- Email: [email protected]
[120] Glutamate decarboxylase (Escherichia coli)
[120]
GLUTAMATEDECARBOXYLASE
857
[120] Glutamate Decarboxylase (Escherichia coli) 1 By
LOWELL
P.
HAGER
NH3 +
l -O2CCH2CH2CHCO2-
+ H +~
CO2 + -O2CCH2CH2CH2NH3
+
Glutamate decarboxylase in Escherichia coli is an inducible enzyme, synthesized in large amounts when the organism is grown in the presence of glutamate. TM T h e crystalline enzyme has been obtained as a byproduct from the purification of pyruvic dehydrogenase (cytochrome bl). 2'3 Assay method Principle. The routine assay of enzymatic activity is performed by determining the rate of release of 14CO2 from glutamate- 1-~4C. The rate of the reaction is proportional to enzyme concentration and the release of CO2 from substrate is linear with respect to time under the conditions described. Reagents
Pyridine-HCl buffer, 0.4 M, pH 4.5 L-Glutamate, 0.052 M, containing in 0.5 ml a total of 40,000 cpm of glutamate- 1-14C Sodium phosphate buffer, 0.01 M, pH 7.0, containing 0.2% bovine serum albumin Scintillation counting fluid containing 200 ml of toluene, 50 ml of methyl Cellosolve, 1 g of 2,5-diphenyloxazole (PPO) and 60 mg of 2,2-p-phenylenebis (5-phenyloxazole) (POPOP) Solution of ethanolamine in ethylene glycol monomethyl ether, 1:2 v/v Sulfuric acid, 5 N Procedure. The reaction is carried out in reaction flasks (K-88230) purchased from the Kontes Glass Company. The center cup in the reaction flask contains 0.15 ml of the ethanolamine-ethylene glycol monomethyl ether solution to absorb CO2. For assay, a concentrated prepara-
1EC4.1.1.15; e-glutamate i-carboxy-lyase. lap. H. Strausbauch, E. H. Fisher, C. Cunningham, and L. P. Hager, Biochem. Biophys. Res. Commun. 28, 525 (1967). 2SeeVol. 1X [51]. SF. R. Williamsand L. P. Hager, Arch.Biochem.Biophys.116, 168 (1966).
858
GLUTAMIC ACID AND GLUTAMINE
[120]
tion of glutamate decarboxylase is diluted in the sodium phosphate buffer-bovine serum albumin buffer mixture to yield a preparation containing approximately 0.02 mg of enzyme per milliliter. The reaction is started by injecting a 0.5-ml aliquot of the diluted enzyme into the reaction vessel which contains 1.0 ml of pyridine-HC1 buffer and 0.5 ml of glutamate-14C. All solutions are preincubated at 38 ° to ensure temperature equilibration. After 5 minutes' incubation with shaking, the reaction is terminated by injecting 0.4 ml of 5 N H2SO4 into the main compartment of the reaction flask. The incubation and shaking is continued for 30 additional minutes at room temperature to allow the 14CO~ to be absorbed by the ethanolamine. The center well of the reaction flask containing the ethanolamine solution is dropped into scintillation vials containing 10 ml of scintillation fluid and counted according to the method of Jeffay and Alverez. 4 Definition of Unit and Specific Activity. One unit is the amount of enzyme required to produce 1 micromole of COs per minute under the specified assay conditions. Specific activity is expressed as units per milligram of protein. Protein is determined by the biuret method of Weichselbaum 5 using crystalline bovine serum albumin as a standard. Purification Procedure The growth conditions for Escherichia coli and the initial steps in the purification are identical to those developed for the isolation of pyruvic dehydrogenase (cytochrome b~).2,z During chromatography on DEAESephadex (step 6), pyruvic dehydrogenase and glutamate decarboxylase are resolved. Glutamate decarboxylase elutes from the DEAE column at an approximate purity of 60%. Crystallization. The DEAE-Sephadex column fractions containing glutamate decarboxylase are pooled and the enzyme is precipitated by the addition of solid ammonium sulfate to a final concentration of 40% w/v. For crystallization, the precipitated enzyme is dissolved in 0.05 M sodium phosphate buffer, pH 6.5, at a level of 40 mg of protein per milliliter. Solid ammonium sulfate is added in small portions to the icecold solution over a period of 5 days. Crystals appear in the form of thin needles or flat plates when the concentration of ammonium sulfate reaches 15 % w/v. Additional ammonium sulfate is slowly added over a 2-day period to a final concentration of 20% w/v. The specific activity of the preparation can be improved by recrystallization using the same procedure as described above. The results of a purification run are shown in the table. 4H. Jeffay and J. Alverez, Anal. Chem. 33, 612 (1961). ST. E. Weichselbaum, Am.J. Clin. Pathol. (Tech. Sect.) 10, 40 (1946).
[ 120]
GLUTAMATE DECARBOXYLASE
~
<
859
v v v l
e. o u:=
Z
~D
=. ~J
O
~8
860
GLUTAMIC ACID AND GLUTAMINE
[121]
Properties The crystalline decarboxylase has a calculated molecular weight of approximately 365,000. The sedimentation constant (s~0,w) in 0.1 M pyridine.HCl, pH 4.5, is 12.7 S. Pyridoxal phosphate analyses indicate that the enzyme contains 1 mole of pyridoxal 5'-phosphate per 75,000 g of protein. The crystalline enzyme has a specific activity of 110 (+5) micromoles of COz released per minute per milligram of protein at pH 4.5, 38 °. TheKm for glutamate is 5 x 10-4M.
[121 ] D-Glutamic Acid Cyclotransferase (Mouse Kidney, Liver) D-Glutamic acid .
B y ALTON MEISTER • D-pyrrolidone carboxylic acid + H20
This enzyme, which catalyzes the optically specific cyclization of v-glutamic acid, has been found in the kidney and liver of the mouse, rat, and man; some activity has also been found in certain other animal tissues. 1"2 This enzyme appears to be responsible for the conversion in certain animals of administered D-glutamic acid to urinary D-pyrrolidone carboxylic acid. 3 D-Pyrrolidone carboxylic acid is a normal urinary excretory product in man. 2
Assay Method Principle. 2 The activity of the enzyme is determined by following the conversion of D-glutamate-14C to D-pyrrolidone carboxylate-14C by measuring the rate of disappearance of a4C-labeled substrate or of the appearance of the a4C product. If v-glutamate-l-~4C is employed, the reaction mixtures (after incubation) may be treated with ninhydrin, thus effecting the oe-decarboxylation of the remaining D-glutamate-1-~4C; the residual 14C due to the pyrrolidone carboxylate-14C is then determined. The reaction may also be followed by determination of the rate of disappearance of D-glutamate as estimated by the colorimetric ninhydrin reaction.
1A. Meister and M. W. Bukenberger, Nature 194, 557 (1962). ~A. Meister, M. W. Bukenberger, and M. Strassburger, Biochem. Z. 338, 217 (1963). aS. Ramer, J. Biol. Chem. 152, 559 (1944).
GLUTAMATEDECARBOXYLASE
857
[120] Glutamate Decarboxylase (Escherichia coli) 1 By
LOWELL
P.
HAGER
NH3 +
l -O2CCH2CH2CHCO2-
+ H +~
CO2 + -O2CCH2CH2CH2NH3
+
Glutamate decarboxylase in Escherichia coli is an inducible enzyme, synthesized in large amounts when the organism is grown in the presence of glutamate. TM T h e crystalline enzyme has been obtained as a byproduct from the purification of pyruvic dehydrogenase (cytochrome bl). 2'3 Assay method Principle. The routine assay of enzymatic activity is performed by determining the rate of release of 14CO2 from glutamate- 1-~4C. The rate of the reaction is proportional to enzyme concentration and the release of CO2 from substrate is linear with respect to time under the conditions described. Reagents
Pyridine-HCl buffer, 0.4 M, pH 4.5 L-Glutamate, 0.052 M, containing in 0.5 ml a total of 40,000 cpm of glutamate- 1-14C Sodium phosphate buffer, 0.01 M, pH 7.0, containing 0.2% bovine serum albumin Scintillation counting fluid containing 200 ml of toluene, 50 ml of methyl Cellosolve, 1 g of 2,5-diphenyloxazole (PPO) and 60 mg of 2,2-p-phenylenebis (5-phenyloxazole) (POPOP) Solution of ethanolamine in ethylene glycol monomethyl ether, 1:2 v/v Sulfuric acid, 5 N Procedure. The reaction is carried out in reaction flasks (K-88230) purchased from the Kontes Glass Company. The center cup in the reaction flask contains 0.15 ml of the ethanolamine-ethylene glycol monomethyl ether solution to absorb CO2. For assay, a concentrated prepara-
1EC4.1.1.15; e-glutamate i-carboxy-lyase. lap. H. Strausbauch, E. H. Fisher, C. Cunningham, and L. P. Hager, Biochem. Biophys. Res. Commun. 28, 525 (1967). 2SeeVol. 1X [51]. SF. R. Williamsand L. P. Hager, Arch.Biochem.Biophys.116, 168 (1966).
858
GLUTAMIC ACID AND GLUTAMINE
[120]
tion of glutamate decarboxylase is diluted in the sodium phosphate buffer-bovine serum albumin buffer mixture to yield a preparation containing approximately 0.02 mg of enzyme per milliliter. The reaction is started by injecting a 0.5-ml aliquot of the diluted enzyme into the reaction vessel which contains 1.0 ml of pyridine-HC1 buffer and 0.5 ml of glutamate-14C. All solutions are preincubated at 38 ° to ensure temperature equilibration. After 5 minutes' incubation with shaking, the reaction is terminated by injecting 0.4 ml of 5 N H2SO4 into the main compartment of the reaction flask. The incubation and shaking is continued for 30 additional minutes at room temperature to allow the 14CO~ to be absorbed by the ethanolamine. The center well of the reaction flask containing the ethanolamine solution is dropped into scintillation vials containing 10 ml of scintillation fluid and counted according to the method of Jeffay and Alverez. 4 Definition of Unit and Specific Activity. One unit is the amount of enzyme required to produce 1 micromole of COs per minute under the specified assay conditions. Specific activity is expressed as units per milligram of protein. Protein is determined by the biuret method of Weichselbaum 5 using crystalline bovine serum albumin as a standard. Purification Procedure The growth conditions for Escherichia coli and the initial steps in the purification are identical to those developed for the isolation of pyruvic dehydrogenase (cytochrome b~).2,z During chromatography on DEAESephadex (step 6), pyruvic dehydrogenase and glutamate decarboxylase are resolved. Glutamate decarboxylase elutes from the DEAE column at an approximate purity of 60%. Crystallization. The DEAE-Sephadex column fractions containing glutamate decarboxylase are pooled and the enzyme is precipitated by the addition of solid ammonium sulfate to a final concentration of 40% w/v. For crystallization, the precipitated enzyme is dissolved in 0.05 M sodium phosphate buffer, pH 6.5, at a level of 40 mg of protein per milliliter. Solid ammonium sulfate is added in small portions to the icecold solution over a period of 5 days. Crystals appear in the form of thin needles or flat plates when the concentration of ammonium sulfate reaches 15 % w/v. Additional ammonium sulfate is slowly added over a 2-day period to a final concentration of 20% w/v. The specific activity of the preparation can be improved by recrystallization using the same procedure as described above. The results of a purification run are shown in the table. 4H. Jeffay and J. Alverez, Anal. Chem. 33, 612 (1961). ST. E. Weichselbaum, Am.J. Clin. Pathol. (Tech. Sect.) 10, 40 (1946).
[ 120]
GLUTAMATE DECARBOXYLASE
~
<
859
v v v l
e. o u:=
Z
~D
=. ~J
O
~8
860
GLUTAMIC ACID AND GLUTAMINE
[121]
Properties The crystalline decarboxylase has a calculated molecular weight of approximately 365,000. The sedimentation constant (s~0,w) in 0.1 M pyridine.HCl, pH 4.5, is 12.7 S. Pyridoxal phosphate analyses indicate that the enzyme contains 1 mole of pyridoxal 5'-phosphate per 75,000 g of protein. The crystalline enzyme has a specific activity of 110 (+5) micromoles of COz released per minute per milligram of protein at pH 4.5, 38 °. TheKm for glutamate is 5 x 10-4M.
[121 ] D-Glutamic Acid Cyclotransferase (Mouse Kidney, Liver) D-Glutamic acid .
B y ALTON MEISTER • D-pyrrolidone carboxylic acid + H20
This enzyme, which catalyzes the optically specific cyclization of v-glutamic acid, has been found in the kidney and liver of the mouse, rat, and man; some activity has also been found in certain other animal tissues. 1"2 This enzyme appears to be responsible for the conversion in certain animals of administered D-glutamic acid to urinary D-pyrrolidone carboxylic acid. 3 D-Pyrrolidone carboxylic acid is a normal urinary excretory product in man. 2
Assay Method Principle. 2 The activity of the enzyme is determined by following the conversion of D-glutamate-14C to D-pyrrolidone carboxylate-14C by measuring the rate of disappearance of a4C-labeled substrate or of the appearance of the a4C product. If v-glutamate-l-~4C is employed, the reaction mixtures (after incubation) may be treated with ninhydrin, thus effecting the oe-decarboxylation of the remaining D-glutamate-1-~4C; the residual 14C due to the pyrrolidone carboxylate-14C is then determined. The reaction may also be followed by determination of the rate of disappearance of D-glutamate as estimated by the colorimetric ninhydrin reaction.
1A. Meister and M. W. Bukenberger, Nature 194, 557 (1962). ~A. Meister, M. W. Bukenberger, and M. Strassburger, Biochem. Z. 338, 217 (1963). aS. Ramer, J. Biol. Chem. 152, 559 (1944).