JOURNAL OF BACTERIOLOGY, JUlY 1975, p. 203-211Copyright 0 1975 American Society for Microbiology

Vol. 123, No. 1Printed in U.S.A.

Purification and Properties of the Glutathione Reductase ofChromatium vinosum

Y. C. CHUNG AND R. E. HURLBERT*

Department ofBacteriology and Public Health, Washington State University, Pullman, Washington 99163

Received for publication 10 February 1975

The Chromatium vinosum glutathione reductase [NAD(P)H:glutathione di-sulfide oxidoreductase, EC 1.6.4.2] was purified to apparent homogeneity. Theenzyme was found to require reduced nicotinamide adenine dinucleotide(NADH) as a reductant and to be specific for oxidized glutathione (GSSG). Thepolypeptide molecular weight in sodium dodecyl sulfate was found to be 52,000.Incubation of enzyme with NADH in the absence of GSSG resulted in a

significant loss in activity. The enzyme was stimulated by phosphate and sulfateion, but was inhibited by chloride ion, heavy metals, and sulfhydryl reagents.Adenylate nucleotides were inhibitory, and the data suggested that they wereacting as competitive inhibitors of flavin adenine dinucleotide (FAD). The Kmvalues of 7 x 10-3 for GSSG and 6 x 10- 5 M for NADH were the highest reportedof any previously investigated glutathione reductase. The order of addition ofcomponents markedly affected the response of the enzyme to FAD. A require-ment for FAD (Km 5.2 x 10-7 M) was seen if the enzyme was incubated withNADH prior to GSSG addition, whereas no FAD was required if the order was

reversed.

Glutathione reductase [NAD(P)H:glutath-ione disulfide oxidoreductase, EC 1.6.4.2] hasbeen demonstrated in higher plants (6, 19), in avariety of animal tissues (10, 12, 15, 24, 26, 27,30, 31), in yeast (4, 20, 24) and filamentousfungi (32), and in a number of bacteria (1, 3,29). In those cases where a detailed study of thenature of the enzyme has been undertaken, ithas been shown that flavin adenine dinucleo-tide (FAD) is present as a tightly bound pros-thetic group and that the enzyme is eitherspecific for reduced nicotinamide adenine dinu-cleotide phosphate (NADPH) as the reductantor has a much lower activity with reducednicotinamide adenine dinucleotide (NADH).The highest previously observed activity withNADH has been for the human erythrocyteenzyme, where the maximal rate of the reactionwith NADH was 23% that of NADPH (26).

In the present paper the purification andpartial characterization of a glutathione reduc-tase from Chromatium vinosum that is highlyspecific for NADH is reported.

MATERIALS AND METHODSC. vinosum (formerly strain D) was grown in

completely filled 12-liter carboys in the liquid thiosul-fate salts medium described by Hurlbert and Las-celles (13). The carboys were sealed with a rubberstopper and were incubated with continuous stirringat 30 C in a light chamber with banks of 75- to 100-W

tungsten lamps. Air was blown across the carboys toprevent overheating. After growth was complete (4 to7 days, as judged by visual inspection), the cells werecollected at 7 C by using a Sharples centrifuge. Thepacked cells were stored without washing, at -70 Cuntil used for the enzyme preparation.The reductase activity was measured by two

methods. For all determinations involving NADHconcentrations giving an optical density at 340 nm ofbetween 0.2 and 1.0 (0.03 to 0.175 mM), the rate ofNADH oxidation was followed by measuring thedecrease in absorption at 340 nm on a Gilford record-ing spectrophotometer. Unless otherwise noted, thereaction mixture contained 125 mM potassium phos-phate buffer, pH 7, 0.014 mM FAD, enzyme, 0.175mM NADH, and 12.5 mM GSSG (oxidized glutathi-one) added in that order. An NADH oxidase controlwas run with each experiment. Crude preparationshad approximated 0.003 units of NADH oxidaseactivity per mg, while pure preparations had nodetectable NADH oxidase activity. It is necessary toadd the FAD to the enzyme before the NADH in orderto prevent inhibition of the enzyme by NADH. TheNADH solution was prepared fresh for all assays. Forkinetic studies involving small quantities of NADH,the method of Beutler and Yeh (2) was followed.This assay is based on measuring the reduced gluta-thione (GSH) produced by linking it to the reductionof 5,5-dithio-bis-2-nitrobenzoate (DTNB) which ismeasured at 412 nm. Unless noted otherwise, the reac-tion mixture contained, in addition to the above,0.02% DTNB. Since prolonged incubation of DTNBwith the enzyme caused inhibition, the DTNB wasadded immediately before initiating the reaction.

203

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204 CHUNG AND HURLBERT

Controls run in the absence of GSSG, NADH, orenzyme showed no activity with either assay.

Kinetic experiments, by using either assay andGSSG as the variable substrate, gave identical re-sults. All kinetic data were subjected to linear regres-sion analysis. A unit of enzyme activity is defined asthe amount of enzyme that oxidized 1 timol ofNADHper min or reduced 1 Mmol of DTNB per min.

Protein was measured by the method of Lowry etal. (16) employing bovine serum albumin as a proteinstandard. Analytical disc gel electrophoresis was per-formed by the method of Davis (8) in 5, 7.5, and 10%polyacrylamide gels. The electrophoresis was carriedout at 3 mA per tube. The gels were stained either in1% Amido Schwartz in 7% acetic acid or in 0.1%Coomassie brilliant blue as described below. Analyti-cal gel electrophoresis in 10% polyacrylamide gelscontaining sodium dodecyl sulfate was done accordingto the method of King and Laemmli (14). The gelswere rinsed twice at 37 C for 1 h each in 50%MeOH-7% acetic acid. The gels were stained for 1 to 2h at 37 C by 0.1% Coomassie brilliant blue in 50%(vol/vol) methyl alcohol to which 0.7 ml of glacialacetic acid per 10 ml of stain was added immediatelybefore use. All gels were destained in 7% acetic acid.Molecular weight estimation in sodium dodecyl sul-fate gels was carried out by the split-gel method ofSchnaitman (25).

All chemicals were obtained from Sigma ChemicalCo. Sephadex G-200 and diethylaminoethyl(DEAE)-A50 were products of Pharmacia, Uppsala,Sweden. The hydroxyapatite was a product of Bio-Rad Laboratories, Richmond, Calif.

RESULTSPurification of glutathione reductase. All

operations were carried out at 0 to 7 C.Step 1. Crude extract. The frozen cells were

suspended in a proportion of 1 g (packed wetweight) per 10 ml of a solution of 0.05 M po-tassium phosphate buffer, pH 7.0, and 2 mMethylenediaminetetraacetic acid (EDTA). Thecells were disrupted by a single pass through aFrench pressure cell at 20,000 lb/in2. Becausesome early samples were inactive after treat-ment the French pressure cell was presoaked for30 to 60 min in 0.01 M EDTA prior to disrup-tion. The suspension was centrifuged at 105,000x g for 2 to 3 h, and the straw-colored superna-tant (crude extract) was collected. Since theenzyme was stable when frozen, it was possibleto store the extract at -20 or -70 C for severalmonths without loss of activity.Step 2. pH fractionation. The crude extract

(fresh or thawed material) was placed in an icebath, and cold M HSPO4 was added dropwisewith vigorous stirring until the pH dropped to4.8. The precipitate which formed was removedby centrifugation at 27,000 x g for 5 min. Thesupernatant was saved, and 0.1 M HPO4 was

added as before until the pH dropped to 4.4.The precipitate was removed as before, and thesupernatant was collected. Potassium hydrox-ide (6 N) was added dropwise with stirring tothe supernatant until the pH was returned to7.0.Step 3. Ammonium sulfate fractionation.

Solid ammonium sulfate crystals (27.4 g/100 mlof solution) were added slowly with stirring tothe material from step 2 in an ice bath. After 10min the precipitate (45% saturation) was re-moved by centrifugation at 8,000 x g for 5 minat 4 C. To this supernatant 3.2 g of ammoniumsulfate per 100 ml of original solution was addedas before. After 10 min the precipitate (50%saturation) was sedimented as before. Afterdecanting the supernatant and wiping down thesides of the centrifuge bottle, the precipitatewas dissolved in a 100-ml solution of 0.05 Mpotassium phosphate buffer, pH 7.0, and 2 mMEDTA.

Step 4. DEAE chromatography. The dis-solved 50% ammonium sulfate precipitate wasadded to a DEAE-Sephadex A-50 column (2 by25 cm) equilibrated with a solution of 0.05 Mpotassium phosphate buffer, pH 7.0, and 2 mMEDTA at the rate of approximately 0.7 ml permin. The protein was eluted with a lineargradient of 0.05 to 0.5 M potassium phosphatebuffer, pH 7.0, containing 2 mM EDTA.Fractions of 5.5 ml were collected. The materialbetween 375 to 460 ml (Fig. 1) contained theactivity. The fractions containing the highest

P.4tI

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FIG. 1. Elution profile of glutathione reductasefrom a DEAE-Sephadex column. Precipitate fromammonium sulfate fractionation dissolved in a solu-tion of 0.05 M phosphate buffer, pH 7.0, and 2 mMEDTA was adsorbed to a DEAE-Sephadex A-50column (2.5 by 24 cm). Elution and assay for glutathi-one reductase activity was carried out as described intext. Symbols: (O) optical density at 280 nm; (0)glutathione reductase activity, (A) 2nd phosphateconcentration. Fractions between bars were used forfurther purification.

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GLUTATHIONE REDUCTASE 205

activity were pooled and concentrated by usingan Amicon ultrafiltration cell with a type PM 10ultrafilter membrane.Step 5. Sephadex G-200 column. The DEAE

concentrate was added to a Sephadex G-200column (2.5 by 31 cm) equilibrated with thephosphate-EDTA buffer. The protein waseluted at the rate of approximately 0.7 ml permin, and 5.5-ml fractions were collected (Fig.2A). The fractions containing the highest activ-ity were pooled and concentrated as above.Step 6. Hydroxyapatite column. The con-

centrated material from step 5 was added to ahydroxyapatite column (2 by 16 cm) equili-brated with the phosphate-EDTA buffer. Thesample was eluted with the same buffer at therate of 0.2 ml per min, and 5.5-ml fractions werecollected. The fractions containing the highestactivity were concentrated as before to a smallvolume and stored at -20 C (Fig. 2B). Theresults of one such purification is seen in Table1.

Criteria of purity. To determine the purityof the isolated material, it was examined byanalytical disc gel electrophoresis at concentra-tions of 5, 7.5, and 10% acrylamide. Only oneprotein band was visible in the final material.To determine that the band corresponded to theenzyme activity, a gel was run in the normalway, and a thin verticle slice of the gel wasstained for 15 min and rapidly destained byusing a Canalco electric gel destainer. Thesection of the unstained gel corresponding to theprotein band was cut out and crushed in a smallvolume of buffer containing 0.05 M phosphate,pH 7.0, and 2 mM EDTA. After 15 to 30 min ofelution at 4 C the gel was removed by briefcentrifugation, and the supernatant was foundto contain glutathione reductase activity.Molecular weight determination. Disc gel

electrophoresis in sodium dodecyl sulfate poly-acrylamide gels was employed to determine theminimum molecular weight. A single band witha molecular weight of 52,000 was observed (Fig.3).

Stability of the enzyme. Repeated freezingand thawing of either the purified or the crudematerial did not cause any significant loss ofactivity. The enzyme could be stored at -20 Cfor over 2 years with no significant loss of activ-ity. The enzyme was rapidly inactivated by tem-

UIE IUMER

FIG. 2. Gel chromatography on Sephadex G-200and hydroxyapatite. (A) Active fractions from DEAEchromatography were pooled, concentrated, andadded to a Sephadex G-200 column (2.5 by 31 cm)equilibrated with a solution of 0.05 M phosphatebuffer, pH 7.0, and 2 mM EDTA. Fractions of 5.5 mlwere eluted at the rate of 0.7 ml/min with the samebuffer. (B) Active fractions from G-200 chromatogra-phy were pooled, concentrated, and applied to ahydroxyapitite column (2 by 16 cm) equilibrated withthe phosphate-EDTA buffer. Fractions of 5.5 ml wereeluted at the rate of 0.2 ml/min with the same buffer.Symbols: (0) glutathione reductase activity; solidlines, optical density at 280 nm. Fractions betweenbars were used for further study.

TABLE 1. Summary of the purification of glutathione reductase from C. vinosum

Protein Total Sp act PurificationStep8s5 Vol (ml) | content (mg) cU) (U/mg) | Recovery (%) factor

Crude extracta 450.0 2336.0 76.8 0.03 100 1pH 4.4 supernatant 473.0 733.0 36.6 0.05 48 ' 1.5245 to 50% (NH4),S04 pre- 100.0 82.0 29.0 0.35 38 10.6

cipitateDEAE-Sephadex chroma- 6.2 2.0 19.6 9.8 26 297tography

Sephadex G-200 chroma- 2.0 1.07 15.0 14.0 20 424tography

Hydroxyapatite 1.8 0.19 5.4 28.4 7 861

a From 48 g of packed cells.b Micromoles of NADH oxidized per minute per milligram of protein.

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206 CHUNG AND HURLBERT

10987

, 60- 5x

r4 302LaJ-J0

0

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@3

@4

2 4DISTANCE IN GEL (cm)

FIG. 3. Electrophoretic migration of glutathionereductase as a function of molecular weight. Gelelectrophoresis in split gels with authentic standardswas performed as described in Materials andMethods. (1) Bovine serum albumin; (2) pyruvatekinase; (3) ovalbumin; (4) chymotrysinogen; (5) tryp-sin; open circle, glutathione reductase.

peratures above 45 C. About 60% of the activitywas lost after 5 min at 50 C, and no activitycould be detected after heating for 5 min at65 C.Substrate specificity. The Chromatium en-

zyme shows a high specificity for NADH as anelectron donor. At equal molar concentrations(2.5 x 10- 4 M) the rate ofDTNB reduction withNADPH as reductant was only 5% that ofNADH.The enzyme is also specific for GSSG, as no

activity was observed when GSSG was replacedwith cystine, oxidized lipoic acid, or oxidizedlipoamide at a concentration of 12.5 mM.FAD requirement and the effect of the

order of addition of components. During earlystudies of the enzyme, NADH was added priorto GSSG. Under these conditions it was foundnecessary to add FAD to a concentration of 5,uM or greater in order to demonstrate thereaction. A double reciprocal plot of FAD con-centration versus activity gave an apparent Kmfor FAD of 5.2 x 10-7 M. Flavin mononucleo-tide did not replace the FAD.Subsequent experiments showed that the re-

quirement of FAD is dependent upon the GSSGconcentration and the order of addition of

components to the reaction mixture. In experi-ments where GSSG is added to initiate thereaction, little or no enzyme activity is detectedin dialyzed extracts (crude and purified enzymebehave similarly) in the absence of added FAD(Fig. 4). However, if GSSG is added first andNADH is used to start the reaction, significantactivity is observed in the absence of any addedFAD (Fig. 4). The extent of the reaction isdependent upon GSSG concentration, in that atGSSG concentrations of 7 and 12.5 mM, NADHreduction ceases after only a small fraction of

5 the GSSG has been utilized, whereas with 50mM GSSG the reaction continues until theNADH is totally oxidized (Fig. 4). Subsequentaddition of FAD to the reaction mixture startedeither with GSSG or with NADH, but with 7 or12.5 mM GSSG, restored the enzyme activity.To determine the role of GSSG concentration

an experiment was run with 7 mM GSSG untilNADH oxidation ceased. At this point GSSGwas added to bring the concentration to 50 mM.Thie arldiLinn riitinnAM initisit1 nnu iniirPnQP in

UTES

FIG. 4. Effect of GSSG concentration and theorder of addition of substrate on the activity ofdialyzed glutathione reductase. The enzyme wasdialyzed against several changes of 2 mM EDTA at7 C for 24 h. The reaction was started either by theaddition of NADH (solid lines) or GSSG (brokenlines). The reaction mixture contained 250 mM phos-phate buffer, pH 7.0, 0.175 mM NADH, and thefollowing concentrations of GSSG: (0) 50 mM; (0)12.5 mM; (x) 7 mM. In one experiment (A) 7 mMGSSG was added initially, followed by the addition ofGSSG to 50 mM. In all cases FAD was added to a finalconcentration of 5 AM.

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GLUTATHIONE REDUCTASE 207

NADH oxidation, but the subsequent additionofFAD did stimulate NADH oxidation (Fig. 4).Experiments in which GSSG (50 mM) was

added to the reaction mixture at various timesafter the addition of NADH show that exposure

of the enzyme to NADH in the absence of GSSGfor 20 s is sufficient to inhibit the reaction by98%. If 5 AM or greater FAD is included in thereaction mixture, no NADH inhibition was

observed. A sample of enzyme mixed with 0.35mM NADH and dialyzed against 2 mM EDTAovernight had no activity when the reaction wasstarted with NADH, and the addition of FADonly restored 1.6% of the activity present in a

duplicate enzyme sample dialyzed in the ab-sence of NADH. Thus it appears that dialysisunder these conditions results in extensive inac-tivation of the enzyme.

pH optimum and the effect of phosphateand sulfate ion. The pH optimum of theenzyme was 7.0 in N-2-hydroxyethyl-pipera-zine-N'-2'-ethanesulfonic acid (HEPES), tris-(hydroxymethyl)aminomethane * hydrochlorideand phosphate buffer (Fig. 5). However, theactivity of the enzyme was higher in phosphatebuffer than in either HEPES or tris(hydroxy-methyl)aminomethane hydrochloride. In 0.25M HEPES buffer, pH 7.0, and 12.5 mMGSSG, the activity was 45% that in a systembuffered with 0.25 M phosphate, pH 7.0. In

pH

FIG. 5. Effect of pH in phosphate buffer on gluta-thione reductase activity. The crude extract was

dialyzed against 12 liters of distilled water for 16 h at7 C. The activity was assayed in 125 mM phosphatebuffer. The pH of the reaction mixture was measuredat the conclusion of the experiment.

the presence of 50 mM GSSG, the HEPESactivity was 65% that of the phosphate-bufferedsystem. The addition of tris(hydroxymethyl)-aminomethane or HEPES buffer in equal molaramounts to a phosphate-buffered system hadno effect on the enzyme activity. The saturatingphosphate concentration with 12.5 mM GSSGwas 0.25 M, and no additional effect was ob-served up to 0.5 M phosphate.

Sulfate ion (Na+ or NH,+) stimulated theactivity, but the activity at saturating sulfateconcentration (0.2 M) was only 77% that ob-tained with optimal phosphate concentrations.However, if 0.2 M sulfate was added to amixture containing a suboptimal phosphateconcentration (0.125 M), activity equal to thatat a saturating phosphate concentration wasobtained. Arsenate had no effect on the enzyme.Pyrophosphate at concentrations of 40 mM orgreater stimulated the activity to about 70%that of maximal phosphate activity.

Inhibition by salts, heavy metals, and sulf-hydryl reagents. The enzyme is inhibited by avariety of heavy metals and sulfhydryl reagents(Tables 2 and 3). The enzyme is particularlysensitive to mercury and mercury-containingreagents.

Chloride salts (K+, Na+, or NH+) are inhibi-

TABLE 2. Effect of salts and heavy metals on theactivity of glutathione reductasea

Compounds Concn (mM) Inhibition (%)

KI 2.5 625.0 20

CUC12 0.25 330.5 100

CaCl2 0.25 130.5 451.25 89

BaCl2 0.5 41.25 56

ZnCl2 0.25 100.5 361.25 100

MgSO;7H2O 12.5 1325.0 45

Fe(NO3)2 .9H20 0.25 350.5 591.25 100

AgNO, 0.25 100MnSO4 0.5 39

1.25 47HgC12 8 x 10-6 10

lx 10-4 442 x 10-4 100

aReagents and enzyme added to salt solutions innormal sequence.

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208 CHUNG AND HURLBERT

TABLE 3. Effect of sulfhydryl reagents on the activityof glutathione reductasea

Compound Concn (GM) Inhibition

Hydroxymercuribenzoate 2.5 104.5 549.0 100

N-ethylmaleimide 50 10125 60250 90

Iodoacetamide 50 13150 57300 87

aThe reaction mixture was incubated for 10 minwith the reagent before initating the reaction withGSSG.

tory, with about 50% of the enzyme activitybeing lost at a concentration of 0.18 M Cl- ion.The inhibition by chloride ion was found to bereversible since the activity of the enzyme canbe completely recovered by dialysis againstdistilled water.

Inhibition by adenylate compounds. Theadenylate nucleotides, adenosine 5'-triphos-phate (ATP), adenosine 5'-diphosphate (ADP),and adenosine 5'-monophosphate (AMP), wereall found to be inhibitory to the enzyme atnonsaturating GSSG concentrations in bothHEPES and phosphate buffer (Fig. 6).Among these three compounds, ADP was the

most effective inhibitor, with 0.2 mM ADPinhibiting the enzyme by 50% in the presence of12.5 mM GSSG. ATP and AMP required about5 and 8 mM, respectively, to obtain the samelevel of inhibition as ADP (Fig. 6).The following compounds were not inhibitory

at the concentrations tested: adenosine, 6.25mM; thymine, 6.25 mM; guanosine diphos-phate, 6.25 mM; guanosine monophosphate,6.25 mM; guanosine triphosphate, 6.25 mM;cytidine diphosphate, 9.7 mM; cytidine mono-phosphate, 9.7 mM; cytosine, 2 mM; and cyclic3',5'- and 2',3'-AMP, 1.25 mM.

Kinetic studies. The Km for GSSG andNADH were determined in two different ways.Figures 7 and 8 represent results obtained whenone substrate is held constant at several concen-trations and the other is varied. The kineticconstants determined according to the methodof Dalziel (7) were 7.4 x 10-3 M for GSSG and6.25 x 10- M for NADH (Fig. 7B and 8B). TheKm values obtained with one substrate saturat-ing were 5 x 10-5 M for NADH and 7 x 10-3 Mfor GSSG (Fig. 9). The Km values were the samein HEPES buffer. The Km of NADPH was3 x 10-3M.

Effect of products and excess substrate.High concentrations of GSSG (75 mM) andNADH (7 mM) had no effect on the activity.The effect of higher concentrations was nottested.Nicotinamide adenine dinucleotide (0.28

mM) was not inhibitory under conditions ofunsaturated GSSG (7 mM) and variable NADH,or under conditions of unsaturated NADH(14 x 10-6 M) and variable GSSG. High con-centrations (3.5 mM maximum) of nicotinamideadenine dinucleotide were not inhibitory in areaction mixture containing 50 mM GSSG and0.175 mM NADH.GSH inhibited the reaction, but only at high

concentrations. A concentration of GSH (pH7.0) of 75 mM inhibited the reaction by 53%when GSSG was below saturating concentration(7 mM). Kinetic analysis of GSH inhibitionunder these conditions indicated that GSH wasan uncompetitive inhibitor of GSSG at 50 mMconcentrations, but became a noncompetitiveinhibitor at higher (75 mM) concentration.With both NADH and GSSG saturating (0.175and 50 mM, respectively), 75 mM GSH inhib-ited the reaction by 32%.

Kinetic analysis of adenine nucleotide

100 *

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FIG. 6. Effect ofATP, ADP, and AMP on glutathi-one reductase activity. The purified enzyme wasdialyzed against 12 liters of distilled water for 24 h.Symbols: (-) ADP; (0) ATP; and (0) AMP.

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GLUTATHIONE REDUCTASE 209

0 005 01 00o 0-2

NADI,iM-' GSSG,m -'

FIG. 7. Double reciprocal plot of glutathione re-ductase activity at a series of constant GSSG concen-trations as influenced by the concentration ofNADH(A) and Dalziel plot of the influence of GSSG concen-tration on the VNADH values. (B) Assays were done in0.125 Mphosphate buffer, pH 7.0, 15 AM FAD, 0.02%DTNB, and the following GSSG concentrations: (0) 5x 10-' M; (A) 7.5 x 10-3 M; (0) 10 x 10-' M; (V)12.5 x 10-3M; (x) 15 x 10- 3M; and (0) 20 x 10-3M.

inhibition. At unsaturating GSSG concentra-tion (12.5 mM) the adenine nucleotides exhib-ited noncompetitive inhibition towards NADH.The inhibition pattern towards GSSG was com-petitive.Because of the NADH inhibition of the en-

zyme at low concentrations of FAD and GSSG,it was not possible to carry out a kinetic analysisof the interaction between FAD and the adenyl-ate nucleotides. However, the effect of FADconcentration on the activity of the enzyme inthe presence of a nonsaturating concentration ofGSSG (7.5 mM) and 0.125 mM ADP wasexamined. In these experiments the reactionswere started by the addition of NADH. In theabsence of ADP and with 5 gM FAD thereaction is linear for over 3 min; however, when0.125 mM ADP was included in the assaymixture the reaction was only linear for 20 to 30s, and the rate decreased within 3 min to 14.5%of the initial rate. The addition ofFAD to 25 ,mafter 5.5 min restored the activity to 50% of theinitial rate. If 0.1 mM FAD was included in thereaction mixture, the reaction rate remainedlinear both in the presence and absence of ADP,and the rate of the reaction in the presence of0.125 mM ADP was 68% that of a control withno ADP.

DISCUSSIONC. vinosum glutathione reductase differs in

several respects from the glutathione reductasesof other organisms. Of these, the most signifi-cant is its specificity for NADH as the reduc-tant. All of the other glutathione reductases

studied in detail either have an absolute speci-ficity for NADPH or show only a fraction of theactivity with NADH that they show withNADPH. It is of interest to note that not only isthis true with organisms evolutionarily distantfrom Chromatium (e.g., peas and mammaliantissues), but it is also the case with the otherbacterial glutathione reductases investigated(1, 2, 3, 6, 12, 15, 19, 20, 21, 22, 23, 24, 26, 29, 30,32).The reason for this difference is unclear, but

it has been suggested that the Chromatiaceae

I0~ ~ ~ ~ ~ ~

A

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GSSG, Mm NADH,aM '

FIG. 8. Double reciprocal plot of glutathione re-ductase activity at a series of constant NADH concen-trations as influenced by the concentration of GSSG(A), and Dalziel plot of the influence of NADHconcentration on the VGssG values (B). Assays weredone as in Fig. 7 in the presence of the followingNADH concentrations: (0) 9.8 x 10-6 M; (x) 1.31 x10-i M; (0) 1.64 x 10-i M; (A) 1.97 x 10-5 M; and(V) 2.6 x 10-i M.

02

oil

A

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0

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0 01 0 01 0-2

NADM,p.M' GSSG, mm'

FIG. 9. Double reciprocal plots of glutathione re-ductase activity at: (A) saturating GSSG concentra-tions as influenced by the concentration of NADH,and (B) saturating NADH concentrations as in-fluenced by the concentration of GSSG. Assays weredone in 0.25 M phosphate buffer, pH 7.0, containing0.05M GSSG, 0.005% DTNB, and 25 AMFAD (A), orin 0.25 M phosphate buffer, pH 7.0, containing 0.175mMNADH, and 25AMFAD (B).

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210 CHUNG AND HURLBERT

represent a very primitive bacterial group andthat NADPH developed as a reductant laterthan NADH (11). If this is the case, the sugges-tion can be made that other Chromatiaceaespecies will also show a specificity for NADH.However, such a conclusion must await addi-tional study.As with the previously investigated glutathi-

one reductases, the C. vinosum enzyme hasFAD as a prosthetic group. However, the natureof the interaction of the FAD with the C.vinosum enzyme is complex. The enzyme(dialyzed crude or purified material) appears torequire FAD, if NADH is incubated with theenzyme prior to GSSG addition or if the concen-tration of GSSG used is below saturating.However, if saturating concentrations of GSSGare employed and if the GSSG is added to theenzyme prior to the NADH, high activity isobtained in the absence of any added FAD. Atlow GSSG concentrations the enzyme activitystops after an initial short period of NADHoxidation, but is restored by the addition ofFAD. The addition of GSSG to saturating levelsdoes not, however, restore the activity. If theenzyme is incubated for 20 s with NADH in theabsence of GSSG, no activity is observed uponthe addition of GSSG (50 mM), but activity isrestored by the addition of FAD.Although the data in the present study are

not sufficient to enable a single explanation forthe above observations, at least three alterna-tives are possible. Glutathione reductases froma variety of sources have been shown to beinhibited by excess NADPH or NADH (3, 4, 20,22, 27, 32), and it has been proposed that thisinhibition is due to an overreduction of theenzyme (3, 20, 32). This explanation can, withsome modifications, account for the inhibitionseen with the C. vinosum enzyme. In our case itis clear that FAD and high concentrations ofGSSG protect the enzyme from inhibition byNADH. In the case of FAD, it is possible thatthe excess reducing potential is trapped asnon-enzyme-associated reduced flavin adeninedinucleotide (FADH). The GSSG may protectby virtue of the reaction being rapid enough toprevent any build up of reduced enzyme.An alternate explanation would be to assume

that oxidized FAD is tightly bound to theenzyme, but that reduced FAD is easily lostunless GSSG is present. Under these conditionsa brief incubation of the dilute enzyme in thereaction cuvette with NADH would produce anapoenzyme lacking FAD. Addition of FAD to asaturating concentration would restore activity.High concentrations of GSSG would, by block-

ing the release of FADH, maintain the activityat a high level, but at lower GSSG concentra-tions the FADH would eventually be lost.A third possible explanation would be that

NADH has two binding sites, the active site anda second site that inhibits activity. In this caseFAD and GSSG (if added in the right sequenceand concentration) would prevent the bindingof NADH to the inhibitory site. Proof of one ofthese (or other) explanations must await-furtherinvestigation.The C. vinosum enzyme is similar to others in

its inhibition by chloride ions (3, 19, 20, 21, 26,32). The nature of the stimulation by certaindivalent anions is unknown. Phosphate ion hasbeen shown to be stimulatory with some gluta-thione reductases (20, 21, 25, 29, 32) and inhibi-tory with others (19). The observation thatphosphate has no effect on the Km of eitherGSSG or NADH in the C. vinosum enzymeindicates that phosphate ion effects only theturnover rate.The Km values of GSSG and NADH are the

highest reported for any glutathione reductase.The pH optimum of 7.0 is similar to that foundfor the majority of glutathione reductases. Theinhibitory effect of heavy metals and sulfhydrylinhibitors suggests that a sulfhydryl group isinvolved in the reaction as has been indicatedfor other glutathione reductases (3, 20, 32).The C. vinosum enzyme is the first glutathi-

one reductase that has been reported to beinhibited by adenylate nucleotides. Since theadenylate nucleotides are structural analoguesof both NADH and FAD, it is possible that theycould compete for the active site of one or bothof these compounds on the enzyme surface. Thefact that they inhibit NADH noncompetitivelyindicates that they either combine with anenzyme form other than the one that NADHcombines with, with a reversible step betweenthe two enzyme forms, or that the inhibitorsbind the same form as NADH but at a differentsite. The competitive inhibition of the adenyl-ate nucleotides with GSSG suggests that thelatter possibility seems more likely, i.e., theinhibitors either bind with the same form of theenzyme, or are separated in the reaction se-quence by a series of reversible steps alongwhich they can interact such that an increase inthe concentration of GSSG can eliminate theinhibition (5).The observation that a high concentration of

FAD overcomes the ADP inhibition suggeststhat the adenylate nucleotides may be acting ascompetitive inhibitors of FAD. Egami and Yagi(9) have reported that adenosine monosulfate

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GLUTATHIONE REDUCTASE 211

can act as a competitive inhibitor of FAD in theD-alanine acid oxidase system.

Several previously investigated glutathionereductases give a steady-state kinetic patternwhich is typical of a ping-pong reaction mecha-nism (26, 28, 32). However, recent analysis ofthe data suggests that a more complex explana-tion may be required (17, 18). The kinetic datareported here is not complete enough to assign areaction mechanism to the Chromatium en-zyme.

ACKNOWLEDGMENT

This work was supported by U. S. Public Health Serviceresearch grant AI09161 from the National Institute of Allergyand Infectious Diseases.

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