging from 5 to 50 PM were prepared, and lo-p1 aliquots were spotted on strips and chromatographed. The portion between 4.5 cm from the start to just beyond the solvent front was cut from the strip and dropped into a counting vial prefilled with 20 ml of the cocktail solution. The radioactivity of the vials containing parts of the strips was compared with the control radioactivity of the vials containing 10 ~1 of the respective ACh solution.
356
GRUBIC, KIAUTA
acetyl
AND BRZIN
CoA
+ acetate
23L56789 0
cm
FIG. 1. Histogram of a typical chromatogram spot on chromatogram; F, front.
F of a reaction mixture.
0, origin
of
Assay procedure. The enzyme preparation: Male Wistar strain albino rats, weighing from 150 to 250 g, were anesthetized with ether and bled to death; their brains were removed and placed in the icecold buffer solution (340 mg of brain tissue/ml). The tissue was homogenized for 3 min in a Sorvall Omnimixer at about 0°C and a speed of 10 scale units. The homogenate was centrifuged in a Sorvall RCZB centrifuge at 13,OOOg and at 0-4°C for 1 hr. The supernatant, which served as the enzyme stock preparation, was removed and stored in the vapor phase of a liquid nitrogen freezer. In order to obtain five concentrations of the enzyme preparation, 5, 10, 15, 20, and 25 ~1 of the enzyme stock preparation were brought up to 50 ~1 with the buffer solution. Physostigmine sulphate (0.4 mM) was included in all the samples. The mixtures were prepared in small glass tubes (length, 4 cm; inner diameter, 4 mm), mixed on a vortex mixer, and preincubated for 1 hr at about 0°C in order to ensure inhibition of cholinesterases present in the enzyme preparation. After preincubation, 10 ~1 of the substrate solution were added to 10 ~1 of each of the preincubation mixtures, mixed on a vortex mixer, and incubated for 20 min at 38°C. In blanks, the buffer solution was substituted for the enzyme solution. The final concentration of [1-14C]acetyl-CoA (sp act 3.0 pCi/pmol) was 0.5 mM; of choline iodide, 30 mM; and of physostigmine sulphate, 0.2 mM. After incubation, the tubes with the samples were cooled in melting ice in order to stop the enzyme reaction. Aliquots (10 ~1) of the samples were
TLC RADIOMETRIC
0
5
10 ~1 of
METHOD
20
15 enzyme
stock
357
FOR ChAc
25
preporatlon
FIG. 2. The synthesis of [I-W]ACh in the presence of increasing concentrations of ChAc. The abscissa represents the volumes of the enzyme stock preparation in 50 ~1 of the preincubation mixtures. Incubation time was 20 min. [I-W]Acetyl-CoA and choline iodide concentrations were 0.5 mM and 30 mM, respectively. Open circles are means of three parallel runs.
spotted on tic strips and chromatographed immediately thereafter. No spot drying was necessary. All measurements were done in triplicate. RESULTS
When the mixture of [l-14C]ACh, [1-14C]acetate, and [1-14C]acetylCoA was spotted and developed, [l-‘*C]ACh separated totally from the other two compounds. The same separation was obtained after enzyme synthesis of [l-14C]ACh (Fig. 1). The R, values at 21°C determined after chromatographing each compound alone, were about 0.1 for acetyl-CoA and acetate, and about 0.9 for ACh. The recovery of [ l-14C]ACh was found to be quantitative (99 to 100%). Synthesis of [l-14C]ACh by ChAc was calculated by dividing the radioactivity present as [1-14C]ACh by the radioactivity of [ l-*4C]acetylCoA added to the chromatogram. There was a linear relationship between percent synthesis of ACh and the concentration of the enzyme preparation present (Fig. 2). Reproducibility was 2 to 4% (SD). Identical results were obtained when this experiment was repeated with Fonnum’s method (6). The blanks amounted to 0.80 2 0.08% (mean of eight parallel runs + SD) and were probably due to labeled impurities in the commercial isotope preparations. DISCUSSION
The presented tic method for the assay of ChAc activity makes use of sodium tetraphenylboron dissolved in acetonitrile as the mobile
358
GRUB&,
KIAUTA
AND BRZIN
phase. This mixture was originally introduced by Fonnum (6) for the liquid cation extraction method for ChAc. The recovery of the reaction product is quantitative and its separation from other labeled compounds of the incubation medium total, The sensitivity of the assay is limited only by the specific activity of the labeled substrate. Reproducibility is good. The method is rapid: Following the basic incubation step, 40 samples can be conveniently analyzed in 40 mitt, using seven beakers for tic. In this method, the enzyme reaction is stopped by cooling the samples to 0°C which according to Big1 (8) is a satisfactory way of stopping the reaction after incubation. The method is also potentially adaptable for the differential assay of ChAc (12), by which other possible acetylated products of the enzyme reaction would be excluded. In conclusion, we feel that the method compares favorably with and might in some instances be a useful alternative of other radiometric methods for ChAc assay. ACKNOWLEDGMENTS The authors wish to thank Dr. L. Kosta of the Department of Analytical Chemistry, University of Ljubljana, for his gift of sodium tetraphenylboron. The skilled technical assistance of Mr. Vasilij Loboda, MrsDarka Ka&Zupan, and Mrs. Sonja TrPek is gratefully acknowledged.
REFERENCES 1. 2. 3. 4.
Schrier, B. K., and Schuster, L. (1967) J. Neurochem. 14, 977-985. McCaman, R. E., and Hunt, J. M. (1%5) J. Neurochem. 12, 253-259. Fonnum, F. (1966) Biochem. J. 100,479-484. Goldberg, A. M., Kaita, A. A., and McCaman, R. E. (1%9) J. Neurochem. 16, 823-824.
5. 6. 7. 8. 9.
Fonnum, F. (1969) Biochem. J. 115, 465-472. Fonnum, F. (1975) J. Neurochem. 24,407-409. Glover, V., and Green, D. P. L. (1972) J. Neurochem. 19, 2465-2466. Bigl, V. (1975)Acta Biol. Med. Germ. 34, 1437-1440. Potter, L. T., Glover, V. A. S., and Saelens, J. K. (1%8) J. Biol. 243, 3864-3870. 10. Lewis, M. K., and Eldefrawi, M. E. (1974)Anal. Biochem. 57, 588-592. Il. Grub& Z., Sketelj, J., and Brzin, M. (1975) Anal. Biochem. 69, 306-309. 12. Hamprecht, B., and Amano, T. (1974) Anal. Biochem. 57, 162-172.
Chem.