JOURNAL OF BACTERIOLOGY, Nov. 1975, p. 775-783Copyright X) 1975 American Society for Microbiology

Vol. 124, No. 2Printed in USA.

Glutamate Dehydrogenase from Escherichia coli: Purificationand Properties

NAOTO SAKAMOTO,' ANN MARIE KOTRE, AND MICHAEL A. SAVAGEAU*

Department of Microbiology, University of Michigan, Ann Arbor, Michigan 48104

Received for publication 29 May 1975

Glutamate dehydrogenase (L-glutamate:NADP+ oxidoreductase [deaminat-ing], EC 1.4.1.4) has been purified from Escherichia coli B/r. The purity of theenzyme preparation has been established by polyacrylamide gel electrophoresis,ultracentrifugation, and gel filtration. A molecular weight of 300,000 + 20,000has been calculated for the enzyme from sedimentation equilibrium measure-

ments. Polyacrylamide gel electrophoresis in sodium dodecyl sulfate and sedi-mentation equilibrium measurements in guanidine hydrochloride have revealedthat glutamate dehydrogenase consists of polypeptide chains with the identicalmolecular weight of 50,000 ± 5,000. The results of molecular weight determina-tion lead us to propose that glutamate dehydrogenase is a hexamer of subunitswith identical molecular weight. We also have studied the stability and kineticsof purified glutamate dehydrogenase. The enzyme remains active when heattreated or when left at room temperature for several months but is inactivatedby freezing. The Michaelis constants of glutamate dehydrogenase are 1,100, 640,and 40 ,uM for ammonia, 2-oxoglutarate, and reduced nicotinamide adeninedinucleotide phosphate, respectively.

Glutamic acid, as the donor of an a-aminogroup in transaminase reactions, is a key me-tabolite for the biosynthesis of amino acids.There are two enzymes in Escherichia coli re-sponsible for the biosynthesis of glutamic acid(13, 14, 16). One is glutamate dehydrogenase (L-glutamate:NADP+ oxidoreductase [deaminat-ing], EC 1.4.1.4) that catalyzes the reversiblereaction:

2-oxoglutarate + NH4+ + NADPH

= L-glutamate + water + NADP+ (i)

The other is glutamate synthase (L-gluta-mate:NADP+ oxidoreductase [deaminating,glutamine-forming], EC 1.4.1.X) that carriesout the virtually irreversible reaction of amidetransfer:

2-oxoglutarate + L-glutamine + NADPH

-* 2 L-glutamate + NADP+ (ii)

In this paper we report the purification ofglutamate dehydrogenase from E. coli B/r andthe characterization of some of its kinetic andmolecular properties. We also have purified glu-tamate synthase and have obtained results thatconfirm those reported by Miller and Stadtmanfor E. coli W (13). Our interest in glutamate

I Present address: Life Science Division, Rikagaku Ken-kyusho, 2-28-8 Honkomagome, Bunkyo-ku, Tokyo 113, Ja-pan.

synthase derives from two earlier observationsthat suggested a possible association of gluta-mate dehydrogenase and glutamate synthaseactivities (14): glutamate formation in the pres-ence of both ammonia and glutamine is notadditive in a cell extract, and a partially puri-fied preparation utilizes either ammonia or glu-tamine as an amino donor. Although we foundthat purified glutamate dehydrogenase and glu-tamate synthase catalyze reactions i and ii,respectively, we were unable to isolate a specieshaving both activities. The explanation for the"nonadditivity" may be that glutamine, whichfor glutamate synthase is the specific aminodonor, inhibits the activity of glutamate dehy-drogenase competitively with respect to ammo-nia.

MATERIALS AND METHODSBacterial growth and assays. The bacterial

strain used for the enzyme purification was E. coliB/r. Cultures were grown aerobically in glucose-minimal medium (6) and were harvested by centrifu-gation in late exponential phase.The ammonia- and glutamine-dependent activi-

ties were determined by measuring the rate of oxida-tion of NADPH, which was monitored continuouslyat 340 nm. The assay system was composed of thefollowing: 50 mM potassium phosphate (pH 8.0), 10mM 2-oxoglutarate, 70 ,uM NADPH, and 20 mMammonium chloride for the ammonia-dependent ac-tivity or 20 mM L-glutamine for the glutamine-de-pendent activity. The activity of glutamate deami-

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776 SAKAMOTO, KOTRE, AND SAVAGEAU

nation was assayed by monitoring NADPH forma-tion at 340 nm in an assay mixture containing 50mM potassium phosphate (pH 8.0), 10 mM L-gluta-mate, and 70,M NADP+. A unit of activity is equiv-alent to the oxidation or formation of 1 Amol ofNADPH per min at 25 C.

Protein concentration was estimated by the colorn-metric method of Lowry et al. (11) with bovine se-rum albumin as the standard.Column chromatography. Diethylaminoethyl

(DEAE)-Sephadex A-50, obtained from PharmaciaFine Chemicals, Inc., was prepared and packed ac-cording to the manual of the manufacturer.

Bio-Gel A-5m (6% agarose), purchased from Bio-Rad Laboratories, was used for gel filtration. Molec-ular weight determinations were performed by themethod of Andrews (1). The column was calibratedwith standard proteins (5 mg in a 0.5- to 1.0-ml vol).Protein peaks were located spectrophotometricallyor by enzymatic assay. The standard proteins wereobtained from Sigma Chemical Co.

Electrophoresis in polyacrylamide gels. We fol-lowed the general method of Davis (7) for gel electro-phoresis, except that spacer and sample gels wereomitted. The samples (10 to 20 ,ug of protein) wereapplied to the surface of gels (5.5 by 0.5 cm; 5%acrylamide and 0.135% N,N'-methylenebisacrylam-ide) and were subjected to a current of 8 mA per gelin 50 mM sodium phosphate (pH 7.2) or 5 mMtris(hydroxymethyl )aminomethane-glycine (pH8.2). In the case of gel electrophoresis in the pres-ence of sodium dodecyl sulfate (18), 10-gg samples ofprotein were treated with 1% sodium dodecyl sulfateand 1% 2-mercaptoethanol at 37 C for 2 h or longerand were applied to the gels (5.5 by 0.5 cm; 8%acrylamide and 0.2% N,N'-methylenebisacrylam-ide).

Gels were stained for 3 h with 0.25% Coomassiebrilliant blue in 45% methanol-9% acetic acid anddestained electrophoretically with a Canalco Quick-Destainer in 7% acetic acid. They were stored in 7%acetic acid. Enzymatic activity in the gels was de-tected by staining as described by Yarrison et al.(20).

Ultracentrifugal analysis. Ultracentrifugationexperiments were done using a Spinco model E ana-lytical ultracentrifuge equipped with an RTIC unitand electronic speed control. Sedimentation velocitywas measured at 20 C with a schlieren optical sys-tem. Sedimentation equilibrium measurementswere made at 20 C by meniscus depletion (21) with aRayleigh interference optical system. In the lattermeasurements, the sample volume was 110 ul.Zone centrifugation in sucrose density gra-

dients. Zone centrifugation experiments were per-formed following the general method of Martin andAmes (12). Linear gradients from 5 to 20% sucrose,total volume 4.2 ml, were prepared. Samples of 0.1or 0.2 ml were layered on the gradients and thenwere centrifuged at 4 C and 26,000 rpm for 16 h in aSpinco model L2-65B ultracentrifuge with an SW65K rotor. About 40 fractions were collected from eachtube.

Paper chromatography. Amino acids (i.e., glu-tamic acid and glutamine) in 2-/l samples were

J. BACTERIOL.

separated on Whatman filter paper no. 1 (20 by 20cm) with a phenol-water (100:20, vol/vol) solventsystem and were detected by ninhydrin.

RESULTSPurification of glutamate dehydrogenase.

(i) Procedure. Table 1 summarizes the purifica-tion of glutamate dehydrogenase from E. coliB/r. The related enzyme glutamate synthasealso was purified in this process. All steps inthe purification were carried out at 0 to 4 Cwith buffers containing 1 mM ethylenediamine-tetraacetic acid and 10 mM 2-mercaptoethanol.The harvested cells (30 g, wet weight) were

suspended in 60 ml of 10 mM potassium phos-phate (pH 7.2) and were disrupted by sonicoscillation with a Branson Sonifier (model W140) at the highest intensity for three periods of3 min each. The suspension of broken cells wascentrifuged at 30,000 x g for 30 min. The super-natant fluid (crude extract) was dialyzedagainst 8 liters of 10 mM potassium phosphate(pH 7.2) for 16 h.

Nucleic acids were removed from the di-alyzed crude extract by the slow addition, withstirring, of a neutralized 10% (wt/vol) solutionof streptomycin sulfate. The volume of this addi-tion was 1/lo the volume of the extract. After 1 hofadditional stirring, the suspension was centri-fuged at 30,000 x g for 30 min. The supernatantfluid was dialyzed against 8 liters of 10 mMpotassium phosphate (pH 7.2) for 16 h.The dialyzed extract was treated with a satu-

rated solution of ammonium sulfate adjusted topH 7.2 with ammonium hydroxide. Proteinsprecipitating between 32.5 and 52.5% of satura-tion were dissolved in 9 ml of buffer A (20 mMpotassium phosphate [pH 7.2] containing 0.1 Mpotassium chloride and 2 mM 2-oxoglutarate)and were dialyzed against 2 liters of the samebuffer for 16 h.The dialyzed fraction was applied to a column

(50 by 2.5 cm) packed with DEAE-Sephadex A-50 and was equilibrated with buffer A. Proteinswere eluted initially with 200 ml of buffer Aand then with a linear gradient of potassiumchloride concentrations from 0.1 to 0.7 M in 20mM potassium phosphate (pH 7.2) containing 2mM 2-oxoglutarate (total vol, 1 liter). The flowrate was 15 ml/h, and the fractions collectedwere 5.5 ml each.The activities of interest were eluted in two

regions of the gradient. Eighty percent of theglutamine-dependent activity (reaction ii) wasfound in peak I of the elution profile (Fig. 1)with the remainder appearing in peak II. Theammonia-dependent activity (reaction i), on theother hand, appeared predominantly in peak IIand was barely detected in peak I. The fractions

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GLUTAMATE DEHYDROGENASE FROM E. COLI 777

TABLE 1. Purification ofglutamate dehydrogenase and glutamate synthase from E. coli Blr

Activity (U) Sp act (U/mg of protein)Fraction Vol Protein(ml) (mg) Ammonia Glutamine Ammonia Glutamine

dependent dependent dependent dependent

Crude extract 74 2,640 555 588 0.212 0.222

Streptomycin treatment 79 2,240 522 553 0.234 0.247

Ammonium sulfate (32.5-52.5% 20 1,090 492 518 0.451 0.475saturation)

DEAE-SephadexPeak Ia 2.0 39.0 2 234 0.011 6.01Peak II 1.6 29.6 396 62.0 13.4 2.10

Heat treatmentPeak I 1.5 14.2 1.5 171 0.106 12.1Peak II 1.2 9.70 388 54.6 40.0 5.64

Bio-Gel A-5mGSb 1.3 6.21 0 138 0 22.2GDHc 1.1 1.21 217 0 180 0

a The data in this line were obtained after ammonium sulfate precipitation (37.5 to 47.5% saturation) ofthe pooled fractions of peak I.

b GS, Glutamate synthase.c GDH, Glutamate dehydrogenase.

'_ 10_ _

_ ZQw

az a_

CL,U5I> a- 0lJ< z

2

6.0

50

40

1I3.0 °

N

2.0

1.0

050 100 150

FRACTION NUMBERFIG. 1. Elution profile of glutamate dehydrogenase and glutamate synthase from a DEAE-Sephadex

column. The preparation ofsamples and the elution procedure are described in the text. Protein is representedby absorbance at280 nm; ammonia- and glutamine-dependent activities were assayed as described in the text.

corresponding to peak I were pooled and treatedfor the further purification of glutamate syn-thase. The pooled fractions of peak II were usedfor the purification of glutamate dehydrogen-ase.The pooled fractions of peak I were treated

with ammonium sulfate. Proteins precipitatingbetween 37.5 and 47.5% of saturation were dis-solved in 2 ml of buffer B (20 mM sodium phos-

phate [pH 7.2] containing 2 mM 2-oxoglutar-ate). The solution was placed in a water bath at60 C for 10 min with shaking, and then centri-fuged at 16,000 x g for 15 min. The resultingsupernatant fluid was applied to a column (90by 1.5 cm) that had been packed with Bio-Gel A-5m, 200 to 400 mesh, and was washed withbuffer B. Proteins were eluted with buffer B ata flow rate of 3.5 ml/h. Fractions (2.7 ml) were

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778 SAKAMOTO, KOTRE, AND SAVAGEAU

collected. Those having specific activity ofgluta-mate synthase greater than 18 U/mg of proteinwere pooled and concentrated in an AmiconDiaflo cell equipped with a PM-10 membrane.The final preparation (glutamate synthase) wasstored at 0 to 4 C.The pooled fractions of peak II were concen-

trated with an Amicon Diaflo cell and weresubjected to heat treatment and gel filtration asdescribed for the fractions of peak I. Fractionshaving specific activity of glutamate dehydro-genase greater than 150 U/mg of protein werepooled and concentrated. The final preparation(glutamate dehydrogenase) was stored at 0 to4 C.

(ii) Purity of enzyme preparations. Purityof the two enzyme preparations was establishedby polyacrylamide gel electrophoresis, ultracen-trifugal analysis, and gel filtration. Samplescontaining 20 jig of protein were applied topolyacrylamide gels as described above. Electro-phoresis at pH 7.2 revealed a single proteinband for each preparation. The protein bandswere shown to be glutamate dehydrogenaseand glutamate synthase by the appropriate ac-tivity stain (20) and by the assay of the enzymeseluted from the sliced gels. Electrophoresis atpH 8.2 yielded a diffuse major band plus minorbands for glutamate dehydrogenase and a dif-fuse major band for glutamate synthase; how-ever, the protein and activity stains corre-sponded exactly, indicating no contaminatingproteins.

Sedimentation equilibrium measurementsalso supported the purity of both enzyme prepa-rations. Yphantis plots, plots of the logarithmof fringe displacement versus the square of thedistance from the axis of rotation, were linear.The purity of the enzyme preparations was fur-ther suggested by the results from gel filtrationon Bio-Gel A-5m; a single protein peak con-tained enzyme with constant specific activitythroughout.

Kinetic properties and stability. (i) Sub-strate specificity and Michaelis constants.Kinetic studies with the purified enzymes estab-lished that glutamate dehydrogenase and gluta-mate synthase catalyze reactions i and ii, re-spectively. An analysis of the reaction productsby paper chromatography also indicated thatglutamate dehydrogenase cannot utilize gluta-mine as the amino donor. No radioactive glu-tamic acid was found when glutamate dehydro-genase was incubated in the assay mixture con-taining radioactive glutamine and samplesfrom the assay mixture were chromatographed.Both enzymes are specific for NADPH; NADHcannot serve as the coenzyme for reaction i or ii.

Table 2 summarizes the Michaelis constants

J. BACTERIOL.

TABLE 2. Michaelis constants ofglutamatedehydrogenase and glutamate synthase

from E. coli Blr

Km (M)Substratea Glutamate Glutamate

dehydrogenase synthase

2-Oxoglutarate ...... 6.4 x 10-4 3.6 x 10-5Ammonia ........... 1.1 x 10-3Glutamine ...... 3.0 x 10-4Glutamic acid ....... 1.3 x 10-3 _ b

NADPH ............ 4.0 x 10-5 8.5 x 10-6NADP+.............. 4.2 x 10-5 _ b

a All other assay conditions are described in Mate-rials and Methods.

b The constants for these substrates were notmeasured since the aminating reaction of glutamatesynthase is virtually irreversible.

(Kin) we obtained for purified glutamate dehy-drogenase from E. coli B/r. Values for gluta-mate synthase, similar to those for the enzymefrom E. coli W (13), also are shown for compari-son. The values for glutamate synthase arefrom 4 to 20 times lower than the correspondingvalues for glutamate dehydrogenase, which isconsistent with the proposed dominant role forglutamate synthase in vivo (16).

(ii) Inhibition by homoserine. L-Homoser-ine has been observed to inhibit both ammonia-and glutamine-dependent activities in cell ex-tracts of E. coli B/r (14). This inhibition also isreported for glutamate synthase from E. coli W(13). These findings were confirmed for purifiedglutamate dehydrogenase and glutamate syn-thase from E. coli B/r. L-Homoserine is a com-petitive inhibitor with respect to both ammoniaand glutamine. The corresponding values of Kiare 25 mM for glutamate dehydrogenase and 13mM for glutamate synthase.

(iii) Inhibition by glutamine. Glutamine,which for glutamate synthase is the specificamino donor, was found to inhibit the activityof glutamate dehydrogenase. The inhibition ofthe aminating reaction by glutamine was com-petitive with respect to ammonia, Ki of 60 mM,whereas the inhibition of the deaminating reac-tion was noncompetitive with respect to gluta-mate. On the other hand, ammonia, which forglutamate dehydrogenase is the specific aminodonor, had no effect on the activity ofglutamatesynthase.

(iv) Stability. Glutamate dehydrogenase isquite stable at 4 C and at room temperature inwater, 20 mM potassium phosphate, or sodiumphosphate (pH 7.2). The enzyme remains fullyactive at room temperature for at least 3months. Storage at 4 C preserves its activityeven longer. On the other hand, the enzyme is

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inactivated by freezing-thawing. The enzymestability in four solvent systems was examinedat different temperatures, and the results aresummarized in Table 3.

Glutamate dehydrogenase is stable whenheat treated. Almost total activity was re-covered after treatment at 60 C for 10 min or at37 C for 5 h; the 60 C treatment was then usedas a step in purification.

(v) Denaturation and renaturation. Gluta-mate dehydrogenase was treated at 37 C for 2 hwith guanidine hydrochloride ofvarious concen-trations (0 to 6.0 M) and 10 mM dithiothreitol in20 mM sodium phosphate (pH 7.2). The remain-ing activity was assayed in a reaction mixturesupplemented with guanidine hydrochloride atthe same concentration used for the treatment.Activity of the treated enzyme also was meas-ured after diluting guanidine hydrochloridewith the reaction mixture (600-fold dilution)(Fig. 2A).Glutamate dehydrogenase was completely in-

activated by guanidine hydrochloride at concen-trations higher than 1.0 M. After the dilution ofguanidine hydrochloride, the enzyme treated atconcentrations lower than 2.0 M recovered al-most total activity. On the other hand, no recov-ery of activity was noted when the enzyme hadbeen treated at concentrations higher than 4.0M. The incubation ofthe enzyme in its appropri-ate assay mixture forl6 h had no effect on recov-ery. Renaturation of the enzyme treated with6.0 M guanidine hydrochloride was attemptedby the removal of the denaturant with gel filtra-tion on Bio-Gel A-5m; the eluted protein, how-ever, had no enzymatic activity. Similar re-sults, but at lower concentrations of guanidinehydrochloride, were obtained with glutamatesynthase (Fig. 2B).

Molecular properties of glutamate dehy-

TABLE 3. Stability ofglutamate dehydrogenase fromE. coli Blr a

Solvent Activity retained (%)system 25C 4C -17C -70C

i 100 100 57 87ii 100 100 70 82

iii 100 100 4 48iv 100 100 7 47

a The activity of the enzyme was assayed after ithad been left overnight at room temperature (25 C),4 C, -17 C, and -70 C in the following solventsystems: (i) water, (ii) 20 mM potassium phosphate(pH 7.2), (iii) 20mM sodium phosphate (pH 7.2), and(iv) 20 mM sodium phosphate (pH 7.2) containing2 mM 2-oxoglutarate, 1 mM ethylenediaminetetra-acetic acid, and 5 mM 2-mercaptoethanol.

S°H

E100 B

z

50

02 3 4 5 6

GUANIDINE HYDROCHLORIDECONCENTRATION (M)

FIG. 2. Effects ofguanidine hydrochloride on theactivities ofglutamate dehydrogenase and glutamatesynthase. The enzymes were treated at 37 C for 2 hwith the denaturant and 10 mM dithiothreitol in 20mM sodium phosphate, pH 7.2. Activity was assayedin the presence and absence of the denaturant. Thedenaturant was removed by 30- to 60()-fold dilutionwith the assay mixture. Relative activity in the pres-ence (a) and after the removal (0) of the denaturantis plotted against the concentration of the denatur-ant. (A) Glutamate dehydrogenase; (B) glutamatesynthase.

drogenase. (i) Molecular weight. Sedimenta-tion equilibrium measurements were per-formed in 50 mM sodium phosphate (pH 7.2)containing 2 mM 2-oxoglutarate, 1 mM ethyl-enediaminetetraacetic acid, and 5 mM 2-mer-captoethanol, against which the sample hadbeen previously dialyzed at 4 C for 16 h. Theprotein concentrations used were 0.2 to 0.4 mg/ml. Photographs were taken after 24 h of cen-trifugation at 20 C and 12,000 rpm. TheYphantis plots were linear for all protein con-centrations used (Fig. 3). The molecular weightwas calculated to be 300,000 + 20,000, with avalue of 0.74 ml/g as the partial specific volumesince it is an average value for globular pro-teins (15). Molecular weights determined atvarious protein concentrations fluctuated ran-domly within experimental error.From gel filtration on Bio-Gel A-5m, 200 to

400 mesh, the molecular weight of glutamatedehydrogenase was estimated to be 300,000 +30,000. The column was calibrated by usingphosphorylase a (molecular weight, 370,000),catalase (molecular weight, 230,000), and yeastalcohol dehydrogenase (molecular weight, 150,-000).

(ii) Subunit structure. Polyacrylamide gel

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780 SAKAMOTO, KOTRE, AND SAVAGEAU

1000 e

2

U)JUf)

(5z

100 -

50 51r2 (cm2)

FIG. 3. Sedimentation equilibrium measurementfor glutamate dehydrogenase. A sample, 03 mglml,previously dialyzed against 50 mM sodium phos-phate (pH 7.2) containing 2 mM 2-oxoglutarate, 1mM ethylenediaminetetraacetic acid, and 5 mM 2-mercaptoethanol, was centrifuged at 12,000 rpm and20 C. The logarithm offringe displacement is plottedwith respect to the square of the radial distance.

electrophoresis in the presence of sodium dode-cyl sulfate yielded a single band for glutamatedehydrogenase. The molecular weight of thesubunit was estimated to be 50,000 + 5,000(Fig. 4). By comparison, purified glutamate syn-thase yielded two distinct bands, and the molec-ular weights of these subunits were estimatedto be 55,000 + 5,000 and 140,000 + 10,000.The molecular weight of the glutamate dehy-

drogenase subunit was also determined by sedi-mentation equilibrium measurements in 6.0M guanidine hydrochloride. The enzyme solu-tions (0.2 to 0.4 mg/ml) had been previouslydialyzed against 6.0 M guanidine hydrochlorideand 0.1 M 2-mercaptoethanol in 50 mM sodiumphosphate (pH 7.2). The Yphantis plots permit-ted the calculation of a molecular weight of48,000 + 3,000, by using a value of 0.73 ml/g forthe partial specific volume of the unfolded poly-peptide chain (Fig. 5).The molecular weights of the native enzyme

and the subunit lead us to postulate that gluta-mate dehydrogenase (molecular weight, 300,-000) is a hexamer composed of identical sub-units (molecular weight, 50,000).

(iii) Sedimentation studies. Although eachof the purified enzymes has only one activityand no species having both ammonia- and glu-tamine-deperdent activities was found, a bi-functional complex of glutamate dehydrogen-ase and glutamate synthase might have been

20

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0

10g'9

ui835cc7

(56LU

45

4

20 40 60

MOBILITY (%)

FIG. 4. Molecular weight estimation ofsubunits ofglutamate dehydrogenase and glutamate synthase bypolyacrylamide gel electrophoresis in sodium dodecylsulfate. The estimated molecular weights for the sub-unit of glutamate dehydrogenase (GDH) and for thesmall (GS-I) and the large (GS-2) subunits ofgluta-mate synthase are 50,000 + 5,000, 55,000 + 5,000,and 140,000 + 15,000, respectively. Standard pro-teins are: (1) /3galactosidase (molecular weight,130,000), (2) phosphorylase a (molecular weight, 94,-000), (3) bovine serum albumin (molecular weight,68,000), (4) ovalbumin (molecular weight, 43,000),and (5) liver alcohol dehydrogenase (molecularweight, 41,000).

3000

(5

z

L 100_/_

,2 (cm2)

FIG. 5. Sedimentation equilibrium measurementfor glutamate dehydrogenase in 6.0 M guanidinehydrochloride. A sample, 03 mg/ml, previously di-alyzed against 6.0 M guanidine hydrochloride and0.1 M2-mercaptoethanol in 50 mM sodiumphosphate(pH 7.2), was centrifuged at 30,000 rpm and 20 C.The logarithm offringe displacement is plotted withrespect to the square of the radial distance.

XGDH -

3 K

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lost during purification. Such a complex wassuggested for the protein contained in peak IIfrom a DEAE-Sephadex column (Fig. 1) sincethe peaks of ammonia- and glutamine-depend-ent activities were superimposable (14). Twoactive forms of glutamate synthase also havebeen suggested (13, 14). To explore these possi-bilities further, we examined the sedimenta-tion behavior of the two enzymes at each step inthe purification.

Sedimentation coefficients for the two en-zymes were first estimated by sedimentationvelocity experiments. Schlieren patterns re-vealed a single symmetrical peak for each ofthe purified enzymes throughout the course ofthe experiment. Glutamate dehydrogenase andglutamate synthase have sedimentation coeffi-cients of 13.3S (at a protein concentration of 2.8mg/ml) and 19.5S (at a protein concentration of4.2 mg/ml), respectively, at 20 C in 50 mM so-dium phosphate (pH 7.2) containing 1 mM ethyl-enediaminetetraacetic acid and 10 mM 2-mer-captoethanol.

Purified glutamate dehydrogenase and gluta-mate synthase, when subjected to ultracentrifu-gation in sucrose density gradients, yielded sin-gle peaks, which on the basis of the above stud-ies were labeled "13S" and "20S," respectively(Fig. 6). The sedimentation pattern of a mix-ture of the purified enzymes was the same asthat obtained when the sedimentation patternof one purified enzyme was superimposed onthat of the other. These sedimentation patternswere used as standards to follow the two en-zymes at each step in the purification proce-dure.

Different sedimentation patterns (Fig. 7athrough e') were observed with samples takenat various steps in the purification of the en-zymes (Table 1). (a) For the crude extract, am-monia-dependent activity appeared at the 13Sposition, whereas glutamine-dependent activityappeared at both the 13S and 20S positions.(b) The same pattern was obtained after strep-

> 'n<>w 2

A 8,0 - 13S 3

>0-

C0-10 20 30

FRACTION NUN

p

20S

I

10 20 30ABER

FIG. 6. Sedimentation in sucrose density gra-dients ofpurified glutamate dehydrogenase and glu-tamate synthase. Gradient preparation, centrifuga-tion, and assays for activity are described in the text.Sedimentation is from right to left. (A) Glutamatedehydrogenase; (B) glutamate synthase.

20

10

09n

0 / 3

20

H105

133S0 d d' 1 30

10

020 e et 13S

I0

010 20 30 10 20 30

FRACTION NUMBERFIG. 7. Sedimentation in sucrose density gra-

dients ofsamples from each step in thepurification ofglutamate dehydrogenase and glutamate synthase.Gradient preparation, centrifugation, and assays foractivity are described in the text. Sedimentation isfrom right to left. Symbols: (0), ammonia-dependentactivity; (a), glutamine-dependent activity. (a)Crude extract; (b) streptomycin-treated extract; (c)ammonium sulfate fraction (32.5 to 52.5% of satura-tion); (d and d') fractions of peaks I and II, respec-tively, from a DEAE-Sephadex column; (e and e')heat-treated fractions ofpeaks I and II, respectively,from a DEAE-Sephadex column.

tomycin treatment. (c) For the ammoniumsulfate fraction (32.5 to 52.5% of saturation)both ammonia- and glutamine-dependent ac-tivities were found only at the 13S position.(d) For the fractions of peak I from a DEAE-Sephadex column, glutamine-dependent activ-ity was located only at the 20s position, corre-sponding to purified glutamate synthase. (d')For the fractions of peak II, on the other hand,a profile similar to that for the ammoniumsulfate fraction was obtained but with lowerglutamine-dependent activity. (e) Heat treat-ment of the fractions from peak I did not changethe profile seen in Fig. 7d. (e') Similarly, heattreatment of the fractions from peak II did notaffect the profile seen in Fig. 7d'.At each stage in the purification, the peak of

ammonia-dependent activity always occurredat 13S, the position of activity for purified gluta-mate dehydrogenase. On the other hand, theglutamine-dependent activity corresponded to apeak at 13S, 20S, or in some cases at both.The possible formation of a complex between

glutamate dehydrogenase and glutamate syn-

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782 SAKAMOTO, KOTRE, AND SAVAGEAU

thase was examined by gel filtration and poly-acrylamide gel electrophoresis, in addition tothe sedimentation experiments reported above.When a crude extract was applied to a column(90 by 1.5 cm) of Bio-Gel A-Sm, a peak of ammo-nia-dependent activity and two peaks of gluta-mine-dependent activity were eluted, each cor-

responding to known forms of the enzymes. Nopeaks having both activities were found at posi-tions corresponding to molecular weights of pos-sible complexes. Gel electrophoresis at pH 7.2 ofa mixture of the purified enzymes revealed nonew bands corresponding to complexes. In addi-tion, sodium dodecyl sulfate electrophoresis of amixture amidinated with dimethyl suberimi-date, by the method of Davies and Stark (5),yielded no bands other than those correspond-ing to the components of the two enzymes.

All the foregoing results indicate that theprotein in peak II was not a complex but a

mixture of the two individual enzymes and thatthere exists no species having both ammonia-and glutamine-dependent activities.

(iv) Two active forms of glutamate syn-thase. Although purified glutamate synthasewas shown to consist of a single molecular spe-cies with nonidentical subunits, under certainconditions this enzyme was converted to a sec-ond active form similar to glutamate dehydro-genase in sedimentation coefficient and chro-matographic behavior on DEAE-Sephadex.Two active forms of glutamate synthase were

suggested by the results of DEAE-Sephadexcolumn chromatography (Fig. 1). When the frac-tions in peak II were reapplied to a DEAE-Sephadex column, the elution profile of the ac-

tivity reproduced exactly that of the firstDEAE-Sephadex column; glutamine-depend-ent activity was found in both peak I and peakII. When the single 13S glutamate synthasespecies resulting from ammonium sulfate treat-ment (Fig. 7c) was chromatographed on a

DEAE-Sephadex column, glutamine-depend-ent activity occurred in both peaks I and II.When the fractions corresponding to either oneof the peaks were rechromatographed on a

DEAE-Sephadex column, glutamine-depend-ent activity again appeared in both peaks I andII. Furthermore, sedimentation experimentsshowed that purified glutamate synthase (20S),when treated with ammonium sulfate, is con-

verted to a 13S species.Gel filtration of a crude extract on Bio Gel A-

5m yielded two peaks of activity correspondingto apparent molecular weights of 800,000 and200,000. Since purified glutamate synthase(20S) has a molecular weight of 800,000, weinferred that the apparent molecular weight of200,000 corresponds to the 13S species. Gel

filtration of the fractions corresponding to peakII from a DEAE-Sephadex column resulted inthe separation of the ammonia-dependent activ-ity from the glutamine-dependent activity thatwas eluted at the position corresponding to anapparent molecular weight of 200,000. These re-sults lead us to propose that glutamate syn-thase has two interconvertible active forms ofdifferent molecular sizes. One form has molecu-lar weight 800,000 and a sedimentation coeffi-cient of 20S, and the other has apparentmolecular weight 200,000 and a sedimentationcoefficient of 13S.

DISCUSSIONWe have isolated glutamate dehydrogenase

from E. coli B/r. The purity of the preparationhas been established by polyacrylamide gel elec-trophoresis, analytical ultracentrifugation, andgel filtration. The molecular weight of purifiedglutamate dehydrogenase is calculated to be300,000 + 20,000 from sedimentation equilib-rium data (Fig. 3). This method has the leastexperimental error among those available forthe determination of molecular weight. Never-theless, the molecular weight estimated fromgel filtration on Bio-Gel A-5m is in close agree-ment with that given above. Glutamate dehy-drogenase from E. coli B/r, then, has a molecu-lar weight similar to that of the homologousenzymes from bovine liver (molecular weight,320,000) (8), Neurospora (NADP+-specific form;molecular weight, 288,400) (2), Salmonella ty-phimurium (molecular weight, 280,000) (3),Clostridium (molecular weight, 275,000) (19),Peptococcus aerogenes (molecular weight, 266,-000) (9), and Mycoplasma (molecular weight,250,000) (20). However, its value is quite differ-ent from that for the enzymes of Thiobacillusnovellus (NADP+- and NAD+-specific forms;molecular weight 120,000) (10) and Neurospora(NAD+-specific form; molecular weight 480,000)(17).The molecular weight of the glutamate dehy-

drogenase subunit is 50,000 + 5,000, as ob-tained from polyacrylamide gel electrophoresisin the presence of sodium dodecyl sulfate (Fig.4). This value is in close agreement with thatdetermined from sedimentation equilibriummeasurements in 6.0 M guanidine hydrochlo-ride (Fig. 5). From the molecular weights of thenative enzyme and its subunit, we propose thatglutamate dehydrogenase is a hexamer com-posed of subunits with identical molecularweight. A similar subunit structure has beenreported for this enzyme from bovine liver (8)and for the NADP+-specific form of this enzymefrom Neurospora (2).

It has been suggested that glutamate syn-

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GLUTAMATE DEHYDROGENASE FROM E. COLI 783

thase from E. coli W has two active forms (13).We have observed two active species for theenzyme from E. coli B/r. Since these formsappeared in the crude extract, it is possible thattwo active forms normally exist in vivo. Apartial characterization of the two forms hasrevealed that the larger one has an apparentmolecular weight of 800,000 and a sedimenta-tion coefficient of 20S whereas the smaller onehas an apparent molecular weight of 200,000and a sedimentation coefficient of 13S. Thesedimentation coefficient of 13S is high for amolecular weight of 200,000, whereas a value of20S is low for a molecular weight of 800,000,suggesting a substantial deviation from spheri-cal shape for these species.

In contrast to the relatively small number ofmicrobial species from which glutamate dehy-drogenase has been purified to homogeneity,there are many species for which the Michaelisconstants of this enzyme have been determinedin cell-free extracts or partially purified prepa-rations. TheKm values for NADPH and NADP+and the relative Km values for glutamate and 2-oxoglutarate are generally similar among spe-cies, whereas there is considerable variationwith respect to the Km for ammonia (16-fold)and the Km for 2-oxoglutarate (10-fold) (4). It isinteresting that our Km values for glutamatedehydrogenase from E. coli appear to differsignificantly from those of the closely relatedspecies S. typhimurium. Our Km values forNADPH, NADP+, and ammonia are approxi-mately 2, 4, and 4 times higher, respectively,than the corresponding Km values reported forthe enzyme from S. typhimurium, whereas ourKm values for glutamate and 2-oxoglutarate,respectively, are about 40 and 6 times lower (4).In S. typhimurium, the Km for glutamate is12.5 times that for 2-oxoglutarate and Coultonand Kapoor (4) have concluded from this that invivo the glutamate dehydrogenase reaction isessentially unidirectional in favor of glutamatebiosynthesis. In E. coli, these Km values onlydiffer by twofold and, thus, if these differencesare real, the reaction in this organism may notbe entirely unidirectional.

ACKNOWLEDGMENTSWe are indebted to D. D. Dziewiatkowski for the use of

his analytical ultracentrifuge.This work was supported by grant GB-27701 from the

National Science Foundation. N. S. was the recipient of anF. G. Novy Postdoctoral Fellowship from the University ofMichigan Department of Microbiology.

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14. Savageau, M. A., A. M. Kotre, and N. Sakamoto. 1972.A possible role in the regulation of primary amina-tion for a complex of glutamine: a-ketoglutarate ami-dotransferase and glutamate dehydrogenase in Esch-erichia coli. Biochem. Biophys. Res. Commun. 48:41-47.

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