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Enzymatic preparation of radiolabeled succinic semialdehyde
ANALYTICAL
BIOCHEMISTRY
135,436-437
(1983)
Enzymatic Preparation of Radiolabeled Succinic Semialdehyde’ KENNETH
M.
GIBSON*‘t
AND
LAWRENCE
SWEETMAN?
*Departments of Chemistry and fPediatrics, University of Cal~ornnia San Diego, La Jolla. California 92093 Received July 14, 1983 [U-‘4C]Succinic semialdehyde was prepared with yields of 30-40% by oxidation of purified [U-‘gC]4-aminobutyric acid with commercially available bovine plasma monoamine oxidase. [U-“C]Succinic semialdehyde was purified by cation-exchange chromatography and quantified as the oxime and methoxime derivatives using liquid partition chromatography on silicic acid. The availability of [U-‘4C]succinic semiaklehyde permits the reliable assayof succinic semial&hyde dehydrogenase in crude cell extracts of lymphocytes isolated from human blood, cultured human lymphoblasts, and other tissues where 4-aminobutyric acid metabolism is known to occur. KEY WORDS: GABA, succinic semialdehyde; succinic semialdehyde dehydrogenase; lymphocyte; lymphoblast; monoamine oxidase.
Succinic semialdehyde dehydrogenase (SSADH,* succinic semialdehyde: NADi oxidoreductase, EC 1.2.1.24) is responsible for the production of succinic acid from succinic semialdehyde derived from the metabolism of the inhibitory neurotransmitter GABA (1). The recent documentation of an inherited deficiency of SSADH (2) and the demonstration of GABA metabolizing enzymes in human lymphocytes and lymphoblasts have prompted the need for a reliable assay of SSADH in crude cell extracts. Spectrophotometric or fluorometric assays in crude extracts were unreliable (3). We report the preparation of [U14C]SSA by oxidation of [U-14C]GABA with monoamine oxidase and purification by cation-exchange chromatography. This provides the opportunity for an accurate radiochemical determination of SSADH activity in crude extracts of many tissues including human lymphocytes and lymphoblasts.
MATERIALS
[U-i4C]GABA (sp act 208-238 mCi/mmol) was purchased from New England Nuclear (Boston, Mass.). Bovine plasma monoamine oxidase (25 units/g protein), pyridoxal 5phosphate, FAD, and nonlabeled SSA were purchased from Sigma (St. Louis, MO.). AG 5OW-X4 (200-400 mesh, H+ form) cationexchange resin was from Bio-Rad Laboratories (Richmond, Calif.). [U-‘4C]GABA and [U-‘4C]SSA were purified using a modification of the procedure of Schmidt (4). Two glass columns (0.9 X 30 cm), each with stopcock and lOO-ml reservoir, were filled to a height of 12- 15 cm with AG 5OW-X4 resin and equilibrated with distilled water. An aliquot of 50-100 &i of stock [U14C]GABA in 0.01 N HCl was loaded onto the first column and rinsed with 100 ml of distilled water. [U-‘4C]GABA was eluted with 60 ml of 2 M NH40H. Ammonia was evaporated under a stream of N2 and the remaining volume was lyophilized to dryness. The [U14C]GABA was redissolved in 2 ml of 100 mM Tris . HCI, pH 8.0, containing 0.1 mM each of FAD and pyridoxal5-phosphate. Insoluble material was removed by centrifugation. Between 1.2 and 1.4 units (final concentration
’ This work was supported in part by U. S. Public Health Services Grant No. HD04608 from the National Institute of Child Health and Human Development, and Grant No. GM-17702 from the National Institute of General Medical Sciences, National Institutes of Health. * Abbreviations used: SSA, succinic semialdehyde; GABA, 4-aminobutyric acid, SSADH, succinic semialdehyde dehydrogenase; LPC, liquid partition chromatography. 0003-2697183 $3.00 Copyright 0 1983 by Academic Press. Inc. All rights of reproduction in any form reserved.
AND METHODS
436
PREPARATION
OF LABELED
0.6-0.7 units/ml) of lyophilized monoamine oxidase was added and the mixture was incubated 6 h at 37°C. The pH was adjusted to less than 5 with 1 M HCl and the reaction mixture was applied to a second AG-50W-X4 column. The column was eluted with 20 ml of distilled water and 2-ml fractions were collected. The peak of radioactivity (usually fractions 5-7), located by liquid scintillation counting of 5~1 aliquots of the fractions, was pooled, adjusted to pH 8.0 with 6 ml of 100 mM Tris . HCl, pH 8.0, and lyophilized. The dried material was dissolved in distilled water and centrifuged to remove insoluble material, and the volume was adjusted with distilled water to obtain approximately 2 Ki/ml. The yield of [U-i4C]SSA from [U-14C]GABA was 30-40%. Unreacted [U-‘4C]GABA could be recovered from the second column by elution with 2 M NH,OH and treated as described above to remove NH3, and the deamination repeated to increase the yield. The purity of oxime and methoxime derivatives of [U-‘4C]SSA was determined by liquid partition chromatography according to Gibson et al. (2) using nonlabeled SSA as carrier. Analysis by LPC showed the [U-‘4C]SSA to be greater than 99% radiochemically pure and free of radiolabeled succinic acid. RESULTS
AND DISCUSSION
Numerous methods for the chemical synthesis of SSA have been described (5-9). However, few of these procedures can be adapted for production of radiolabeled SSA. Maitre et al. (10) generated [3H]SSA from [3H]GABA using purified rat brain GABA-transaminase with 2% yields. In the present procedure, bovine plasma monoamine oxidase was found to give adequate yields of [U-i4C]SSA of high radiochemical purity. Buffer salts, cofactors, and residual protein in the [U-‘4C]SSA preparation could be removed by diethyl ether extraction as described by Bruce et al. (8). Our method for analysis of [U-‘4C]SSA purity is
SUCCINIC
SEMIALDEHYDE
437
not commonly available, although the thinlayer procedure of Bruce et al. (8) could be easily employed. By avoiding extremes of pH and obtaining a dilute final concentration of [UL4C]SSA (9-10 PM), we have avoided difficulties with the polymerization of SSA experienced by other authors (6,7). Assuming the SSADH activity in white cells reflects the brain enzyme, then, as suggested by White (11) the radiochemical assay of enzymes that metabolize GABA in accessible tissues such as platelets (1 l), lymphocytes, and lymphoblasts may provide new avenues of investigation for convulsive, psychological, and behavioral disorders where GABA metabolism is thought to be involved (12). ACKNOWLEDGMENT The authors wish to thank M. Carter-Biikman for typing the manuscript.
REFERENCES 1. Cooper, J. R., Bloom, F. E., and Roth, R. H. (1982) The Biochemical Basis of Neuropharmacology, pp. 249-294, Oxford University Press, New York/ London. 2. Gibson, K. M., Sweetman, L., Nyhan, W. L., Jakobs, C., Rating, D., Siemes, H., and Hanefeld, F. (1983) Clin. Chim. Acta 133, 33-42. 3. Cash, C. D., Maitre, M., Ossola, L., and Mandel, P. (1978) Biochim. Biophys. Acta 524, 26-36. 4. Schmidt, L. ( 1974) in Heritable Disorders of Amino Acid Metabolism (Nyhan, W. L., ed.), pp. 675697, Wiley, New York. 5. Santaniello, E., and Manzocchi, A. (1980) Anal. Biochem. 108, 190-192. 6. Wennuth, C. G. (1979) J. Org. Chem. 44, 24062408. 7. Tabemer, P. V., Bamett, J. E. G., and Kerkut, G. A. (1972) J. Neurochem. 19, 95-99. 8. Bruce, R., Sims, K., and Pitts, F. N., Jr. (1971) Anal. Biochem. 41, 271-273. 9. Carrier, E. (1972) Ann. Chim. (Paris) 17, 43. 10. Maitre, M., Ossola, L., and Mandel, P. (1976) FEB.9 Lett. 72, 53-57. 11. White, H. L. (1979) Science 205, 696-698. 12. Tower, D. B. (1976) in GABA in Nervous System Function (Roberts, E., Chase, T. N., and Tower, D. B., eds.), pp. 461-478, Raven Press, New York.
BIOCHEMISTRY
135,436-437
(1983)
Enzymatic Preparation of Radiolabeled Succinic Semialdehyde’ KENNETH
M.
GIBSON*‘t
AND
LAWRENCE
SWEETMAN?
*Departments of Chemistry and fPediatrics, University of Cal~ornnia San Diego, La Jolla. California 92093 Received July 14, 1983 [U-‘4C]Succinic semialdehyde was prepared with yields of 30-40% by oxidation of purified [U-‘gC]4-aminobutyric acid with commercially available bovine plasma monoamine oxidase. [U-“C]Succinic semialdehyde was purified by cation-exchange chromatography and quantified as the oxime and methoxime derivatives using liquid partition chromatography on silicic acid. The availability of [U-‘4C]succinic semiaklehyde permits the reliable assayof succinic semial&hyde dehydrogenase in crude cell extracts of lymphocytes isolated from human blood, cultured human lymphoblasts, and other tissues where 4-aminobutyric acid metabolism is known to occur. KEY WORDS: GABA, succinic semialdehyde; succinic semialdehyde dehydrogenase; lymphocyte; lymphoblast; monoamine oxidase.
Succinic semialdehyde dehydrogenase (SSADH,* succinic semialdehyde: NADi oxidoreductase, EC 1.2.1.24) is responsible for the production of succinic acid from succinic semialdehyde derived from the metabolism of the inhibitory neurotransmitter GABA (1). The recent documentation of an inherited deficiency of SSADH (2) and the demonstration of GABA metabolizing enzymes in human lymphocytes and lymphoblasts have prompted the need for a reliable assay of SSADH in crude cell extracts. Spectrophotometric or fluorometric assays in crude extracts were unreliable (3). We report the preparation of [U14C]SSA by oxidation of [U-14C]GABA with monoamine oxidase and purification by cation-exchange chromatography. This provides the opportunity for an accurate radiochemical determination of SSADH activity in crude extracts of many tissues including human lymphocytes and lymphoblasts.
MATERIALS
[U-i4C]GABA (sp act 208-238 mCi/mmol) was purchased from New England Nuclear (Boston, Mass.). Bovine plasma monoamine oxidase (25 units/g protein), pyridoxal 5phosphate, FAD, and nonlabeled SSA were purchased from Sigma (St. Louis, MO.). AG 5OW-X4 (200-400 mesh, H+ form) cationexchange resin was from Bio-Rad Laboratories (Richmond, Calif.). [U-‘4C]GABA and [U-‘4C]SSA were purified using a modification of the procedure of Schmidt (4). Two glass columns (0.9 X 30 cm), each with stopcock and lOO-ml reservoir, were filled to a height of 12- 15 cm with AG 5OW-X4 resin and equilibrated with distilled water. An aliquot of 50-100 &i of stock [U14C]GABA in 0.01 N HCl was loaded onto the first column and rinsed with 100 ml of distilled water. [U-‘4C]GABA was eluted with 60 ml of 2 M NH40H. Ammonia was evaporated under a stream of N2 and the remaining volume was lyophilized to dryness. The [U14C]GABA was redissolved in 2 ml of 100 mM Tris . HCI, pH 8.0, containing 0.1 mM each of FAD and pyridoxal5-phosphate. Insoluble material was removed by centrifugation. Between 1.2 and 1.4 units (final concentration
’ This work was supported in part by U. S. Public Health Services Grant No. HD04608 from the National Institute of Child Health and Human Development, and Grant No. GM-17702 from the National Institute of General Medical Sciences, National Institutes of Health. * Abbreviations used: SSA, succinic semialdehyde; GABA, 4-aminobutyric acid, SSADH, succinic semialdehyde dehydrogenase; LPC, liquid partition chromatography. 0003-2697183 $3.00 Copyright 0 1983 by Academic Press. Inc. All rights of reproduction in any form reserved.
AND METHODS
436
PREPARATION
OF LABELED
0.6-0.7 units/ml) of lyophilized monoamine oxidase was added and the mixture was incubated 6 h at 37°C. The pH was adjusted to less than 5 with 1 M HCl and the reaction mixture was applied to a second AG-50W-X4 column. The column was eluted with 20 ml of distilled water and 2-ml fractions were collected. The peak of radioactivity (usually fractions 5-7), located by liquid scintillation counting of 5~1 aliquots of the fractions, was pooled, adjusted to pH 8.0 with 6 ml of 100 mM Tris . HCl, pH 8.0, and lyophilized. The dried material was dissolved in distilled water and centrifuged to remove insoluble material, and the volume was adjusted with distilled water to obtain approximately 2 Ki/ml. The yield of [U-i4C]SSA from [U-14C]GABA was 30-40%. Unreacted [U-‘4C]GABA could be recovered from the second column by elution with 2 M NH,OH and treated as described above to remove NH3, and the deamination repeated to increase the yield. The purity of oxime and methoxime derivatives of [U-‘4C]SSA was determined by liquid partition chromatography according to Gibson et al. (2) using nonlabeled SSA as carrier. Analysis by LPC showed the [U-‘4C]SSA to be greater than 99% radiochemically pure and free of radiolabeled succinic acid. RESULTS
AND DISCUSSION
Numerous methods for the chemical synthesis of SSA have been described (5-9). However, few of these procedures can be adapted for production of radiolabeled SSA. Maitre et al. (10) generated [3H]SSA from [3H]GABA using purified rat brain GABA-transaminase with 2% yields. In the present procedure, bovine plasma monoamine oxidase was found to give adequate yields of [U-i4C]SSA of high radiochemical purity. Buffer salts, cofactors, and residual protein in the [U-‘4C]SSA preparation could be removed by diethyl ether extraction as described by Bruce et al. (8). Our method for analysis of [U-‘4C]SSA purity is
SUCCINIC
SEMIALDEHYDE
437
not commonly available, although the thinlayer procedure of Bruce et al. (8) could be easily employed. By avoiding extremes of pH and obtaining a dilute final concentration of [UL4C]SSA (9-10 PM), we have avoided difficulties with the polymerization of SSA experienced by other authors (6,7). Assuming the SSADH activity in white cells reflects the brain enzyme, then, as suggested by White (11) the radiochemical assay of enzymes that metabolize GABA in accessible tissues such as platelets (1 l), lymphocytes, and lymphoblasts may provide new avenues of investigation for convulsive, psychological, and behavioral disorders where GABA metabolism is thought to be involved (12). ACKNOWLEDGMENT The authors wish to thank M. Carter-Biikman for typing the manuscript.
REFERENCES 1. Cooper, J. R., Bloom, F. E., and Roth, R. H. (1982) The Biochemical Basis of Neuropharmacology, pp. 249-294, Oxford University Press, New York/ London. 2. Gibson, K. M., Sweetman, L., Nyhan, W. L., Jakobs, C., Rating, D., Siemes, H., and Hanefeld, F. (1983) Clin. Chim. Acta 133, 33-42. 3. Cash, C. D., Maitre, M., Ossola, L., and Mandel, P. (1978) Biochim. Biophys. Acta 524, 26-36. 4. Schmidt, L. ( 1974) in Heritable Disorders of Amino Acid Metabolism (Nyhan, W. L., ed.), pp. 675697, Wiley, New York. 5. Santaniello, E., and Manzocchi, A. (1980) Anal. Biochem. 108, 190-192. 6. Wennuth, C. G. (1979) J. Org. Chem. 44, 24062408. 7. Tabemer, P. V., Bamett, J. E. G., and Kerkut, G. A. (1972) J. Neurochem. 19, 95-99. 8. Bruce, R., Sims, K., and Pitts, F. N., Jr. (1971) Anal. Biochem. 41, 271-273. 9. Carrier, E. (1972) Ann. Chim. (Paris) 17, 43. 10. Maitre, M., Ossola, L., and Mandel, P. (1976) FEB.9 Lett. 72, 53-57. 11. White, H. L. (1979) Science 205, 696-698. 12. Tower, D. B. (1976) in GABA in Nervous System Function (Roberts, E., Chase, T. N., and Tower, D. B., eds.), pp. 461-478, Raven Press, New York.