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

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

Purification and Properties of the Glutathione Reductase of Chromatium vinosum Y. C. CHUNG AND R. E. HURLBERT* Department of Bacteriology and Public Health, Washington State University, Pullman, Washington 99163 Received for publication 10 February 1975

The Chromatium vinosum glutathione reductase [NAD(P)H:glutathione disulfide oxidoreductase, EC 1.6.4.2] was purified to apparent homogeneity. The enzyme was found to require reduced nicotinamide adenine dinucleotide (NADH) as a reductant and to be specific for oxidized glutathione (GSSG). The polypeptide 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 sulfate ion, but was inhibited by chloride ion, heavy metals, and sulfhydryl reagents. Adenylate nucleotides were inhibitory, and the data suggested that they were acting as competitive inhibitors of flavin adenine dinucleotide (FAD). The Km values of 7 x 10-3 for GSSG and 6 x 10- 5 M for NADH were the highest reported of any previously investigated glutathione reductase. The order of addition of components markedly affected the response of the enzyme to FAD. A requirement for FAD (Km 5.2 x 10-7 M) was seen if the enzyme was incubated with NADH prior to GSSG addition, whereas no FAD was required if the order was reversed.

Glutathione reductase [NAD(P)H:glutath- tungsten lamps. Air was blown across the carboys to ione disulfide oxidoreductase, EC 1.6.4.2] has prevent overheating. After growth was complete (4 to by visual inspection), the cells were been demonstrated in higher plants (6, 19), in a 7 days, as judged at 7 C by using a Sharples centrifuge. The variety of animal tissues (10, 12, 15, 24, 26, 27, collected packed cells were stored without washing, at -70 C 30, 31), in yeast (4, 20, 24) and filamentous until used for the enzyme preparation. fungi (32), and in a number of bacteria (1, 3, The reductase activity was measured by two 29). In those cases where a detailed study of the methods. For all determinations involving NADH nature of the enzyme has been undertaken, it concentrations giving an optical density at 340 nm of has been shown that flavin adenine dinucleo- between 0.2 and 1.0 (0.03 to 0.175 mM), the rate of tide (FAD) is present as a tightly bound pros- NADH oxidation was followed by measuring the thetic group and that the enzyme is either decrease in absorption at 340 nm on a Gilford recording spectrophotometer. Unless otherwise noted, the specific for reduced nicotinamide adenine dinu- reaction mixture contained 125 mM potassium phoscleotide phosphate (NADPH) as the reductant phate buffer, pH 7, 0.014 mM FAD, enzyme, 0.175 with reduced lower or has a much activity mM NADH, and 12.5 mM GSSG (oxidized glutathinicotinamide adenine dinucleotide (NADH). one) added in that order. An NADH oxidase control The highest previously observed activity with was run with each experiment. Crude preparations NADH has been for the human erythrocyte had approximated 0.003 units of NADH oxidase enzyme, where the maximal rate of the reaction activity per mg, while pure preparations had no detectable NADH oxidase activity. It is necessary to with NADH was 23% that of NADPH (26). In the present paper the purification and add the FAD to the enzyme before the NADH in order prevent inhibition of the enzyme by NADH. The partial characterization of a glutathione reduc- to was prepared fresh for all assays. For NADH tase from Chromatium vinosum that is highly kinetic solution studies involving small quantities of NADH, specific for NADH is reported. the method of Beutler and Yeh (2) was followed. MATERIALS AND METHODS C. vinosum (formerly strain D) was grown in completely filled 12-liter carboys in the liquid thiosulfate salts medium described by Hurlbert and Lascelles (13). The carboys were sealed with a rubber stopper and were incubated with continuous stirring at 30 C in a light chamber with banks of 75- to 100-W

This assay is based on measuring the reduced glutathione (GSH) produced by linking it to the reduction of 5,5-dithio-bis-2-nitrobenzoate (DTNB) which is measured at 412 nm. Unless noted otherwise, the reaction mixture contained, in addition to the above, 0.02% DTNB. Since prolonged incubation of DTNB with the enzyme caused inhibition, the DTNB was added immediately before initiating the reaction. 203

204

CHUNG AND HURLBERT

J. BACTERIOL.

Controls run in the absence of GSSG, NADH, or enzyme showed no activity with either assay. Kinetic experiments, by using either assay and GSSG as the variable substrate, gave identical results. All kinetic data were subjected to linear regression analysis. A unit of enzyme activity is defined as the amount of enzyme that oxidized 1 timol of NADH per min or reduced 1 Mmol of DTNB per min. Protein was measured by the method of Lowry et al. (16) employing bovine serum albumin as a protein standard. Analytical disc gel electrophoresis was performed by the method of Davis (8) in 5, 7.5, and 10% polyacrylamide gels. The electrophoresis was carried out at 3 mA per tube. The gels were stained either in 1% Amido Schwartz in 7% acetic acid or in 0.1% Coomassie brilliant blue as described below. Analytical gel electrophoresis in 10% polyacrylamide gels containing sodium dodecyl sulfate was done according to the method of King and Laemmli (14). The gels were rinsed twice at 37 C for 1 h each in 50% MeOH-7% acetic acid. The gels were stained for 1 to 2 h at 37 C by 0.1% Coomassie brilliant blue in 50% (vol/vol) methyl alcohol to which 0.7 ml of glacial acetic acid per 10 ml of stain was added immediately before use. All gels were destained in 7% acetic acid. Molecular weight estimation in sodium dodecyl sulfate gels was carried out by the split-gel method of Schnaitman (25). All chemicals were obtained from Sigma Chemical Co. Sephadex G-200 and diethylaminoethyl (DEAE)-A50 were products of Pharmacia, Uppsala, Sweden. The hydroxyapatite was a product of BioRad Laboratories, Richmond, Calif.

added as before until the pH dropped to 4.4. The precipitate was removed as before, and the supernatant was collected. Potassium hydroxide (6 N) was added dropwise with stirring to the supernatant until the pH was returned to 7.0. Step 3. Ammonium sulfate fractionation. Solid ammonium sulfate crystals (27.4 g/100 ml of solution) were added slowly with stirring to the material from step 2 in an ice bath. After 10 min the precipitate (45% saturation) was removed by centrifugation at 8,000 x g for 5 min at 4 C. To this supernatant 3.2 g of ammonium sulfate per 100 ml of original solution was added as before. After 10 min the precipitate (50% saturation) was sedimented as before. After decanting the supernatant and wiping down the sides of the centrifuge bottle, the precipitate was dissolved in a 100-ml solution of 0.05 M potassium phosphate buffer, pH 7.0, and 2 mM EDTA. Step 4. DEAE chromatography. The dissolved 50% ammonium sulfate precipitate was added to a DEAE-Sephadex A-50 column (2 by 25 cm) equilibrated with a solution of 0.05 M potassium phosphate buffer, pH 7.0, and 2 mM EDTA at the rate of approximately 0.7 ml per min. The protein was eluted with a linear gradient of 0.05 to 0.5 M potassium phosphate buffer, pH 7.0, containing 2 mM EDTA. Fractions of 5.5 ml were collected. The material RESULTS between 375 to 460 ml (Fig. 1) contained the Purification of glutathione reductase. All activity. The fractions containing the highest 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 wet weight) per 10 ml of a solution of 0.05 M potassium phosphate buffer, pH 7.0, and 2 mM 'I ethylenediaminetetraacetic acid (EDTA). The tP.4 cells were disrupted by a single pass through a I French pressure cell at 20,000 lb/in2. Because z11I 3some early samples were inactive after treatment the French pressure cell was presoaked for ua 30 to 60 min in 0.01 M EDTA prior to disruption. The suspension was centrifuged at 105,000 x g for 2 to 3 h, and the straw-colored supernaTubeMusBER tant (crude extract) was collected. Since the enzyme was stable when frozen, it was possible FIG. 1. Elution profile of glutathione reductase to store the extract at -20 or -70 C for several from a DEAE-Sephadex column. Precipitate from ammonium sulfate fractionation dissolved in a solumonths without loss of activity. Step 2. pH fractionation. The crude extract tion of 0.05 M phosphate buffer, pH 7.0, and 2 mM was adsorbed to a DEAE-Sephadex A-50 (fresh or thawed material) was placed in an ice EDTA column (2.5 by 24 cm). Elution and assay for glutathibath, and cold M HSPO4 was added dropwise one activity was carried out as described in with vigorous stirring until the pH dropped to text.reductase Symbols: (O) optical density at 280 nm; (0) 4.8. The precipitate which formed was removed glutathione reductase activity, (A) 2nd phosphate by centrifugation at 27,000 x g for 5 min. The concentration. Fractions between bars were used for supernatant was saved, and 0.1 M HPO4 was further purification. 0

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GLUTATHIONE REDUCTASE

activity were pooled and concentrated by using an Amicon ultrafiltration cell with a type PM 10 ultrafilter membrane. Step 5. Sephadex G-200 column. The DEAE concentrate was added to a Sephadex G-200 column (2.5 by 31 cm) equilibrated with the phosphate-EDTA buffer. The protein was eluted at the rate of approximately 0.7 ml per min, and 5.5-ml fractions were collected (Fig. 2A). The fractions containing the highest activity were pooled and concentrated as above. Step 6. Hydroxyapatite column. The concentrated material from step 5 was added to a hydroxyapatite column (2 by 16 cm) equilibrated with the phosphate-EDTA buffer. The sample was eluted with the same buffer at the rate of 0.2 ml per min, and 5.5-ml fractions were collected. The fractions containing the highest activity were concentrated as before to a small volume and stored at -20 C (Fig. 2B). The results of one such purification is seen in Table

205

electrophoresis in sodium dodecyl sulfate polyacrylamide gels was employed to determine the minimum molecular weight. A single band with a molecular weight of 52,000 was observed (Fig. 3). Stability of the enzyme. Repeated freezing and thawing of either the purified or the crude material did not cause any significant loss of activity. The enzyme could be stored at -20 C for over 2 years with no significant loss of activity. The enzyme was rapidly inactivated by tem-

1.

Criteria of purity. To determine the purity of the isolated material, it was examined by analytical disc gel electrophoresis at concentrations of 5, 7.5, and 10% acrylamide. Only one protein band was visible in the final material. To determine that the band corresponded to the enzyme activity, a gel was run in the normal way, and a thin verticle slice of the gel was stained for 15 min and rapidly destained by using a Canalco electric gel destainer. The section of the unstained gel corresponding to the protein band was cut out and crushed in a small volume of buffer containing 0.05 M phosphate, pH 7.0, and 2 mM EDTA. After 15 to 30 min of elution at 4 C the gel was removed by brief centrifugation, and the supernatant was found to contain glutathione reductase activity. Molecular weight determination. Disc gel

UIE IUMER

FIG. 2. Gel chromatography on Sephadex G-200 and hydroxyapatite. (A) Active fractions from DEAE chromatography were pooled, concentrated, and added to a Sephadex G-200 column (2.5 by 31 cm) equilibrated with a solution of 0.05 M phosphate buffer, pH 7.0, and 2 mM EDTA. Fractions of 5.5 ml were eluted at the rate of 0.7 ml/min with the same buffer. (B) Active fractions from G-200 chromatography were pooled, concentrated, and applied to a hydroxyapitite column (2 by 16 cm) equilibrated with the phosphate-EDTA buffer. Fractions of 5.5 ml were eluted at the rate of 0.2 ml/min with the same buffer. Symbols: (0) glutathione reductase activity; solid lines, optical density at 280 nm. Fractions between bars were used for further study.

TABLE 1. Summary of the purification of glutathione reductase from C. vinosum Protein | content (mg)

Total

cU)

(U/mg)

450.0 473.0 100.0

2336.0 733.0 82.0

76.8 36.6 29.0

0.03 0.05 0.35

100 48 ' 38

6.2

2.0

19.6

9.8

26

297

2.0

1.07

15.0

14.0

20

424

1.8

0.19

5.4

28.4

7

861

Step8s5 Vol (ml)

Crude extracta pH 4.4 supernatant

(NH4),S04 precipitate DEAE-Sephadex chromatography Sephadex G-200 chromatography Hydroxyapatite a From 48 g of packed cells. b Micromoles of NADH oxidized 45 to 50%

per minute per milligram of protein.

Sp act

| Recovery (%)

Purification

factor

1 1.52 10.6

206

2

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CHUNG AND HURLBERT

10 9 8 7

, 6 0 5 x

\1 @2 0

@3

r

4 3

@4

L-JaJ

0

0

5

0

2 4 DISTANCE IN GEL (cm) FIG. 3. Electrophoretic migration of glutathione reductase as a function of molecular weight. Gel electrophoresis in split gels with authentic standards was performed as described in Materials and Methods. (1) Bovine serum albumin; (2) pyruvate kinase; (3) ovalbumin; (4) chymotrysinogen; (5) trypsin; open circle, glutathione reductase.

peratures above 45 C. About 60% of the activity was lost after 5 min at 50 C, and no activity could be detected after heating for 5 min at 65 C. Substrate specificity. The Chromatium enzyme shows a high specificity for NADH as an electron donor. At equal molar concentrations (2.5 x 10- 4 M) the rate of DTNB reduction with NADPH as reductant was only 5% that of NADH. The enzyme is also specific for GSSG, as no activity was observed when GSSG was replaced with cystine, oxidized lipoic acid, or oxidized lipoamide at a concentration of 12.5 mM. FAD requirement and the effect of the order of addition of components. During early studies of the enzyme, NADH was added prior to GSSG. Under these conditions it was found necessary to add FAD to a concentration of 5 ,uM or greater in order to demonstrate the reaction. A double reciprocal plot of FAD concentration versus activity gave an apparent Km for FAD of 5.2 x 10-7 M. Flavin mononucleotide did not replace the FAD. Subsequent experiments showed that the requirement of FAD is dependent upon the GSSG concentration and the order of addition of

components to the reaction mixture. In experiments where GSSG is added to initiate the reaction, little or no enzyme activity is detected in dialyzed extracts (crude and purified enzyme behave similarly) in the absence of added FAD (Fig. 4). However, if GSSG is added first and NADH is used to start the reaction, significant activity is observed in the absence of any added FAD (Fig. 4). The extent of the reaction is dependent upon GSSG concentration, in that at GSSG concentrations of 7 and 12.5 mM, NADH reduction ceases after only a small fraction of the GSSG has been utilized, whereas with 50 mM GSSG the reaction continues until the NADH is totally oxidized (Fig. 4). Subsequent addition of FAD to the reaction mixture started either with GSSG or with NADH, but with 7 or 12.5 mM GSSG, restored the enzyme activity. To determine the role of GSSG concentration an experiment was run with 7 mM GSSG until NADH oxidation ceased. At this point GSSG was added to bring the concentration to 50 mM. Thie innAM t1 ttuuitliuii riit Ulu livtl initisi lllitlltttlt: nnu 11MI-CLIbc I illb arldiLinn ctily iniirPnQP

in III

UTES

FIG. 4. Effect of GSSG concentration and the order of addition of substrate on the activity of dialyzed glutathione reductase. The enzyme was dialyzed against several changes of 2 mM EDTA at 7 C for 24 h. The reaction was started either by the addition of NADH (solid lines) or GSSG (broken lines). The reaction mixture contained 250 mM phosphate buffer, pH 7.0, 0.175 mM NADH, and the following concentrations of GSSG: (0) 50 mM; (0) 12.5 mM; (x) 7 mM. In one experiment (A) 7 mM GSSG was added initially, followed by the addition of GSSG to 50 mM. In all cases FAD was added to a final concentration of 5 AM.

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GLUTATHIONE REDUCTASE

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NADH oxidation, but the subsequent addition of FAD did stimulate NADH oxidation (Fig. 4). Experiments in which GSSG (50 mM) was added to the reaction mixture at various times after the addition of NADH show that exposure of the enzyme to NADH in the absence of GSSG for 20 s is sufficient to inhibit the reaction by 98%. If 5 AM or greater FAD is included in the reaction mixture, no NADH inhibition was observed. A sample of enzyme mixed with 0.35 mM NADH and dialyzed against 2 mM EDTA overnight had no activity when the reaction was started with NADH, and the addition of FAD only restored 1.6% of the activity present in a duplicate enzyme sample dialyzed in the absence of NADH. Thus it appears that dialysis under these conditions results in extensive inactivation of the enzyme. pH optimum and the effect of phosphate and sulfate ion. The pH optimum of the enzyme was 7.0 in N-2-hydroxyethyl-piperazine-N'-2'-ethanesulfonic acid (HEPES), tris(hydroxymethyl)aminomethane * hydrochloride and phosphate buffer (Fig. 5). However, the activity of the enzyme was higher in phosphate buffer than in either HEPES or tris(hydroxymethyl)aminomethane hydrochloride. In 0.25 M HEPES buffer, pH 7.0, and 12.5 mM GSSG, the activity was 45% that in a system buffered with 0.25 M phosphate, pH 7.0. In

the presence of 50 mM GSSG, the HEPES activity was 65% that of the phosphate-buffered system. The addition of tris(hydroxymethyl)aminomethane or HEPES buffer in equal molar amounts to a phosphate-buffered system had no effect on the enzyme activity. The saturating phosphate concentration with 12.5 mM GSSG was 0.25 M, and no additional effect was observed up to 0.5 M phosphate. Sulfate ion (Na+ or NH,+) stimulated the activity, but the activity at saturating sulfate concentration (0.2 M) was only 77% that obtained with optimal phosphate concentrations. However, if 0.2 M sulfate was added to a mixture containing a suboptimal phosphate concentration (0.125 M), activity equal to that at a saturating phosphate concentration was obtained. Arsenate had no effect on the enzyme. Pyrophosphate at concentrations of 40 mM or greater stimulated the activity to about 70% that of maximal phosphate activity. Inhibition by salts, heavy metals, and sulfhydryl reagents. The enzyme is inhibited by a variety of heavy metals and sulfhydryl reagents (Tables 2 and 3). The enzyme is particularly sensitive to mercury and mercury-containing reagents. Chloride salts (K+, Na+, or NH+) are inhibiTABLE 2. Effect of salts and heavy metals on the activity of glutathione reductasea Compounds

KI

CUC12 CaCl2 BaCl2

ZnCl2 MgSO;7H2O

Fe(NO3)2 .9H20 AgNO, pH

FIG. 5. Effect of pH in phosphate buffer on glutathione reductase activity. The crude extract was dialyzed against 12 liters of distilled water for 16 h at 7 C. The activity was assayed in 125 mM phosphate buffer. The pH of the reaction mixture was measured at the conclusion of the experiment.

MnSO4

HgC12

Concn (mM)

Inhibition (%)

2.5 25.0 0.25 0.5 0.25 0.5 1.25 0.5 1.25 0.25 0.5 1.25 12.5 25.0 0.25 0.5 1.25 0.25 0.5 1.25 8 x 10-6 lx 10-4 2 x 10-4

6 20 33 100 13 45 89 4 56 10 36 100 13 45 35 59 100 100 39 47 10 44 100

aReagents and enzyme added to salt solutions in normal sequence.

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CHUNG AND HURLBERT

TABLE 3. Effect of sulfhydryl reagents on the activity of glutathione reductasea Compound

Concn (GM)

Inhibition

Hydroxymercuribenzoate

2.5 4.5 9.0 50 125 250 50 150 300

10 54 100 10 60 90 13 57 87

N-ethylmaleimide Iodoacetamide

aThe reaction mixture was incubated for 10 min with the reagent before initating the reaction with GSSG.

Effect of products and excess substrate. High concentrations of GSSG (75 mM) and NADH (7 mM) had no effect on the activity. The effect of higher concentrations was not tested. Nicotinamide adenine dinucleotide (0.28 mM) was not inhibitory under conditions of unsaturated GSSG (7 mM) and variable NADH, or under conditions of unsaturated NADH (14 x 10-6 M) and variable GSSG. High concentrations (3.5 mM maximum) of nicotinamide adenine dinucleotide were not inhibitory in a reaction mixture containing 50 mM GSSG and 0.175 mM NADH. GSH inhibited the reaction, but only at high concentrations. A concentration of GSH (pH 7.0) of 75 mM inhibited the reaction by 53% when GSSG was below saturating concentration (7 mM). Kinetic analysis of GSH inhibition under these conditions indicated that GSH was an uncompetitive inhibitor of GSSG at 50 mM concentrations, but became a noncompetitive inhibitor at higher (75 mM) concentration. With both NADH and GSSG saturating (0.175 and 50 mM, respectively), 75 mM GSH inhibited the reaction by 32%. Kinetic analysis of adenine nucleotide

tory, with about 50% of the enzyme activity being lost at a concentration of 0.18 M Cl- ion. The inhibition by chloride ion was found to be reversible since the activity of the enzyme can be completely recovered by dialysis against distilled water. Inhibition by adenylate compounds. The adenylate nucleotides, adenosine 5'-triphosphate (ATP), adenosine 5'-diphosphate (ADP), and adenosine 5'-monophosphate (AMP), were all found to be inhibitory to the enzyme at nonsaturating GSSG concentrations in both * 100 HEPES and phosphate buffer (Fig. 6). Among these three compounds, ADP was the most effective inhibitor, with 0.2 mM ADP inhibiting the enzyme by 50% in the presence of 12.5 mM GSSG. ATP and AMP required about 0~~~~ 5 and 8 mM, respectively, to obtain the same 0~~~~ level of inhibition as ADP (Fig. 6). The following compounds were not inhibitory 50~~~~~~~ .!S at the concentrations tested: adenosine, 6.25 mM; thymine, 6.25 mM; guanosine diphos- z0 phate, 6.25 mM; guanosine monophosphate, m 50 0 6.25 mM; guanosine triphosphate, 6.25 mM; cytidine diphosphate, 9.7 mM; cytidine monophosphate, 9.7 mM; cytosine, 2 mM; and cyclic I TO O 3',5'- and 2',3'-AMP, 1.25 mM. Kinetic studies. The Km for GSSG and NADH were determined in two different ways. Figures 7 and 8 represent results obtained when one substrate is held constant at several concentrations and the other is varied. The kinetic constants determined according to the method of Dalziel (7) were 7.4 x 10-3 M for GSSG and 6.25 x 10- M for NADH (Fig. 7B and 8B). The CONC (mM) Km values obtained with one substrate saturating were 5 x 10-5 M for NADH and 7 x 10-3 M FIG. 6. Effect of A TP, ADP, and AMP on glutathifor GSSG (Fig. 9). The Km values were the same one reductase activity. The purified enzyme was in HEPES buffer. The Km of NADPH was dialyzed against 12 liters of distilled water for 24 h. 3 x 10-3M. Symbols: (-) ADP; (0) ATP; and (0) AMP.

oa.

"

VOL. 123, 1975

0

GLUTATHIONE REDUCTASE

005

NADI,iM-'

01

0 o 0 GSSG,m -'

0-2

FIG. 7. Double reciprocal plot of glutathione reductase activity at a series of constant GSSG concentrations as influenced by the concentration of NADH (A) and Dalziel plot of the influence of GSSG concentration on the VNADH values. (B) Assays were done in 0.125 Mphosphate buffer, pH 7.0, 15 AM FAD, 0.02% DTNB, and the following GSSG concentrations: (0) 5 x 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 10-3 M.

209

studied in detail either have an absolute specificity for NADPH or show only a fraction of the activity with NADH that they show with NADPH. It is of interest to note that not only is this true with organisms evolutionarily distant from Chromatium (e.g., peas and mammalian tissues), but it is also the case with the other bacterial 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

S

I-

v6L,-~~~~~~~~~~~~~~~~~~4

V 0z inhibition. At unsaturating GSSG concentration (12.5 mM) the adenine nucleotides exhibited noncompetitive inhibition towards NADH. The inhibition pattern towards GSSG was com2 petitive. Because of the NADH inhibition of the enzyme at low concentrations of FAD and GSSG, GSSG, Mm NADH,aM ' it was not possible to carry out a kinetic analysis of the interaction between FAD and the adenylFIG. 8. Double reciprocal plot of glutathione reate nucleotides. However, the effect of FAD ductase activity at a series of constant NADH concenconcentration on the activity of the enzyme in trations as influenced by the concentration of GSSG the presence of a nonsaturating concentration of (A), and Dalziel plot of the influence of NADH concentration on the VGssG values (B). Assays were GSSG (7.5 mM) and 0.125 mM ADP was done in Fig. 7 in the presence of the following In examined. these experiments the reactions NADHasconcentrations: (0) 9.8 x 10-6 M; (x) 1.31 x were started by the addition of NADH. In the 10-i M; (0) 1.64 x 10-i M; (A) 1.97 x 10-5 M; and absence of ADP and with 5 gM FAD the (V) 2.6 x 10-i M. reaction is linear for over 3 min; however, when 0.125 mM ADP was included in the assay 02 A a mixture the reaction was only linear for 20 to 30 0 s, and the rate decreased within 3 min to 14.5% 0of of the initial rate. The addition of FAD to 25 ,m /0 oil after 5.5 min restored the activity to 50% of the ,0 Ar 0 initial rate. If 0.1 mM FAD was included in the v reaction mixture, the reaction rate remained linear both in the presence and absence of ADP, 0 01 0 01 0-2 and the rate of the reaction in the presence of GSSG, mm' NADM,p.M' 0.125 mM ADP was 68% that of a control with FIG. 9. Double reciprocal plots of glutathione reno ADP.

ye

ductase activity at: (A) saturating GSSG concentra-

tions as influenced by the concentration of NADH, DISCUSSION and (B) saturating NADH concentrations as inC. vinosum glutathione reductase differs in fluenced by the concentration of GSSG. Assays were several respects from the glutathione reductases done in 0.25 M phosphate buffer, pH 7.0, containing of other organisms. Of these, the most signifi- 0.05 M GSSG, 0.005% DTNB, and 25 AM FAD (A), or cant is its specificity for NADH as the reduc- in 0.25 M phosphate buffer, pH 7.0, containing 0.175 tant. All of the other glutathione reductases mMNADH, and 25AM FAD (B).

210

CHUNG AND HURLBERT

represent a very primitive bacterial group and that NADPH developed as a reductant later than NADH (11). If this is the case, the sugges-

tion can be made that other Chromatiaceae species will also show a specificity for NADH. However, such a conclusion must await additional study. As with the previously investigated glutathione reductases, the C. vinosum enzyme has FAD as a prosthetic group. However, the nature of the interaction of the FAD with the C. vinosum enzyme is complex. The enzyme (dialyzed crude or purified material) appears to require FAD, if NADH is incubated with the enzyme prior to GSSG addition or if the concentration of GSSG used is below saturating. However, if saturating concentrations of GSSG are employed and if the GSSG is added to the enzyme prior to the NADH, high activity is obtained in the absence of any added FAD. At low GSSG concentrations the enzyme activity stops after an initial short period of NADH oxidation, but is restored by the addition of FAD. The addition of GSSG to saturating levels does not, however, restore the activity. If the enzyme is incubated for 20 s with NADH in the absence of GSSG, no activity is observed upon the addition of GSSG (50 mM), but activity is restored by the addition of FAD. Although the data in the present study are not sufficient to enable a single explanation for the above observations, at least three alternatives are possible. Glutathione reductases from a variety of sources have been shown to be inhibited by excess NADPH or NADH (3, 4, 20, 22, 27, 32), and it has been proposed that this inhibition is due to an overreduction of the enzyme (3, 20, 32). This explanation can, with some modifications, account for the inhibition seen with the C. vinosum enzyme. In our case it is clear that FAD and high concentrations of GSSG protect the enzyme from inhibition by NADH. In the case of FAD, it is possible that the excess reducing potential is trapped as non-enzyme-associated reduced flavin adenine dinucleotide (FADH). The GSSG may protect by virtue of the reaction being rapid enough to prevent any build up of reduced enzyme. An alternate explanation would be to assume that oxidized FAD is tightly bound to the enzyme, but that reduced FAD is easily lost unless GSSG is present. Under these conditions a brief incubation of the dilute enzyme in the reaction cuvette with NADH would produce an apoenzyme lacking FAD. Addition of FAD to a saturating concentration would restore activity. High concentrations of GSSG would, by block-

J. BACTERIOL.

ing the release of FADH, maintain the activity at a high level, but at lower GSSG concentrations the FADH would eventually be lost. A third possible explanation would be that NADH has two binding sites, the active site and a second site that inhibits activity. In this case FAD and GSSG (if added in the right sequence and concentration) would prevent the binding of NADH to the inhibitory site. Proof of one of these (or other) explanations must await-further investigation. 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 certain divalent anions is unknown. Phosphate ion has been shown to be stimulatory with some glutathione reductases (20, 21, 25, 29, 32) and inhibitory with others (19). The observation that phosphate has no effect on the Km of either GSSG or NADH in the C. vinosum enzyme indicates that phosphate ion effects only the turnover 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 found for the majority of glutathione reductases. The inhibitory effect of heavy metals and sulfhydryl inhibitors suggests that a sulfhydryl group is involved in the reaction as has been indicated for other glutathione reductases (3, 20, 32). The C. vinosum enzyme is the first glutathione reductase that has been reported to be inhibited by adenylate nucleotides. Since the adenylate nucleotides are structural analogues of both NADH and FAD, it is possible that they could compete for the active site of one or both of these compounds on the enzyme surface. The fact that they inhibit NADH noncompetitively indicates that they either combine with an enzyme form other than the one that NADH combines with, with a reversible step between the two enzyme forms, or that the inhibitors bind the same form as NADH but at a different site. The competitive inhibition of the adenylate nucleotides with GSSG suggests that the latter possibility seems more likely, i.e., the inhibitors either bind with the same form of the enzyme, or are separated in the reaction sequence by a series of reversible steps along which they can interact such that an increase in the concentration of GSSG can eliminate the inhibition (5). The observation that a high concentration of FAD overcomes the ADP inhibition suggests that the adenylate nucleotides may be acting as competitive inhibitors of FAD. Egami and Yagi (9) have reported that adenosine monosulfate

VOL. 123, 1975

GLUTATHIONE REDUCTASE

can act as a competitive inhibitor of FAD in the D-alanine acid oxidase system. Several previously investigated glutathione reductases give a steady-state kinetic pattern which is typical of a ping-pong reaction mechanism (26, 28, 32). However, recent analysis of the data suggests that a more complex explanation may be required (17, 18). The kinetic data reported here is not complete enough to assign a reaction mechanism to the Chromatium enzyme. ACKNOWLEDGMENT This work was supported by U. S. Public Health Service research grant AI09161 from the National Institute of Allergy and Infectious Diseases.

LfTERATURE CITED 1. Asnis, R. E. 1955. A glutathione reductase from Escherichia coli. J. Biol. Chem. 213:77-85. 2. Beutler, E., and M. K. Y. Yeh. 1963. Erythrocyte glutathione reductase. Blood 21:573-577. 3. Boll, M. 1969. Glutathione reductase from Rhodospirillum rubrum. Arch. Mikrobiol. 66:374-389. 4. Buzard, J. A., and F. Kopko. 1963. The flavin requirement and some inhibition characteristics of rat tissue glutathione reductase. J. Biol. Chem. 238:464-468. 5. Cleland, W. W. 1963. The kinetics of enzyme-catalyzed reactions with two or more substrates or products. Im. Prediction of initial velocity and inhibition patterns by

6. 7. 8. 9.

10.

11. 12.

13.

inspections. Biochim. Biophys. Acta 67:188-196. Conn, E. E., and B. Vennesland. 1951. Glutathione reductase of wheat germ. J. Biol. Chem. 192:17-28. Dalziel, K. 1957. Initial steady-state velocities in the evaluation of enzyme-coenzymes-substrate reaction mechanisms. Acta Chem. Scand. 11:1706-1723. Davis, B. J. 1964. Disk electrophoresis. II. Method and application to human serum proteins. Ann. N. Y. Acad. Sci. 121:404-427. Egami, F., and K. Yagi. 1956. Biochemical studies on coenzyme sulfate analogs (I) effects of adenosine monosulfate and riboflavine monosulfate on D-alanine acid oxidase. J. Biochem. (Tokyo) 43:153-160. Francoeur, M., and 0. F. Denstedt. 1954. Metabolism of mammalian erythrocyte. VII. The glutathione reductase of the mammalian erythrocyte. Can. J. Biochem. Physiol. 32:663-669. Horecker, B. L. 1965. Pathways of carbohydrate metabolism and their physiological significance. J. Chem. Educ. 42:244-253. Horn, H. D., and F. H. Bruns, 1958. DPNH and TPNHglutathion-reduktase im serum des menschen. Biochem. Z. 331:58-64. Hurlbert, R. E., and J. Lascelles. 1963. Ribulose diphos-

14.

15. 16. 17. 18.

19. 20. 21. 22.

23. 24.

25. 26. 27.

28.

29. 30. 31. 32.

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phate carboxylase in Thiorhodaceae. J. Gen. Microbiol. 33:445-458. King, J., and U. K. Laemmli. 1971. Polypeptides of the tail fibres of bacteriophage T-4. J. Mol. Biol. 62:465-477. Langdon, R. C. 1958. Properties and mechanism of action of purified glutathione reductase. Biochim. Biophys. Acta 30:432-433. Lowry, 0. H., M. J. Rosebrough, A. L. Farr, and R. J. Randall. 1951. Protein measurements with the Folin phenol reagent. J. Biol. Chem. 193:265-275. Mannervik, B. 1969. On the kinetics of glutathione reductase. Acta Chem. Scand. 23:2912-2914. Mannervik, B. 1973. A branching reaction mechanism of glutathione reductase. Biochem. Biophys. Res. Commun. 53:1151-1158. Mapson, L. W., and D. R. Goddard. 1951. The reduction of glutathione by plant tissues. Biochem. J. 49:592-601. Massey, V., and C. H. Williams, Jr. 1965. On the reaction mechanism of yeast glutathione reductase. J. Biol. Chem. 240:4470-4480. Mize, C. E., and R. G. Langdon. 1962. Hepatic glutathione reductase. I. Purification and general kinetic properties. J. Biol. Chem. 237:1589-1595. Moroff, G., and K. G. Brandt. 1973. Steady-state kinetic investigation of specific anion effects on the catalytic activity of yeast glutathione reductase. Arch. Biochem. Biophys. 159:468-474. Powning, R. F., and H. Irzykiewicz. 1960. Cystine and glutathione reductase in the clothes moth. J. Biol. Sci. 13:59-68. Racker, E. 1955. Glutathione reductase from bakers yeast and beef liver. J. Biol. Chem. 217:855-865. Schnaitman, C. A. 1970. Comparison of the envelope protein compositions of several gram-negative bacteria. J. Bacteriol. 104:1404-1405. Scott, E. M., I. W. Duncan, and V. Ekstrand. 1963. Purification and properties of glutathione reductase of human erythrocytes. J. Biol. Chem. 238:3928-3933. Staal, G. E. J., P. W. Helleman, J. DeWael, and C. Veeger. 1969. Purification and properties of glutathione reductase of human erythrocytes. Biochim. Biophys. Acta 185:63-69. Staal, G. E. J., and C. Veeger. 1969b. The reaction mechanism of glutathione reductase from human erythrocytes. Biochim. Biophys. Acta 185:49-62. Suzuki, J., and C. H. Werkman. 1960. Glutathione reductase of thiobacillus thio-oxidans. Biochem. J. 74:359-362. Van Heyningen, R., and A. Pirie. 1953. Reduction of glutathione coupled with oxidative decarboxylation of malate in cattle lens. Biochem. J. 53:436-444. Williams-Ashman, H. G. 1953. Studies on the Ehrlich ascites tumor. II. Oxidation of hexose phosphates. Cancer Res. 13:721-725. Woodin, T. S., and I. H. Segel. 1968. Isolation and characterization of glutathione reductase from Penicillium chrysogenum. Biochim. Biophys. Acta 167:64-77.

Purification and properties of the glutathione reductase of Chromatium vinosum.

Vol. 123, No. 1 Printed in U.S.A. JOURNAL OF BACTERIOLOGY, JUlY 1975, p. 203-211 Copyright 0 1975 American Society for Microbiology Purification and...
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