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[65] Ethanolamine oxidase
354
D~rZDaOO~.NAS~.S AND OXIDASZS
[55]
act as electron acceptor. In the purified state 2,6-dichlorophenolindophenol, ferricyanide, phenazine metbosulfate, thionine, and methylene blue are efficient electron aeceptors. Coenzyme. No evidence was found for the participation of NAD, NADP, FAD, FMN, the usual ubiquinones, and heavy-metal ions. All preparations display the spectrum of a reduced cytochrome-553 on addition of ethanol, meso-2,3-butanediol, or hydrosulfite. In all purification steps the ratio specific activity:specific hemoprotein content remained constant, indicating that enzyme activity is probably associated with this cytochrome-553. The purified enzyme in the oxidized form displays the following maxima: 530 m/~ (diffuse), 412 m/~, and 279 m~. In the reduced form the following maxima were observed: 553 m~, 522 m#, 418 m/~, 315 m/~, 279 n~, and a shoulder ~t 345 m/~. The spectral properties of this enzyme are identical with the ethanol-cytochrome-553 reductase from Acetobacter aceti (rancens).6 Inhibitors. The primary alcohol dehydrogenase is completely inhibited by 10-a M p-chloromercuribenzoate. The primary and secondary alcohol dehydrogenases are not inhibited by the following compounds (10-~M): arsenite, semicarbazide, hydroxylamine, cyanide, azide, ophenanthroline, 8-hydroxyquinoline, and EDTA. ST. Nakayama, J. Biochem. (Tokyo) 49, 240 (1961).
[ 65] E t h a n o l a m i n e O x i d a s e By STUARTA. NARRODand WILLIAM B. JAKOBY Ethanolamine + 1/2 02 --* glycolaldehyde + NH3 Assay Method 1 Principle. The assay is based on measuring the rate of formation of glycolaldehyde. This is accomplished by formation of the bis-2,4-dinitrophenylhydrazone of glycolaldehyde followed by alkali treatment and measurement of the resultant purple color. Reagents Sodium borate, 0.05 M, adjusted to pH 8.0 Ethanolamine, 0.025 M, adjusted to pH 8.0 with HC1 just prior to use. Commercial ethanolamine is redistilled and the fraction boiling between 171 ° and 173 ° is collected. IS. A. Narrod and W. B. Jakoby, J. Biol. Chem. 239, 2189 (1964).
[55]
ETHANOLAMINE OXIDASE
355
2,4-Dinitrophenylhydrazine, 0.1%, in 2 N HC1 Alcoholic KOH, 1 N, prepared just prior to use by diluting 4 N aqueous KOH with 95% ethanol Procedure. Appropriate dilutions of enzyme are incubated at 37 ° in a vessel containing 1 ml of ethanolamine and 1 ml of sodium borate in a total volume of 5 ml. At 1, 15, 30, and 60 minutes, 1-ml aliquots are removed and added to 1 ml of 2,4-dinitrophenylhydrazine solution. The mixture is boiled for 10 minutes and cooled; 5 ml of alcoholic KOH is added. The absorption of the resulting purple bis-2,4-dinitrophenylhydrazone is measured at 550 m# (E55o: 6.9 X 104). Under these conditions, the reaction is linear with respect to time and protein concentration when absorbancies of less than 1.0 are measured. The formation of glycolaldehyde may also be determined with 3methyl-2-benzothiozolone hydrazine.2 Definition of Unit. One unit is defined as the amount of enzyme which forms 1.0 millimicromole of glycolaldehyde per minute under the above conditions. Specific activity is in units per milligram of protein; protein was measured by the method of Lowry et al. 3 with bovine serum albumin as standard.
Purification Procedure Ethanolamine oxidase was found in a bacterium 1 originally isolated by the enrichment culture technique. The organism is now available from the American Type Culture Collection as No. 13796. Large quantities of cells may be obtained by growth in 6-liter flasks containing 1 liter of medium on a reciprocating shaker at 25 °. The medium contains 0.1% ethanolamine, 0.1% MgSo4-7 H:0, 0.2% KH~P04, 0.05% yeast extract and is adjusted to pH 7.0. It was convenient to neutralize the ethanolamine in a 1:20 dilution prior to addition to the other medium components. A 0.1% inoculum, grown for 24 hours in the same medium, is used. Cells are harvested after 18 hours, washed with 0.85% saline solution, and dried in vacuo. Approximately 1 g of dried cells per 4 liters of medium is usually obtained by this procedure. All subsequent manipulations are conducted at approximately 2 ° . 1. Crude Extract. Cell-free extracts are prepared by suspending dried cells, 50 mg/ml, in 0.005 M Tris at pH 7.3 and passing the suspension 2E. Sawicki, T. R. Hauser, T. W. Stanley, and W. Elbert, Anal. Chem. 33, 93 (1961). 30. H. Lowry, N. J. Rosebrough, A. L Farr, and R J. Randall, J. Biol. Chem. 193, 265 (1951).
356
DEHYDROGENASES AND OXIDASES
[55]
twice through a French pressure cell. Cell debris is removed by eentrifugation at 20,000 g, resulting irt a yellow, opalescent solution. 2. Ammonium Sul]ate. Thirty milliliters of 1 M MnC12 is slowly added with stirring to each 100 ml of crude extract, and the precipitate is removed by centrifugation. After the addition of 53 g of ammonium sulfate, the precipitate formed is collected by centrifugation and dissolved in 70 ml of 0.02 M Tris at pH 8.5. This solution is then dialyzed for 18 hours at 5 ° against distilled water. At this stage the absorbance ratio, A280:A260, is approximately 1.0. 3. Second Ammonium Sul]ate. To each 100 ml of fraction 2 is added 17.4 g of ammonium sulfate; the precipitate is discarded. The precipitate formed after the further addition of 17.4 g of ammonium sulfate is suspended in 30 ml of 0.02 M Tris at pH 8.5 and dialyzed against distilled water for 5 hours. ~. DEAE-Cellulose. A DEAE-cellulose column (3 cm2X 14 cm), exhaustively washed with 5 mM Tris at pH 7.2, is charged with 15 ml of fraction 3. Protein is eluted with a linear gradient of sodium chloride, achieved by mixing 500 ml of 5 mM Tris at pH 7.2 with 500 ml of 0.5 M sodium chloride in 5 mM Tris at pH 7.2. Activity is eluted in the fractions between 550 ml and 750 ml. The combined eluates of active material are concentrated by precipitation with ammonium sulfate (70 g/100 ml). The resulting precipitate is dissolved in 7 ml of 0.02 M Tris at pH 8.2 and dialyzed against distilled water for 5 hours. The results of such a course of purification are summarized in the table. SUMMARY OF PURIFICATION PROCEDURE
Fraction I. Cell-free extract II. MnCI~ -t- (NH4)SO~ III. (NI-I4)~SO4 IV. DEAE-cellulose
Volume (ml) 128 65 20 10
Activity (units)
Protein (mg)
Specific activity (units/mg)
1150 2960 2120 435
1130 325 176 7
1.0 9.1 12.1 62.1
Properties I T h e e n z y m e is active with ethanolamine, 3-amino-l-propanol, or 1-amino-2-propanol as substrate at maximal relative velocities of 100, 56, and 18, respectively. Other amines such as ethylamine, n-propylamine, putrescine, choline, and phosphoethanolamine were inactive at a concentration of 0.1 M. The K• for ethanolamine is 5 )~ 10-3 M at pH 8.0.
[66]
FORMALDEHYDE DEHYDROGENASE
357
The enzyme is active at pH values between 7 and 10.5, with optimum activity at pH 8.0. Preparations of purified enzyme have been stored at --5 ° for 6 months without any appreciable loss of activity. Inhibition was obtained with the following reagents at the concentrations noted: 0.2 mM quinacrine, 48%; 1 mM hydrazine, 97%; 0.1 mM hydroxylamine, 100%; 10 mM iproniazid, 64%; 10 mM isoniazid, 8%; 0.1 mM semicarbazide, 96%; 20 mM sodium bisulfite, 68%; 10 mM cysteine, 80%; 10 mM glutathione, 25%; 10 mM mercaptoethanol, 68%.
[ 66 ] F o r m a l d e h y d e D e h y d r o g e n a s e
By
ZELDA B. ROSE a n d EFRAIM RACKER
Formaldehyde + DPN + ----~ formate + DPNH + H +
Assay Method
Princ@le. The assay of the enzyme in crude extracts is based on the formation of acid. 1 Alcohol dehydrogenase present in the extracts reacts with formaldehyde and D P N H to yield methanol and DPN. The overall reaction catalyzed by formaldehyde dehydrogenase and alcohol dehydrogenase is a dismutation of formaldehyde to methanol and formic acid. Catalytic amounts of D P N (NAD) and GSH are required. The reaction is followed by measuring manometrically the rate at which the acid produced releases carbon dioxide from bicarbonate buffer. After acetone treatment (step 2), the enzyme can be assayed spectrophotometrically by observing the initial rate of increase of absorbancy at 340 n ~ due to the reduction of D P N (NAD). Residual alcohol dehydrogenase is inhibited by hydroxylamine. 2 Manometric Assay ]or Crude Extracts Reagents Formaldehyde, 0.12 M Glutathione, 0.067 M D P N (NAD), 0.012 M KHCO~, 2 M (freshly prepared)
Procedure. To the main compartment of a Warburg flask the following are added to a final volume of 2.67 ml: 0.10 ml of GSH, 0.10 ml of 'Z. B. Rose and E. Racker, J. Biol. Chem. 237, 3279 (1962). N. O. Kaplan and M. M. Ciotti, J. Biol. Chem. 201, 785 (1953).
D~rZDaOO~.NAS~.S AND OXIDASZS
[55]
act as electron acceptor. In the purified state 2,6-dichlorophenolindophenol, ferricyanide, phenazine metbosulfate, thionine, and methylene blue are efficient electron aeceptors. Coenzyme. No evidence was found for the participation of NAD, NADP, FAD, FMN, the usual ubiquinones, and heavy-metal ions. All preparations display the spectrum of a reduced cytochrome-553 on addition of ethanol, meso-2,3-butanediol, or hydrosulfite. In all purification steps the ratio specific activity:specific hemoprotein content remained constant, indicating that enzyme activity is probably associated with this cytochrome-553. The purified enzyme in the oxidized form displays the following maxima: 530 m/~ (diffuse), 412 m/~, and 279 m~. In the reduced form the following maxima were observed: 553 m~, 522 m#, 418 m/~, 315 m/~, 279 n~, and a shoulder ~t 345 m/~. The spectral properties of this enzyme are identical with the ethanol-cytochrome-553 reductase from Acetobacter aceti (rancens).6 Inhibitors. The primary alcohol dehydrogenase is completely inhibited by 10-a M p-chloromercuribenzoate. The primary and secondary alcohol dehydrogenases are not inhibited by the following compounds (10-~M): arsenite, semicarbazide, hydroxylamine, cyanide, azide, ophenanthroline, 8-hydroxyquinoline, and EDTA. ST. Nakayama, J. Biochem. (Tokyo) 49, 240 (1961).
[ 65] E t h a n o l a m i n e O x i d a s e By STUARTA. NARRODand WILLIAM B. JAKOBY Ethanolamine + 1/2 02 --* glycolaldehyde + NH3 Assay Method 1 Principle. The assay is based on measuring the rate of formation of glycolaldehyde. This is accomplished by formation of the bis-2,4-dinitrophenylhydrazone of glycolaldehyde followed by alkali treatment and measurement of the resultant purple color. Reagents Sodium borate, 0.05 M, adjusted to pH 8.0 Ethanolamine, 0.025 M, adjusted to pH 8.0 with HC1 just prior to use. Commercial ethanolamine is redistilled and the fraction boiling between 171 ° and 173 ° is collected. IS. A. Narrod and W. B. Jakoby, J. Biol. Chem. 239, 2189 (1964).
[55]
ETHANOLAMINE OXIDASE
355
2,4-Dinitrophenylhydrazine, 0.1%, in 2 N HC1 Alcoholic KOH, 1 N, prepared just prior to use by diluting 4 N aqueous KOH with 95% ethanol Procedure. Appropriate dilutions of enzyme are incubated at 37 ° in a vessel containing 1 ml of ethanolamine and 1 ml of sodium borate in a total volume of 5 ml. At 1, 15, 30, and 60 minutes, 1-ml aliquots are removed and added to 1 ml of 2,4-dinitrophenylhydrazine solution. The mixture is boiled for 10 minutes and cooled; 5 ml of alcoholic KOH is added. The absorption of the resulting purple bis-2,4-dinitrophenylhydrazone is measured at 550 m# (E55o: 6.9 X 104). Under these conditions, the reaction is linear with respect to time and protein concentration when absorbancies of less than 1.0 are measured. The formation of glycolaldehyde may also be determined with 3methyl-2-benzothiozolone hydrazine.2 Definition of Unit. One unit is defined as the amount of enzyme which forms 1.0 millimicromole of glycolaldehyde per minute under the above conditions. Specific activity is in units per milligram of protein; protein was measured by the method of Lowry et al. 3 with bovine serum albumin as standard.
Purification Procedure Ethanolamine oxidase was found in a bacterium 1 originally isolated by the enrichment culture technique. The organism is now available from the American Type Culture Collection as No. 13796. Large quantities of cells may be obtained by growth in 6-liter flasks containing 1 liter of medium on a reciprocating shaker at 25 °. The medium contains 0.1% ethanolamine, 0.1% MgSo4-7 H:0, 0.2% KH~P04, 0.05% yeast extract and is adjusted to pH 7.0. It was convenient to neutralize the ethanolamine in a 1:20 dilution prior to addition to the other medium components. A 0.1% inoculum, grown for 24 hours in the same medium, is used. Cells are harvested after 18 hours, washed with 0.85% saline solution, and dried in vacuo. Approximately 1 g of dried cells per 4 liters of medium is usually obtained by this procedure. All subsequent manipulations are conducted at approximately 2 ° . 1. Crude Extract. Cell-free extracts are prepared by suspending dried cells, 50 mg/ml, in 0.005 M Tris at pH 7.3 and passing the suspension 2E. Sawicki, T. R. Hauser, T. W. Stanley, and W. Elbert, Anal. Chem. 33, 93 (1961). 30. H. Lowry, N. J. Rosebrough, A. L Farr, and R J. Randall, J. Biol. Chem. 193, 265 (1951).
356
DEHYDROGENASES AND OXIDASES
[55]
twice through a French pressure cell. Cell debris is removed by eentrifugation at 20,000 g, resulting irt a yellow, opalescent solution. 2. Ammonium Sul]ate. Thirty milliliters of 1 M MnC12 is slowly added with stirring to each 100 ml of crude extract, and the precipitate is removed by centrifugation. After the addition of 53 g of ammonium sulfate, the precipitate formed is collected by centrifugation and dissolved in 70 ml of 0.02 M Tris at pH 8.5. This solution is then dialyzed for 18 hours at 5 ° against distilled water. At this stage the absorbance ratio, A280:A260, is approximately 1.0. 3. Second Ammonium Sul]ate. To each 100 ml of fraction 2 is added 17.4 g of ammonium sulfate; the precipitate is discarded. The precipitate formed after the further addition of 17.4 g of ammonium sulfate is suspended in 30 ml of 0.02 M Tris at pH 8.5 and dialyzed against distilled water for 5 hours. ~. DEAE-Cellulose. A DEAE-cellulose column (3 cm2X 14 cm), exhaustively washed with 5 mM Tris at pH 7.2, is charged with 15 ml of fraction 3. Protein is eluted with a linear gradient of sodium chloride, achieved by mixing 500 ml of 5 mM Tris at pH 7.2 with 500 ml of 0.5 M sodium chloride in 5 mM Tris at pH 7.2. Activity is eluted in the fractions between 550 ml and 750 ml. The combined eluates of active material are concentrated by precipitation with ammonium sulfate (70 g/100 ml). The resulting precipitate is dissolved in 7 ml of 0.02 M Tris at pH 8.2 and dialyzed against distilled water for 5 hours. The results of such a course of purification are summarized in the table. SUMMARY OF PURIFICATION PROCEDURE
Fraction I. Cell-free extract II. MnCI~ -t- (NH4)SO~ III. (NI-I4)~SO4 IV. DEAE-cellulose
Volume (ml) 128 65 20 10
Activity (units)
Protein (mg)
Specific activity (units/mg)
1150 2960 2120 435
1130 325 176 7
1.0 9.1 12.1 62.1
Properties I T h e e n z y m e is active with ethanolamine, 3-amino-l-propanol, or 1-amino-2-propanol as substrate at maximal relative velocities of 100, 56, and 18, respectively. Other amines such as ethylamine, n-propylamine, putrescine, choline, and phosphoethanolamine were inactive at a concentration of 0.1 M. The K• for ethanolamine is 5 )~ 10-3 M at pH 8.0.
[66]
FORMALDEHYDE DEHYDROGENASE
357
The enzyme is active at pH values between 7 and 10.5, with optimum activity at pH 8.0. Preparations of purified enzyme have been stored at --5 ° for 6 months without any appreciable loss of activity. Inhibition was obtained with the following reagents at the concentrations noted: 0.2 mM quinacrine, 48%; 1 mM hydrazine, 97%; 0.1 mM hydroxylamine, 100%; 10 mM iproniazid, 64%; 10 mM isoniazid, 8%; 0.1 mM semicarbazide, 96%; 20 mM sodium bisulfite, 68%; 10 mM cysteine, 80%; 10 mM glutathione, 25%; 10 mM mercaptoethanol, 68%.
[ 66 ] F o r m a l d e h y d e D e h y d r o g e n a s e
By
ZELDA B. ROSE a n d EFRAIM RACKER
Formaldehyde + DPN + ----~ formate + DPNH + H +
Assay Method
Princ@le. The assay of the enzyme in crude extracts is based on the formation of acid. 1 Alcohol dehydrogenase present in the extracts reacts with formaldehyde and D P N H to yield methanol and DPN. The overall reaction catalyzed by formaldehyde dehydrogenase and alcohol dehydrogenase is a dismutation of formaldehyde to methanol and formic acid. Catalytic amounts of D P N (NAD) and GSH are required. The reaction is followed by measuring manometrically the rate at which the acid produced releases carbon dioxide from bicarbonate buffer. After acetone treatment (step 2), the enzyme can be assayed spectrophotometrically by observing the initial rate of increase of absorbancy at 340 n ~ due to the reduction of D P N (NAD). Residual alcohol dehydrogenase is inhibited by hydroxylamine. 2 Manometric Assay ]or Crude Extracts Reagents Formaldehyde, 0.12 M Glutathione, 0.067 M D P N (NAD), 0.012 M KHCO~, 2 M (freshly prepared)
Procedure. To the main compartment of a Warburg flask the following are added to a final volume of 2.67 ml: 0.10 ml of GSH, 0.10 ml of 'Z. B. Rose and E. Racker, J. Biol. Chem. 237, 3279 (1962). N. O. Kaplan and M. M. Ciotti, J. Biol. Chem. 201, 785 (1953).