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[131] ATP: Glutamine synthetase adenylyltransferase (Escherichia coli B)
922
GLUTAMIC ACID AND GLUTAMINE
[ 131 ]
60 mM Mg ~+. Therefore the n u m b e r of adenylylated subunits per mole, n, can be calculated from the ratio of the y-glutamyltransferase activities at pH 7.15 in the presence and absence of 60 mM Mg 2+, as follows: (activity in 0.2 mM Mn z+ plus 60 mM Mg ~+ ) n= _ activity in 0.3 mM Mn 2+ × 12 This method of determining the E, value has the advantage that only catalytic amounts of enzyme are required and the enzyme need not be absolutely pure, so long as interfering activities (viz., glutaminase) are absent. Feedback Inhibitors. T h e biosynthetic activity of unadenylylated enzyme is less sensitive to inhibition by histidine, tryptophan, or CTP than is the adenylylated enzyme, but it is more susceptible to inhibition by glycine, alanine, and AMP. Quantitatively similar relationships exist for the yglutamyltransferase activity except with respect to AMP, which does not inhibit the Mn 2+ d e p e n d e n t activity of the unadenylylated enzyme but does inhibit this form of the enzyme when Mg 2+ is also present. Tryptophan, histidine, and CTP do not inhibit the transferase activity of the unadenylylated enzyme, but the transferase activity of the adenylylated enzyme is very sensitive to inhibition by these effectors, a4 A review of the physical and kinetic properties of glutamine synthetase, containing also a description and model of the specific cumulative feedback inhibition exhibited by the enzyme, has been published. 24 ~4E. R. Stadtman, B. M. Shapiro, H. S. Kingdon, C. A. Woolfolk, and J. S. Hubbard,
Advan.EnzymeRegulation6, 257 (1968).
[ 131] ATP: Glutamine Synthetase Adenylyltransferase (Escherichia coli B) By EBERHARD EBNER, CARLOS GANCEDO a n d HELMUT HOLZER Glutamine synthetase + 12 ATP -~ glutamine synthetase-(AMP)12 + 12 PPl
Assay Method Principle. Glutamine synthetase is adenylylated upon incubation with ATP:glutamine synthetase adenylyltransferase (adenylylating enzyme), ATP, Mg *+, and glutamine. T h e adenylylation of glutamine synthetase is paralleled by a decrease of its biosynthetic activity. T h e extent of this inactivation is used to determine the activity of the adenylylating
[131]
ATP: GLUTAMINE SYNTHETASE ADENYLYLTRANSFERASE
923
enzyme? In addition to the biosynthesis of glutamine, glutamine synthetase catalyzes the transfer of the y-glutamyl moiety of glutamine to hydroxylamine. This transfer activity (measured in the presence of ADP) is not substantially affected by adenylylation.
Reagents Tris-HCl, 0.1 M, pH 7.6 L-Glutamine, 0.1 M, in 0.1 M Tris HCI, pH 7.6 ATP, 0.02 M, neutralized with 2 N NaOH MgCI2, 0.5 M Glutamine synthetase, 2 a solution containing at least 4000 units/ml, a dialyzed against 0.1 M Tris HCI, pH 7.6.
Assay Mixture. The assay mixture has a final volume of 0.1 ml. It contains the preparation to be assayed, e.g., 10 or 20 pA of extract, 80 units of glutamine synthetase, and the other reagents at the following final concentrations: 1.0 mM ATP, 20 mM L-glutamine, 25 mM MgCI2, and 50 mM Tris-HC1, pH 7.6. Procedure. The enzyme preparation and 30/~1 of Tris-HCl, pH 7.6, are brought with water to a final volume of 50/~1. The reaction is started by addition of a freshly prepared solution containing the other components of the assay mixture. After 10 minutes incubation at 30°, the reaction is stopped by addition of 1.9 ml of glutamine synthetase test solution and the remaining glutamine synthetase is determined. 3 A reference test without adenylylating enzyme and a blank without glutamine synthetase are run in parallel. Definition of Units. One unit is defined as the amount of enzyme that produces a 50% decrease of the initial glutamine synthetase activity. For routine work a calibration curve representing the logarithm of the quotient initial/final glutamine synthetase units against units of adenylylating enzyme is used. T h e r e is a linear relationship between these two values in the range from 0.5 to 3.5 units of adenylylating enzyme. 4 XAn alternative can be used that involves the measurement of the incorporation of radioactivity from 14C-labeled ATP into glutamine synthetase [H. S. Kingdon, B. M. Shapiro, and E. R. Stadtman, Proc. Natl. Acad. Sci. U.S. 58, 1703 (1967); K. Wulff, D. Mecke, and H. Holzer, Biochem. Biophys. Res. Commun. 28, 740 (1967)]. ~Glutamine synthetase is purified from E. coli B according to C. A. Woolfolk, B. Shapiro, and E. R. Stadtman, Arch. Biochem. Biophys. 116, 177 (1966); see also this volume, article
[130]. 3Units and assay as described by G. Kohlhaw, W. Dr~/gert, and H. Holzer, Bioche~ Z. 341, 224 (1965). A similar assay is presented in this volume, article [ 130]. 4It is recommended that a calibration curve be drawn for each glutamine synthetase preparation since different batches exhibit different slopes in the calibration curve.
924
GLUTAMIC ACID AND GLUTAMINE
[ 131]
Purification Procedure
Growth of the Cells. Escherichia coli B is grown aerobically at 37 ° in a synthetic medium containing per liter 13.6 g KH2PO4, 0.2 g MgSO4 • 7 H20, 0.5 mg FeSO4 • 7 H20, 10.0 g glycerol, 2.0 g sodium glutamate, and KOH to adjust the pH to 7.0. Cells are harvested at the end of the log phase by centrifugation in a continuous-flow centrifuge (Christ, Junior 15000). About 2 g of cells (wet weight) per liter of culture medium are obtained. The cells can be stored at - 1 5 ° for at least 3 months without loss of activity. Notes on the Purification Procedure. The following purification steps are performed at 1-4 °. All solutions contain 10 mM 2-mercaptoethanol and 2 mM EDTA unless otherwise stated. DEAE-cellulose (Serva Entwicklungslabor, Heidelberg, Germany; capacity 0.86 meq/g) is pretreated according to the Whatman Technical Bulletin IE 2~s Hydroxylapatite is prepared as described by Levin. 6 For gradient etutions a Beckman Model No. 131 gradient pump is used. Before chromatography all adsorbents are equilibrated with the starting elution buffer. Chromatography is finished when the effluent of the column shows the salt concentration of the limiting elution buffer. The volume of the fractions collected is 20 ml in step 2, and 10 ml in steps 4 and 5. Step I. Preparation of the Extract. Frozen cells, 360 g, are thawed, ground with Alcoa (A-305 bacteriological grade, Aluminum Corporation of Americay and extracted with 20 mM Tris-HCl, pH 7.5; 3.5 g Alcoa and 10 ml buffer are used per gram of cells (wet weight). The suspension is centrifuged at 27,000 g for 30 minutes; the yield is about 3500 ml of a yellow, slightly turbid solution containing 5-10 mg of protein per milliliter~ Step 2: Elution from DEAE-Cellulose at pH 7.5. The extract is stirred for 1 hour with DEAE-cellulose, the extract:DEAE-cellulose ratio being about 3 : 1 (milliliters : column volume). In order to measure the amount of DEAE-cellulose required, it is recommended that the preparation be equilibrated in the column that will later be used for gradient elution (5 × 80 cm). The DEAE-cellulose 4s~then removed from the column, mixed with the enzyme extract, and repacked'into the column. The enzyme is then eluted by a linear 0-0.4 M NaC1 gradient in 20 mM TrisHC1 buffer, p H 7.5. The gradient volume should be approximately equal to the volume of the extract of step 1 (4 liters). The flow rate is 5Avaita~e from-H. Reeve Angel and Co. Ltd., 14~:N°ewBridge Street:~:London EC 4, England. e(~. Levin, Vol. V [2].
[131]
ATP" GLUTAMINE SYNTHETASE ADENYLYLTRANSFERASE
925
maintained at 60-80 ml per hour. Activity appears at a NaCI concentration of 0.18 M. The bulk of the enzyme, which is completely separated from glutamine synthetase, is found in fractions 140-200 (20 ml each). The active fractions are pooled. About 1000 ml of a clear yellow solution are obtained. Step 3. Fractionation with Ammonium Sulfate. To the combined fractions of step 2, solid ammonium sulfate is added to a final concentration of 2.53 M (65% saturation). After 15 minutes, the suspension is centrifuged (27,000 g, 15 minutes). The supernatant is discarded, and the precipitate is stirred for 1 hour in 100 ml (10% by volume of the solution of step 2) of 20 mM potassium phosphate buffer, pH 7.0, containing 1.56 M ammonium sulfate (40% saturation). The suspension obtained by stirring has a final volume of 115 ml and an ammonium sulfate concentration of 1.68 M (43% saturation). If the volume change after the precipitate is suspended differs from that indicated (> 15%) the final ammonium sulfate concentration should be adjusted to 1.68 M. After centrifugation (27,000 g, 15 minutes) the supernatant is discarded and the precipitate is stirred for 1 hour in 100 ml of 20 mM potassium phosphate buffer, pH 7.0, but with 1.09 M ammonium sulfate (28% saturation). The suspension is centrifuged (48,000 g, 10 minutes), and the precipitate is discarded. To the supernatant, solid ammonium sulfate is added to a final concentration of 1.95 M (50% saturation). After 15 minutes the mixture is centrifuged (27,000 g, 5 minutes) and the precipitate is dissolved in 10 ml of 20 mM imidazole-HCl buffer, pH 7.0 (1% by volume of the solution of step 2). The dark greenish-yellow solution is dialyzed overnight against 5 liters of the same buffer. Step 4. Chromatography on DEAE-Cellulose at pH 7.0. The dialyzate of step 3 is stirred for 3 hours with DEAE-cellulose, the protein:cellulose ratio being 1:2 (milligrams:dry weight). W h e n n o more activity is detectable in the supernatant, unadsorbed protein is removed by washing the loaded DEAE-cellulose twice with 20 ml of 20 mM imidazole-HC1 buffer, pH 7.0. The slurry is layered on a prepacked 2.5 × 70 cm column of unloaded DEAE-cellulose. 7 Elution of the activity is performed with 500 ml of 20 mM imidazole-HCl buffer, pH 7.0, containing NaCI in a linear gradient from 0.04 to 0.4 M. The flow rate is maintained at 20 ml/ hour. The activity appears between fractions 50 and 75 (10 ml each). Fractions with the same or higher specific activity than that of the solution of step 3 are poured together. About 150 ml of a slightly yellow solution is obtained. 7A similar procedure of loading the adsorbent has been described by M. G. Sarngadharan, A. Watanabe, and B. M. Pogell, Federation Proc. 27,791 (1968).
926
GLUTAMIC ACID AND GLUTAMINE
[ 131]
Step 5. Chromatography on Hydroxylapatite at pH 7.0. EDTA is omitted from the solutions used in this step. The solution of step 4 is added by means of a p u m p to a 2.5 × 80 cm hydroxylapatite column, the flow rate being 20 ml/hour, The activity is eluted by a linear potassium phosphate gradient from 0 to 0.2 M in 500 ml of 20 mM imidazole-HCl, final pH 7.0. The flow rate is 30 ml/hour. Activity appears in the range of 0.05-0.1 M phosphate (between fractions 65 and 90, each 10 ml). Fractions with a specific activity 2-fold higher than that of the material of step 4 are pooled, resulting in about 75 ml of a colorless solution. Concentration of the protein solution can be achieved as follows. The pH is adjusted to 4.7 by addition of 1 M acetic acid-NaOH buffer, pH 4.0. After 20 minutes the mixture is centrifuged at 27,000 g for 15 minutes. The precipitate is resuspended in 1 ml of 0.1 M imidazoleHC1 buffer, pH 7.6. The table summarizes the purification procedure. TM PURIFICATION OF ATP: GLUTAMINESYNTHETASEADENYLYLTRANSFERASEFROM Escherichia coli
Step 1. Extract 2. Elution from DEAE-cellulose pH 7.5 3. A m m o n i u m sulfate fractionation 4. Chromatography on DEAEcellulose, pH 7.0 5. Chromatography on hydroxyl apatite, pH 7.0
Volume (ml)
Activity (units/ml)
Protein a (mg/ml)
Yield (%)
Purification (fold)
7.4 3.5
100 51
1 4.2
33
8.8
3460 870
230 462
15
18,000
162
1,000
2.3
20
14
75
1,070
0.8
10
43
64
aProtein is determined by the method of Lowry et al. [O. H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chem. 193, 265 (1951); see also Vol. III [73]] with bovine serum albumin as standard.
Properties Stability. At a protein concentration of 10 mg/ml, the final preparation loses half of its activity in about 4 weeks upon storage at 4 °. pH Optimum. Maximal activity of the enzyme is observed at about pH 7.6 either in imidazole-HCl or Tris-HCl buffers. Temperature Dependence. The reaction shows an activation energy of 13,000 cal/mole measured between 5° and 30 °. Specificity. The enzyme shows an absolute requirement for ATP; :aSince the submission of this manuscript, a 300-fold purification of the adenylylating enzyme has been achieved; for details, see footnote 11.
[ 132]
5%a4C-ADENYLYL GLUTAMINESYNTHETASE
927
UTP, CTP, and ITP cannot replace ATP. The Km for ATP is 0.15 mM. Concentrations of ATP higher than 1 mM inhibit the enzyme. This inhibition is complete at about 15 mM. For half maximal activity, 10 mM Mg 2+ is required. At the same concentration, Mn 2+ can substitute for Mg 2+ with a 30% effectivity.9 Ba 2+, Ca 2+, Zn 2+, and Cu 2+ produced complete precipitation of the enzyme already at concentrations of 5 mM, thus resulting in abolition of activity. Metabolic Effectors. The reaction is stimulated by glutamine. Addition of 20 mM glutamine at 2.5 mM ATP produces a 20-fold increase of the reaction rate. 8 Half-maximal activity is attained at 1.3 mM glutamine. Some other amino acids also show stimulatory effect, but to a lesser extent than glutamine, a°,la ot-Ketoglutarate, 0.8 mM, phosphate, 20 mM, pyrophosphate, 5 mM, or sulfate, 6 mM, causes a 50% decrease in the activity of the enzyme.
Acknowledgment The able technical assistance of Miss S. Elsaesser during the purification is gratefully acknowledged. SH. Schutt, unpublished results. 9When Mn~+ is employed instead of Mg~+ in the adenylylating reaction, Mn2+ interferes with the test normally employed; therefore the remaining glutamine synthetase activity should be assayed by the method of P. D. Boyer, R. C. Mills, and H.J. Fromm, Arch. Biochem. B iophys. 81,249 (1959). l°D. Mecke, K. Wulff, and H. Holzer, Biochim. Biophys. Acta 128,559 (1966). laE. Ebner, D. Wolf, C. Gancedo, S. EIs~isser,and H. Holzer, EuropeanJ. Biochem., in press.
[132]
The Preparation Glutamine
of 5'-14C-Labeled Adenylyl S y n t h e t a s e (Escherichia coli) By ANN GINSBURG
Unadenylylated GlnSyn + 14C-ATP
MS'+,glutamine )
adenylyhransferase
t4C-Adenylyl GlnSyn + PP,
The glutamine synthetase (GlnSyn) from Escherichia coli is a dodecameric aggregate that may contain zero to twelve equivalents of 5'adenylyl groups covalently bound through phosphodiester linkage to a specific tyrosyl residue of the protein subunit, t-e The preparation IC. A. Woolfolk, B. M. Shapiro, and E. R. Stadtman, Arch. Biochem. Biophys. 116, 177 (1966). ~H. K. Kingdon, B. M. Shapiro, and E. R. Stadtman, Proc. Natl. Acad. Sci. U. S. 58, 1703 (1967). SB. M. Shapiro, H. S. Kingdon, and E. R. Stadtman, Proc. Natl. Acad. Sci. U. S. 58, 642 (1967).
GLUTAMIC ACID AND GLUTAMINE
[ 131 ]
60 mM Mg ~+. Therefore the n u m b e r of adenylylated subunits per mole, n, can be calculated from the ratio of the y-glutamyltransferase activities at pH 7.15 in the presence and absence of 60 mM Mg 2+, as follows: (activity in 0.2 mM Mn z+ plus 60 mM Mg ~+ ) n= _ activity in 0.3 mM Mn 2+ × 12 This method of determining the E, value has the advantage that only catalytic amounts of enzyme are required and the enzyme need not be absolutely pure, so long as interfering activities (viz., glutaminase) are absent. Feedback Inhibitors. T h e biosynthetic activity of unadenylylated enzyme is less sensitive to inhibition by histidine, tryptophan, or CTP than is the adenylylated enzyme, but it is more susceptible to inhibition by glycine, alanine, and AMP. Quantitatively similar relationships exist for the yglutamyltransferase activity except with respect to AMP, which does not inhibit the Mn 2+ d e p e n d e n t activity of the unadenylylated enzyme but does inhibit this form of the enzyme when Mg 2+ is also present. Tryptophan, histidine, and CTP do not inhibit the transferase activity of the unadenylylated enzyme, but the transferase activity of the adenylylated enzyme is very sensitive to inhibition by these effectors, a4 A review of the physical and kinetic properties of glutamine synthetase, containing also a description and model of the specific cumulative feedback inhibition exhibited by the enzyme, has been published. 24 ~4E. R. Stadtman, B. M. Shapiro, H. S. Kingdon, C. A. Woolfolk, and J. S. Hubbard,
Advan.EnzymeRegulation6, 257 (1968).
[ 131] ATP: Glutamine Synthetase Adenylyltransferase (Escherichia coli B) By EBERHARD EBNER, CARLOS GANCEDO a n d HELMUT HOLZER Glutamine synthetase + 12 ATP -~ glutamine synthetase-(AMP)12 + 12 PPl
Assay Method Principle. Glutamine synthetase is adenylylated upon incubation with ATP:glutamine synthetase adenylyltransferase (adenylylating enzyme), ATP, Mg *+, and glutamine. T h e adenylylation of glutamine synthetase is paralleled by a decrease of its biosynthetic activity. T h e extent of this inactivation is used to determine the activity of the adenylylating
[131]
ATP: GLUTAMINE SYNTHETASE ADENYLYLTRANSFERASE
923
enzyme? In addition to the biosynthesis of glutamine, glutamine synthetase catalyzes the transfer of the y-glutamyl moiety of glutamine to hydroxylamine. This transfer activity (measured in the presence of ADP) is not substantially affected by adenylylation.
Reagents Tris-HCl, 0.1 M, pH 7.6 L-Glutamine, 0.1 M, in 0.1 M Tris HCI, pH 7.6 ATP, 0.02 M, neutralized with 2 N NaOH MgCI2, 0.5 M Glutamine synthetase, 2 a solution containing at least 4000 units/ml, a dialyzed against 0.1 M Tris HCI, pH 7.6.
Assay Mixture. The assay mixture has a final volume of 0.1 ml. It contains the preparation to be assayed, e.g., 10 or 20 pA of extract, 80 units of glutamine synthetase, and the other reagents at the following final concentrations: 1.0 mM ATP, 20 mM L-glutamine, 25 mM MgCI2, and 50 mM Tris-HC1, pH 7.6. Procedure. The enzyme preparation and 30/~1 of Tris-HCl, pH 7.6, are brought with water to a final volume of 50/~1. The reaction is started by addition of a freshly prepared solution containing the other components of the assay mixture. After 10 minutes incubation at 30°, the reaction is stopped by addition of 1.9 ml of glutamine synthetase test solution and the remaining glutamine synthetase is determined. 3 A reference test without adenylylating enzyme and a blank without glutamine synthetase are run in parallel. Definition of Units. One unit is defined as the amount of enzyme that produces a 50% decrease of the initial glutamine synthetase activity. For routine work a calibration curve representing the logarithm of the quotient initial/final glutamine synthetase units against units of adenylylating enzyme is used. T h e r e is a linear relationship between these two values in the range from 0.5 to 3.5 units of adenylylating enzyme. 4 XAn alternative can be used that involves the measurement of the incorporation of radioactivity from 14C-labeled ATP into glutamine synthetase [H. S. Kingdon, B. M. Shapiro, and E. R. Stadtman, Proc. Natl. Acad. Sci. U.S. 58, 1703 (1967); K. Wulff, D. Mecke, and H. Holzer, Biochem. Biophys. Res. Commun. 28, 740 (1967)]. ~Glutamine synthetase is purified from E. coli B according to C. A. Woolfolk, B. Shapiro, and E. R. Stadtman, Arch. Biochem. Biophys. 116, 177 (1966); see also this volume, article
[130]. 3Units and assay as described by G. Kohlhaw, W. Dr~/gert, and H. Holzer, Bioche~ Z. 341, 224 (1965). A similar assay is presented in this volume, article [ 130]. 4It is recommended that a calibration curve be drawn for each glutamine synthetase preparation since different batches exhibit different slopes in the calibration curve.
924
GLUTAMIC ACID AND GLUTAMINE
[ 131]
Purification Procedure
Growth of the Cells. Escherichia coli B is grown aerobically at 37 ° in a synthetic medium containing per liter 13.6 g KH2PO4, 0.2 g MgSO4 • 7 H20, 0.5 mg FeSO4 • 7 H20, 10.0 g glycerol, 2.0 g sodium glutamate, and KOH to adjust the pH to 7.0. Cells are harvested at the end of the log phase by centrifugation in a continuous-flow centrifuge (Christ, Junior 15000). About 2 g of cells (wet weight) per liter of culture medium are obtained. The cells can be stored at - 1 5 ° for at least 3 months without loss of activity. Notes on the Purification Procedure. The following purification steps are performed at 1-4 °. All solutions contain 10 mM 2-mercaptoethanol and 2 mM EDTA unless otherwise stated. DEAE-cellulose (Serva Entwicklungslabor, Heidelberg, Germany; capacity 0.86 meq/g) is pretreated according to the Whatman Technical Bulletin IE 2~s Hydroxylapatite is prepared as described by Levin. 6 For gradient etutions a Beckman Model No. 131 gradient pump is used. Before chromatography all adsorbents are equilibrated with the starting elution buffer. Chromatography is finished when the effluent of the column shows the salt concentration of the limiting elution buffer. The volume of the fractions collected is 20 ml in step 2, and 10 ml in steps 4 and 5. Step I. Preparation of the Extract. Frozen cells, 360 g, are thawed, ground with Alcoa (A-305 bacteriological grade, Aluminum Corporation of Americay and extracted with 20 mM Tris-HCl, pH 7.5; 3.5 g Alcoa and 10 ml buffer are used per gram of cells (wet weight). The suspension is centrifuged at 27,000 g for 30 minutes; the yield is about 3500 ml of a yellow, slightly turbid solution containing 5-10 mg of protein per milliliter~ Step 2: Elution from DEAE-Cellulose at pH 7.5. The extract is stirred for 1 hour with DEAE-cellulose, the extract:DEAE-cellulose ratio being about 3 : 1 (milliliters : column volume). In order to measure the amount of DEAE-cellulose required, it is recommended that the preparation be equilibrated in the column that will later be used for gradient elution (5 × 80 cm). The DEAE-cellulose 4s~then removed from the column, mixed with the enzyme extract, and repacked'into the column. The enzyme is then eluted by a linear 0-0.4 M NaC1 gradient in 20 mM TrisHC1 buffer, p H 7.5. The gradient volume should be approximately equal to the volume of the extract of step 1 (4 liters). The flow rate is 5Avaita~e from-H. Reeve Angel and Co. Ltd., 14~:N°ewBridge Street:~:London EC 4, England. e(~. Levin, Vol. V [2].
[131]
ATP" GLUTAMINE SYNTHETASE ADENYLYLTRANSFERASE
925
maintained at 60-80 ml per hour. Activity appears at a NaCI concentration of 0.18 M. The bulk of the enzyme, which is completely separated from glutamine synthetase, is found in fractions 140-200 (20 ml each). The active fractions are pooled. About 1000 ml of a clear yellow solution are obtained. Step 3. Fractionation with Ammonium Sulfate. To the combined fractions of step 2, solid ammonium sulfate is added to a final concentration of 2.53 M (65% saturation). After 15 minutes, the suspension is centrifuged (27,000 g, 15 minutes). The supernatant is discarded, and the precipitate is stirred for 1 hour in 100 ml (10% by volume of the solution of step 2) of 20 mM potassium phosphate buffer, pH 7.0, containing 1.56 M ammonium sulfate (40% saturation). The suspension obtained by stirring has a final volume of 115 ml and an ammonium sulfate concentration of 1.68 M (43% saturation). If the volume change after the precipitate is suspended differs from that indicated (> 15%) the final ammonium sulfate concentration should be adjusted to 1.68 M. After centrifugation (27,000 g, 15 minutes) the supernatant is discarded and the precipitate is stirred for 1 hour in 100 ml of 20 mM potassium phosphate buffer, pH 7.0, but with 1.09 M ammonium sulfate (28% saturation). The suspension is centrifuged (48,000 g, 10 minutes), and the precipitate is discarded. To the supernatant, solid ammonium sulfate is added to a final concentration of 1.95 M (50% saturation). After 15 minutes the mixture is centrifuged (27,000 g, 5 minutes) and the precipitate is dissolved in 10 ml of 20 mM imidazole-HCl buffer, pH 7.0 (1% by volume of the solution of step 2). The dark greenish-yellow solution is dialyzed overnight against 5 liters of the same buffer. Step 4. Chromatography on DEAE-Cellulose at pH 7.0. The dialyzate of step 3 is stirred for 3 hours with DEAE-cellulose, the protein:cellulose ratio being 1:2 (milligrams:dry weight). W h e n n o more activity is detectable in the supernatant, unadsorbed protein is removed by washing the loaded DEAE-cellulose twice with 20 ml of 20 mM imidazole-HC1 buffer, pH 7.0. The slurry is layered on a prepacked 2.5 × 70 cm column of unloaded DEAE-cellulose. 7 Elution of the activity is performed with 500 ml of 20 mM imidazole-HCl buffer, pH 7.0, containing NaCI in a linear gradient from 0.04 to 0.4 M. The flow rate is maintained at 20 ml/ hour. The activity appears between fractions 50 and 75 (10 ml each). Fractions with the same or higher specific activity than that of the solution of step 3 are poured together. About 150 ml of a slightly yellow solution is obtained. 7A similar procedure of loading the adsorbent has been described by M. G. Sarngadharan, A. Watanabe, and B. M. Pogell, Federation Proc. 27,791 (1968).
926
GLUTAMIC ACID AND GLUTAMINE
[ 131]
Step 5. Chromatography on Hydroxylapatite at pH 7.0. EDTA is omitted from the solutions used in this step. The solution of step 4 is added by means of a p u m p to a 2.5 × 80 cm hydroxylapatite column, the flow rate being 20 ml/hour, The activity is eluted by a linear potassium phosphate gradient from 0 to 0.2 M in 500 ml of 20 mM imidazole-HCl, final pH 7.0. The flow rate is 30 ml/hour. Activity appears in the range of 0.05-0.1 M phosphate (between fractions 65 and 90, each 10 ml). Fractions with a specific activity 2-fold higher than that of the material of step 4 are pooled, resulting in about 75 ml of a colorless solution. Concentration of the protein solution can be achieved as follows. The pH is adjusted to 4.7 by addition of 1 M acetic acid-NaOH buffer, pH 4.0. After 20 minutes the mixture is centrifuged at 27,000 g for 15 minutes. The precipitate is resuspended in 1 ml of 0.1 M imidazoleHC1 buffer, pH 7.6. The table summarizes the purification procedure. TM PURIFICATION OF ATP: GLUTAMINESYNTHETASEADENYLYLTRANSFERASEFROM Escherichia coli
Step 1. Extract 2. Elution from DEAE-cellulose pH 7.5 3. A m m o n i u m sulfate fractionation 4. Chromatography on DEAEcellulose, pH 7.0 5. Chromatography on hydroxyl apatite, pH 7.0
Volume (ml)
Activity (units/ml)
Protein a (mg/ml)
Yield (%)
Purification (fold)
7.4 3.5
100 51
1 4.2
33
8.8
3460 870
230 462
15
18,000
162
1,000
2.3
20
14
75
1,070
0.8
10
43
64
aProtein is determined by the method of Lowry et al. [O. H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chem. 193, 265 (1951); see also Vol. III [73]] with bovine serum albumin as standard.
Properties Stability. At a protein concentration of 10 mg/ml, the final preparation loses half of its activity in about 4 weeks upon storage at 4 °. pH Optimum. Maximal activity of the enzyme is observed at about pH 7.6 either in imidazole-HCl or Tris-HCl buffers. Temperature Dependence. The reaction shows an activation energy of 13,000 cal/mole measured between 5° and 30 °. Specificity. The enzyme shows an absolute requirement for ATP; :aSince the submission of this manuscript, a 300-fold purification of the adenylylating enzyme has been achieved; for details, see footnote 11.
[ 132]
5%a4C-ADENYLYL GLUTAMINESYNTHETASE
927
UTP, CTP, and ITP cannot replace ATP. The Km for ATP is 0.15 mM. Concentrations of ATP higher than 1 mM inhibit the enzyme. This inhibition is complete at about 15 mM. For half maximal activity, 10 mM Mg 2+ is required. At the same concentration, Mn 2+ can substitute for Mg 2+ with a 30% effectivity.9 Ba 2+, Ca 2+, Zn 2+, and Cu 2+ produced complete precipitation of the enzyme already at concentrations of 5 mM, thus resulting in abolition of activity. Metabolic Effectors. The reaction is stimulated by glutamine. Addition of 20 mM glutamine at 2.5 mM ATP produces a 20-fold increase of the reaction rate. 8 Half-maximal activity is attained at 1.3 mM glutamine. Some other amino acids also show stimulatory effect, but to a lesser extent than glutamine, a°,la ot-Ketoglutarate, 0.8 mM, phosphate, 20 mM, pyrophosphate, 5 mM, or sulfate, 6 mM, causes a 50% decrease in the activity of the enzyme.
Acknowledgment The able technical assistance of Miss S. Elsaesser during the purification is gratefully acknowledged. SH. Schutt, unpublished results. 9When Mn~+ is employed instead of Mg~+ in the adenylylating reaction, Mn2+ interferes with the test normally employed; therefore the remaining glutamine synthetase activity should be assayed by the method of P. D. Boyer, R. C. Mills, and H.J. Fromm, Arch. Biochem. B iophys. 81,249 (1959). l°D. Mecke, K. Wulff, and H. Holzer, Biochim. Biophys. Acta 128,559 (1966). laE. Ebner, D. Wolf, C. Gancedo, S. EIs~isser,and H. Holzer, EuropeanJ. Biochem., in press.
[132]
The Preparation Glutamine
of 5'-14C-Labeled Adenylyl S y n t h e t a s e (Escherichia coli) By ANN GINSBURG
Unadenylylated GlnSyn + 14C-ATP
MS'+,glutamine )
adenylyhransferase
t4C-Adenylyl GlnSyn + PP,
The glutamine synthetase (GlnSyn) from Escherichia coli is a dodecameric aggregate that may contain zero to twelve equivalents of 5'adenylyl groups covalently bound through phosphodiester linkage to a specific tyrosyl residue of the protein subunit, t-e The preparation IC. A. Woolfolk, B. M. Shapiro, and E. R. Stadtman, Arch. Biochem. Biophys. 116, 177 (1966). ~H. K. Kingdon, B. M. Shapiro, and E. R. Stadtman, Proc. Natl. Acad. Sci. U. S. 58, 1703 (1967). SB. M. Shapiro, H. S. Kingdon, and E. R. Stadtman, Proc. Natl. Acad. Sci. U. S. 58, 642 (1967).