Planta 9 by Springer-Verlag 1978

Planta 139, 9 17 (1978)

Properties and Physiological Function of a Glutathione Reductase Purified from Spinach Leaves by Affinity Chromatography B. Halliwell and C.H. Foyer Department of Biochemistry, University of London King's College, Strand, London WC2R 2LS, U.K.

Abstract. Glutathione reductase (EC 1.6.4.2) was purified from spinach (Spinacia oleracea L.) leaves by affinity chromatography on ADP-Sepharose. The purified enzyme has a specific activity of 246 enzyme units/mg protein and is homogeneous by the criterion of polyacrylamide gel electrophoresis on native and SDS-gels. The enzyme has a molecular weight of 145,000 and consists of two subunits of similar size. The pH optimum of spinach glutathione reductase is 8.5-9.0, which is related to the function it performs in the chloroplast stroma. It is specific for oxidised glutathione (GSSG) but shows a low activity with N A D H as electron donor. The pH optimum for NADH-dependent GSSG reduction is lower than that for NADPH-dependent reduction. The enzyme has a low affinity for reduced glutathione (GSH) and for N A D P +, but GSH-dependent N A D P § reduction is stimulated by addition of dithiothreitol. Spinach glutathione reductase is inhibited on incubation with reagents that react with thiol groups, or with heavymetal ions such as Zn 2 +. GSSG protects the enzyme against inhibition but N A D P H does not. Pre-incubation of the enzyme with N A D P H decreases its activity, so kinetic studies were performed in which the reaction was initiated by adding N A D P H or enzyme. The K m for GSSG was approximately 200 gM and that for N A D P H was about 3 gM. N A D P § inhibited the enzyme, assayed in the direction of GSSG reduction, competitively with respect to N A D P H and noncompetitively with respect to GSSG. In contrast, GSH inhibited non-competitively with respect to both N A D P H and GSSG. Illuminated chloroplasts, or chloroplasts kept in the dark, contain equal activities of glutathione reductase. The kinetic properties of the enzyme (listed above) suggest that G S H / G S S G ratios in chloroplasts will be very high under both

Abbreviations: GSH =reduced form of the tripel~tide glutathione; GSSG=oxidised form of glutathione

light and dark conditions. This prediction was confirmed experimentally. G S H or GSSG play no part in the light-induced activation of chloroplast fructose diphosphatase or NADP+-glyceraldehyde-3 phosphate dehydrogenase. We suggest that GSH helps to stabilise chloroplast enzymes and may also play a role in removing H202. Glucose-6-phosphate dehydrogenase activity may be required in chloroplasts in the dark in order to provide N A D P H for glutathione reductase. Key words: Affinity chromatography - Ascorbic acid - Calvin cycle - Chloroplasts - Glutathione -

Spinacia.

Introduction Work in this laboratory has shown that intact spinach chloroplast fractions (type A in the classification of Hall, 1972) contain glutathione and glutathione reductase activity. Experiments involving washing techniques, controlled disruption and the use of marker enzymes showed that the glutathione reductase activity was not because contamination of the chloroplasts by other organelles, but that the enzyme was located in the stroma (Foyer and Halliwell, 1976). Since then, two other groups have independently reported the presence of glutathione reductase in chloroplast fractions (Schaedle and Bassham, 1977; Wolosiuk and Buchanan, 1977). Intact chloroplasts produce H202 during photosynthesis but have little, if any, catalase activity (for a review see Halliwell, 1978). Foyer and Halliwell (1976) suggested that glutathione and glutathione reductase, together with the high concentrations of ascorbic acid present in chloroplasts, constitute a system for removing H;O2 as shown b e l o w -

10 H202 + ascorbate

B. Halliwell and C.H. Foyer: Properties and Function of Glutathione Reductase , dehydroascorbate + 2 H20

dehydroascorbate + 2 GSH

~ GSSG + ascorbate

GSSG+NADPH+H § g~ymh~o.e,2 GSH+NADP +.

(1) (2) (3)

reductase

This view has recently been s t r e n g t h e n e d by the disc o v e r y o f an e n z y m e that catalyses r e a c t i o n (1) in chloroplasts ( G r o d e n and Beck, 1977). R e a c t i o n (2) proceeds rapidly at the p H o f the s t r o m a in the illuminated c h l o r o p l a s t ( F o y e r and Halliwell, 1976, 1977). F o y e r and Halliwell (1976) also suggested that G S H m i g h t help to stabilise enzymes o f the C a l v i n cycle. The activity o f several enzymes o f the Calvin cycle is increased w h e n c h l o r o p l a s t s are i l l u m i n a t e d , ' and the m e c h a n i s m o f a c t i v a t i o n seems to involve f o r m a tion o f thiol groups within the c h l o r o p l a s t ( A n d e r s o n and A v r o n , 1976; Halliwell, 1978). W o l o s i u k and Buc h a n a n (1977) h a v e c l a i m e d that g l u t a t h i o n e plays a role in such activation, and they have shown that high c o n c e n t r a t i o n s o f oxidised g l u t a t h i o n e ( G S S G ) inhibit c h l o r o p l a s t fructose d i p h o s p h a t a s e . T h e y have p r o p o s e d that in the dark G S H / G S S G ratios in the c h l o r o p l a s t fall a n d deactivate the enzymes, whereas in the light G S H is r e - f o r m e d f r o m G S S G due to the increased availability o f N A D P H for g l u t a t h i o n e reductase. Despite the i m p o r t a n c e o f g l u t a t h i o n e reductase, no studies have as yet been carried out on the p r o p erties o f an e n z y m e purified f r o m leaf tissues. G l u t a t h i o n e reductase has been purified to h o m o g e n e i t y f r o m h u m a n erythrocytes ( W o r t h i n g t o n and Rosem e y e r , 1974) and f r o m yeast ( M a v i s and Stellwagen, 1968). H o w e v e r , the p r o c e d u r e s used were t i m e - c o n s u m i n g and tedious, i n v o l v i n g m a n y different stages o f purification. N A D P + - d e p e n d e n t enzymes can s o m e t i m e s be purified by affinity c h r o m a t o g r a p h y on c o l u m n s o f A D P - S e p h a r o s e (Brodelius et al. 1974) and this t e c h n i q u e was used by M a n n e r v i k et al. (1976) to achieve a partial p u r i f i c a t i o n o f e r y t h r o c y t e g l u t a t h i o n e reductase. In the present paper, we have d e v e l o p e d a p u r i f i c a t i o n t e c h n i q u e based on c h r o m a t o g r a p h y on A D P - S e p h a r o s e that enables p u r i f i c a t i o n o f s p i n a c h - l e a f g l u t a t h i o n e reductase to h o m o g e n e i t y . The p r o p e r t i e s o f this e n z y m e have been e x a m i n e d in relation to its p r o p o s e d m e t a b o l i c functions. Experiments have also been carried out on isolated (type A) spinach c h l o r o p l a s t s in order to test the p r o p o s a l s o f W o l o s i u k a n d B u c h a n a n (1977).

Material and Methods Reagents GSH, GSSG, NADP + and NADPH were of the highest quality available from Boehringer Ltd., London, U.K. ADP-Sepharose

was obtained from Pharmacia Fine Chemicals Ltd., London. Other reagents were of the highest quality available from Sigma Chemical Corp. or from BDH Chemicals Ltd. (both of London, U.K). Spinach leaves were obtained from New Covent Garden Market, London, U.K. or grown in a greenhouse (variety "Resistoflay"). Only mature, undamaged leaves were used. No difference in the properties of glutathione reductase purified from leaves from these two different sources was observed.

Enzyme Purification All operations were carried out at 0 4~ C. Spinach leaves were washed and de-ribbed. Laminar tissue (35 g) was homogenised in a Waring blender in i00 ml of 0.1 M KH2PO4-KOH buffer, pH 7.5, containing EDTA (1 mg/ml). The homogenate was filtered through two layers of muslin and centrifuged at 25,000 g for 15 rain. 35 ml of the resulting supernatant was applied to a column of ADP-Sepharose (bed volume 12.7 ml) that had been reconstituted in 0.1 M KH2PO4- KOH buffer, pH 7 and then equilibrated with 50 mM KHzPO4-KOH buffer, pH 7.5, containing EDTA (0.5 mg/ml). The column was eluted with 200 ml of 50 mM KHaPO4-KOH buffer, pH 7.5, containing 25 mM KC1; a flow rate of 5.0 ml/cmZ/h was used. Then a 2 ml pulse of NADP + (10 mM) dissolved in the above buffer was passed through the column to remove NADP+-dependent enzymes. The eluate so obtained was applied to a column of DEAE-Sephadex (12 x 1.2 cm) equilibrated with 50 mM KH2PO4-KOH buffer, pH 7.5, and was then eluted with 100 ml of this buffer containing 75 mM KC1. This procedure eluted all NADP +-dependent enzymes except for glutathione reductase and glucose-6-phosphate dehydrogenase. The DEAE-Sephadex column was then eluted with buffer containing 100 mM KC1, to remove both enzymes: the glucose-6-phosphate dehydrogenase was eluted just before the glutathione reductase. Those glutathione reductase fractions that were not contaminated with glucose-6-phosphate dehydrogenase were pooled and concentrated by molecular filtration on a Millipore Pellicon membrane, type PTGC, with a cut-off molecular weight of 10,000. The concentrated enzyme was stable for weeks if kept at 0 4 ~ C.

Chloroplast Isolation Type A spinach chloroplasts were prepared as described by Foyer and Halliwell (1976). The pellets were re-suspended gently in homogenising medium. Unless otherwise stated, they were diluted 10 x with buffer and allowed to stand for 30 s to rupture the envelopes (Walker, 1971) immediately before assay of enzyme activities.

Assay of Chloroplast Fructose Diphosphatase Type A chloroplasts were prepared as described above. Immediately before assay of fructose diphosphatase, they were ruptured by re-suspension in 0.1 M Tris-HC1 buffer, pH 8.5. The assay mixture contained, in a total volume of 1 ml, Tris-HC1 buffer pH 8.5 (100 gmol), MgC12 (16 gmoi), EDTA (0.1 gmol), neutralised fructose 1,6-diphosphate (6 gmol), and, where indicated, QSH or GSSG. Reactions were initiated by adding chloroplast extract and carried out at 25~ C. After 30 min 0.5 ml 10% (w/v) trichloroacetic acid was added and the extract centrifuged at top speed in a bench centrifuge for 10 min. The supernatant was assayed for inorganic phosphate by the method of Fiske and Subba Row (1925). The enzyme activity detected was proportional to the amount offchloroplast extract added. Controls without chloroplast extract and without fructose diphosphate were carried out. Neither GSH, nor GSSG, interfered in the phosphate estimation.

B. Halliwell and C.H. Foyer: Properties and Function of Glutathione Reductase

Other Enzyme Assays Glutathione reductase (EC. 1.6.4.2) was assayed at 20 ~ C by the fall in absorbance at 340 nm as N A D P H was oxidised. Reaction mixtures contained, in a final volume of 3.00 mI, the following reagents at the final concentrations stated: EDTA (0.1 raM), GSSG (5.44 mM), N A D P H (0.2 mM) and Tris-HCl buffer, pH 9 (0.1 M). Reactions were usually started by adding enzyme. Activity was not increased by including F A D in the reaction mixture. Corrections were made for any N A D P H oxidation in the absence of GSSG. N A D P + -glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.9) was assayed by the fall in absorbance at 340 nm as N A D P H is oxidised (Bradbeer, 1969). Malic enzyme (EC 1.1.1.40), glucose6-phosphate dehydrogenase (EC 1.1.1.49) and 6-phosphogluconate dehydrogenase (EC 1.1.1.44) were assayed by measuring N A D P H formation at 340 nm (Macrae, 1971 ; Schnarrenberger et al., 1973). NADP+-malate dehydrogenase (EC 1.1.1.82) was assayed by following oxidation of N A D P H (Johnson and Hatch, 1970).

Assay of Thiol Compounds GSH and GSSG were assayed as described by Tietze (1969). After assay, each fraction was internally standardised by addition of a known amount of GSSG, followed by re-assay.

Protein and Chlorophyll Determinations Protein was determined by the Folin method after contaminants had been removed by precipitation with trichloroacetic acid (Halliwell, 1974). Bovine serum albumin, dessicated before use, was employed as a standard. Chlorophyll was determined spectrophotometrically (Arnon, 1949).

Electrophoresis Disc electrophoresis in 7.5% polyacrylamide gels was carried out in glass tubes using a modification of the methods of Ornstein (1964) and Davis (1964). 6.75 ml of 30% acrylamide 0.8% bis acrylamide, 6.25 ml of 0.5 M Tris-HC1 pH 8.8 and 0.03 mI of tetramethyl-ethylenediamine were mixed with 12 ml of water and degassed. Polymerisation was started by adding 0.2 ml 10% (w/v) ammonium persulphate (freshly prepared). The gels were poured and overlaid with water. They polymerised within 20 min. The electrode buffer was 0.2 M Tris-glycine, pH 8.5. The protein to be examined (20-200 gg in 0.1 ml) was mixed with 0.1 ml of 0.1 M Tris-HC1 buffer pH 6.7 containing 0.1% (w/v) bromophenol blue, and a few grains of sucrose were added. 0.1 ml of the mixture (10-100 lag protein) was applied to the gel and electrophoresis carried out for 2.5 h at 3 mA per tube. After electrophoresis, the gels were stained for enzyme activity or for protein. Protein was detected with 0.2% (w/v) Coomassie brilliant blue in methanol-acetic acid-water (20:10:70; v/v/v). The gels were destained either by washing in the same solvent or in solvent in which methanol had been replaced by isopropanol. For electrophoresis of glutathione reductase all solutions contained 1% (v/v) 2-mercaptoethanol to prevent enzyme aggregation. Omission of the thiol caused formation of broad protein bands due to thiol-disulphide interchange (Worthington and Rosemeyer, 1976). Gels used to test the purity of this enzyme were loaded with 10 50 gg of protein. Gels were stained for glutathione reductase activity by the procedure of Kaplan (1968), except that MTT was replaced by

11

nitro-blue tetrazolium. Duplicate gels were stained in the absence of GSSG to detect any bands due to diaphorase activity. Electrophoresis in the presence of sodium dodecyl sulphate was carried out in 12.5% gels are described by Weber and Osborn (1969).

Results

Purification of the Enzyme Affinity chromatography on ADP-Sepharose enabled a rapid and extensive purification of glutathione reductase from spinach-leaf extracts (Table 1). After washing the column with buffer to remove unbound proteins, NADP § enzymes were dislodged by a single pulse of NADP § The glutathione reductase activity eluted as a single symmetrical peak. The most active fractions were subjected to polyacrylamide gel electrophoresis as described in the Materials and Methods section. The gels were stained both for glutathione reductase activity and for protein: only one band of activity, but several protein bands, were obtained. The fractions were also assayed for other NADP+-dependent enzymes: those detected were glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, NADP +-glyceraldehyde-3phosphate dehydrogenase, NADP§ enzyme and NADP*-malate dehydrogenase. Chromatography on DEAE-Sephadex separated all these contaminating enzymes (and NADP +) from the glutathione reductase activity Gel electrophoresis carried out on the glutathione reductase fractions so obtained showed only a single band of protein whose position corresponded exactly to a single band of activity on

Table 1. Purification of glutathione reductase from spinach leaves.

Purification was carried out by the techniques described in the Materials and Methods section Stage of purification

Total enzyme activity gmol NADPH oxidised/ rain

% recovery of enzyme activity

Specific Purifiactivity cation lamol factor NADPH oxidised/ min/mg protein

Crude leaf homogenate

36

100

0.21

1

Supernatant after 25,000 spin

36

100

0.39

1.8

Eluate from ADPSepharose

24

67

62.3

297

Eluate obtained from DEAE-Sephadex by 100 m M KCI wash

15

42

246.0

1171

12

B. Halliwell and C.H. Foyer: Properties and Function of Glutathione Reductase 5.0 a

4.9

t

4.8

9

C

s z+.7 o

-

e

4.6

450

L 0,1

O ,.2 Mobility

0~.3

0 ,.Z-

Fig. 1. Determination of the subunit molecular weight of spinachleaf glutathione reductase by electrophoresis in the presence of sodium dodecyl sulphate. Electrophoresis was carried out on 12.5% polyacrylamide gels are described by Weber and Osborn (1969). The subunit molecular weights were taken from the literature. Proteins: a-E. coli RNA polymerase (sigma chain); b-bovine serum albumin; c-catalase; d~yeast glutathione reductase; e E. coli RNA polymerase (alpha chain). The arrow shows the position of spinach glutathione reductase

Assay of Glutathione Reductase

200

E

o~ ~oo % W

OZ-

(Mavis and Stellwagen, 1968; Carlberg and Mannervik, 1975). The purified leaf enzyme was stable for several weeks at 4 ~ C, but activity was completely lost after heating at 60 ~ C for 10 min. Spinach-leaf glutathione reductase also gave a single protein band on electrophoresis in the presence of sodium dodecyl sulphate. From the position of this band, a subunit molecular weight of approximately 72,000 could be deduced for the enzyme (Fig. 1). The molecular weight of the native enzyme was found to be about 145,000 from its position of elution upon gel filtration through Sephadex G200 (Fig. 2). These results indicate that spinach-leaf glutathione reductase is a dimer with subunits of very similar, if not identical, size. The purification procedure described in Table 1 could easily be adapted to purify any of the other NADP+-dependent enzymes detected. For example, glucose-6-phosphate dehydrogenase eluted from the DEAE-Sephadex column just before glutathione reductase and was obtained with a specific activity of 130 units/rag protein,

I

5 [Oglo (Molecular weight)

t

6

Fig. 2. Determination of the molecular weight of spinach-leaf glutathione reductase by gel filtration. A Sephadex G200 column (49x2.5 cm) was prepared and equilibrated with 50 mM KH2PO,~ - KOH buffer pH 7.5 containing 0.1% (v/v) 2-mercaptbethanol as described by Andrews (1964). The mercaptoethanol is necessary to prevent aggregation of glntathione reductase (Worthington and Rosemeyer, 1976). The arrow shows the position of elution ofglutathione reductase. Proteins: a-cytochrome c; b ovalbumin; c-bovine serum albumin; d-aldolase; e~zatalase; f ferritin

duplicate gels run simultaneously. It thus seems that the enzyme is completely pure. Further chromatography on various columns did not increase the specific activity of the glutathione reductase above 246 units/mg protein: the completely-pure yeast and animal enzymes have specific activities close to this figure

Glutathione reductase activity was usually assayed by following GSSG-dependent oxidation of NADPH. Since the activity of glutathione reductases is considerably affected by the salt concentration in the reaction mixture (Worthington and Rosemeyer, 1976; Moroff and Brandt, 1975) the concentration of the buffer was adjusted to give the maximum reaction rate. In most experiments, NADPH and GSSG were present initially in the assay mixture and the reaction was started by adding enzyme. An alternative procedure, which gave identical results, was to include GSSG and enzyme in the assay mixture and then start the reaction by adding NADPH. However, if the enzyme was mixed with NADPH and the reaction started by adding GSSG, lower activities were usually obtained. A similar inhibition of erythrocyte glutathione reductase by pre-incubation with NADPH has been observed previously (Worthington and Rosemeyer, 1976). It is possible that some earlier studies upon glutathione reductases failed to appreciate the importance of the order of addition of substrates. Figure 3 shows the effect of pH on the activity of spinach-leaf glutathione reductase. The pH optimum in a range of buffers was 8.5-9.0 with considerable activity in the range 7.5-9.5. The lack of activity at pH values below 6.5 was not due to inactivation of the enzyme by acid pH, since an enzyme sample maintained at pH 5.6 for 30 rain retained full activity in a subsequent assay at pH 8.5.

B. Halliwell and C.H. Foyer: Properties and Function of Glutathione Reductase

Table2. inhibition of spinach-leaf glutathione reductase. The enzyme was pre-incubated in buffer at pH 9 for 5rain with the reagents specified. It was then assayed for activity as described in the Materials and Methods section. The GSSG was not contaminated with GSH, since it produced no colour on prolonged incubation with Ellman's reagent (Owens and BeIcher, 1965)

0.35

._c E

0.30

o.2s u~

c~ < z

13

0,20

Reagents present in pre-incubation medium

Enzyme % Loss of activity enzyme ixmol activity NADPH oxidised/min

None N-ethyl maleimide (5 mM) GSSG (5 mM) N-ethylmaleimide (5 mM) + GSSG (5 raM) Iodoacetate (0.5 mM) Iodoacetate (0.5 mM) + G S S G (5 mM) p-Hydroxymercuribenzoate (1 mM) p-Hydroxymercuribenzoate (1 mM) + GSSG (5 mM) ZnSOr (0.2 mM) ZnSO 4 (0.2 m M ) + GSSG (5 mM) ZnSO 4 ( 0 . 3 m M ) + G S S G (5 mM) ZnSO~ ( 0 . 2 m M ) + N A D P H (0.2 mM)

0.44 0 0.44 0.22

0 100 0 50

0 0.24 0 0.21

100 45 100 52

0 0.4I 0.35 0

100 7 20 100

0.15 0.10

E -t 0.05

pH Fig. 3. Effect of pH on the activity of spinach-leaf glutathione reductase. Assays were carried out as described in the Materials and Methods section, using the buffers stated below at a final concentration of 0.1 M. 9 KH2PO4 buffer adjusted to the required pH with K O H ; 9 Tris buffer adjusted to the required pH with HC1; 9 Glycine buffer adjusted to the required pH with K O H

dithiothreitol increased the rate of the reverse reaction, but only to a maximum of 3.3 gmol N A D P H produced/min/mg protein. It thus seems that the physiological function of the enzyme is to reduce GSSG rather than to oxidise GSH, i.e. its equilibrium position greatly favours GSSG reduction.

It was possible to demonstrate the reverse reaction catalysed by glutathione reductase, but very high concentrations of N A D P § and GSH (1 mM and 10 mM respectively) were necessary to allow significant N A D P H formation. As has been reported for the yeast enzyme (Icen 1971), the presence of 10 mM

35

35

30

30

25

25

500

20

200 Rrv/

M

20

b.

!5

0

15 10

5O

0

1.O

2.0

1/[GSSG] (ram-1)

5

0o b

11o

E

2.0

slo

4'.o

i

s.o

I/[NADPH] (laM ~)

Fig. 4a and b. Effect of N A D P + on the activity of spinach-leaf glutathione reductase. Assays were carried out as described in Materials and Methods section, a The N A D P H concentration was 10 gM. GSSG concentration was varied in the presence of the fixed concentrations of N A D P + shown. The graph shows non-competitive inhibition of the enzyme with respect to GSSG. b The GSSG concentration was 1 mM. N A D P H concentration was varied in the presence of the fixed concentrations of N A D P + shown. The graph shows competitive inhibition of the enzyme with respect to N A D P H

14

B. Halliwell and C.H. Foyer: Properties and Function of Glutathione Reductase

24

20 mM GSH

20 16 5 mM

~2

OmM 8

incubation of the enzyme for 5 rain with N-ethylmaleimide, iodoacetate or p-hydroxymercuribenzoate caused complete loss of activity. Inhibition was much less marked if GSSG was also present in the preincubation mixtures, whereas pre-incubation with GSSG alone had no effect on enzyme activity (Table 2). Heavy-metal ions, especially Zn 2+ , were powerful inhibitors of the enzyme. Again, GSSG could protect against inhibition by Zn 2§ (Table 2). N A D P H did not protect glutathione reductase against inhibition by either Zn 2§ or by thiol reagents.

o13 o14 o'.s

o

Kinetics of Glutathione Re&tctase

1/[NADPH] (pM -~) 12 F

] 1

~

~

6 .~..-.-~

20 mM GSH

lOmM 5ram OmM

O ' 1.O ' '210 ' 1/[GSSG] (ram-~)

Fig. 5a and b. Effect of GSH

on the activity of spinach-leaf glutathione reductase. Assays were carried out as described in the Materials and Methods section, a The GSSG concentration was 0.5 mM. NADPH concentration was varied in the presence of the fixed concentrations of GSH shown. The graph shows non-competitive inhibition of the enzyme with respect to NADPH. b The NADPH concentration was 10gM. GSSG concentration was varied in the presence of the fixed concentrations of GSH shown. The graph shows non-competitive inhibition of the enzyme with respect to GSSG

Specificity of Glutathione Reductase The enzyme showed only barely detectable activity when N A D P H was replaced by N A D H : NADH-dependent GSSG reduction was a maximum at pH 8.0. No N A D P H oxidation was observed when GSSG was replaced by L-cystine or lipoic acid, tested at concentrations up to those representing saturation of the reaction mixture with these substrates.

Inhibitors of Glutathione Reductase The spinach enzyme was highly susceptible to inhibition by reagents that react with thiol groups. Pre-

The Kms of glutathione reductase for N A D P H and GSSG were each determined in the present of a saturating concentration of the other substrate. The enzyme obeyed the Michaelis-Menten equation : K m for GSSG was found to be 196_+40 ~tM and Km for NADPH was found to be 2.78_+0.34 ~M (mean_+ S.D. of 5 determinations on different batches of enzyme) by using direct linear plots (Eisenthal and Cornish-Bowden, 1974) and also Lineweaver-Burk plots. As might be expected, the presence of high concentrations of the products GSH and N A D P + inhibited the enzyme when assayed by following GSSG reduction. Figure 4 shows that inhibition by N A D P + was competitive with respect to N A D P H and noncompetitive with respect to GSSG. In contrast inhibition by GSH was noncompetitive with respect to both substrates (Fig. 5).

Measurement of GSH/GSSG Ratios in Chloroplasts Type A spinach chloroplasts were isolated and then illuminated for 10 rain under the conditions listed in the legend to Table 3, or kept in the dark for 10 min. The chloroplasts were then disrupted by re-suspension in ice-cold buffer (0.1 M KHzPO4 plus 5 mM EDTA, adjusted to pH 7.5 with K O H ) and centrifuged at 6000 g for 10 rain. The supernatant was assayed for G S H and GSSG by the sensitive and specific enzymic method described by Tietze (1969). 90% or more of the total glutathione present in chloroplasts was found to be GSH, whether the chloroplasts had been illuminated or kept in the dark before disruption. Samples of GSH or GSSG added to the chloroplasts before disruption were quantitatively recovered in the correct form in the subsequent assay, and so the extraction procedure does not seem to cause interconversion of GSH and GSSG. It may be concluded that G S H / G S S G ratios in chloroplasts are kept high in both light and darkness.

B. HalliwelI and C.H. Foyer: Properties and Function of Glutathione Reductase T:ible3. Activation of enzymes by illumination of type A spinach chloroplasts. Chloroplasts were prepared, and enzymeassays carried out, as described in the Experimental section. The re-suspended chloroplasts were either kept in the dark at room temperature for 10mira or kept in the dark for 8rain, followed by 2min illumination by a 60W incandescent lamp at a distance of 10cm, separated from the chloroplasts by a 3cm water heat-filter. The chloroplasts were then disrupted by mixing with the appropriate buffer and assayed immediately for the enzymes below. Where indicated, thiols at the concentrations stated were included in the chloroplast preqncubation medium. The amount of these thiols carried over into the final assay was not sufficient to affect the enzyme activities measured. An illumination time of 2min was found to give the greatest activation of glyceraldehyde-3-phosphate dehydrogenase, but no change in glutathione reductase activity could be detected using illumination times ranging from 1 to 10 min. Reduced lipoic acid could replace dithiothreitol in activating glyeeraldehyde-3-phosphatedehydrogenase Treatment of chloroplasts before assay

Dark (10 min) Dark (8 min)+light (2 rain) Dark (10 min) with 2.4 mM dithiothreitoI Dark (i0min) with 2.4raM GSH Dark (8 rain) with 2.4mM dithiothreitol + light (2 rain) Dark (8 min) with 2.4raM GSH + light (2 min)

NADP +-glyceraldehyde 3phosphate dehydrogenase I~molNADPH oxidised/min/ml chloroplast suspension

Glutathione reductase gmol NADPH oxidised/ min/ml chloroplast suspension

0.57 0.80 1.23

0.102 0.101

0.58 1.22 0.76

Light Activation of Chloroplast Enzymes In agreement with the results obtained by previous workers (for a review see Halliwell, 1978), illumination of isolated type A spinach chloroplasts caused an increase in the amount of N A D P +-glyceraldehyde3-phosphate dehydrogenase detected on subsequent assay. In contrast, the glutathione reductase activity of the chloroplasts was unaffected by light or dark treatment (Table 3). As expected (Anderson, 1975; Anderson and Avron, 1976) the effect of light in activating N A D P § glyceraldehyde-3-phosphate dehydrogenase could be mimicked by incubating the chloroplasts in the dark with dithiothreitol. Reduced lipoic acid was also found to be effective. However, G S H could not substitute for dithiol compounds in such experiments (Table 3). Light activation could also be demonstrated in disrupted chloroplasts made by re-suspending t h e chloroplast pellet in medium from which sorbitol had

15

been omitted. Again, G S H could not replace dithiol compounds in activating NADP+-glyceraldehyde-3 phosphate dehydrogenase in disrupted chloroplasts kept in the dark. Chloroplast alkaline fructose diphosphatase can be regulated in vitro by reduced ferredoxin and "protein factors" (Buchanan et al., 1971; Wolosiuk and Buchanan, 1977), but the control of enzyme activity by light-induced increases in the p H and concentration of Mg 2 + in the stroma may be of more physiological significance (Kelly et al., 1976; Halliwell, 1978). The effects of G S H and G S S G on fructose diphosphatase activity of chloroplast extracts, assayed at high p H and Mg 2+ ion concentration (see the Experimental section) were therefore examined. Neither G S H nor GSSG, tested at concentrations up to 4 m M (which is the concentration of glutathione present in chloroplasts in v i v o - Foyer a n d Halliwell, 1976) had any effect on enzyme activity.

Discussion The results reported in the present paper have illustrated the great efficiency of affinity chromatography on ADP-Sepharose in the purification of N A D P +dependent enzymes. A 165-fold purification of spinach-leaf glutathione reductase was obtained by this procedure alone, and completely pure enzyme was obtained in one further chromatographic step. This rapid and simple purification procedure may be contrasted with the large number of stages employed by previous workers to obtain the animal enzymes in pure form. Although we have 0nly u s e d a small column of ADP-Sepharose, which provided sufficient enzyme for our needs, the preparation could be easily scaled up to provide much greater quantities of enzyme, if required. Spinach-leaf glutathione reductase has a molecular weight of about 145,000. Although this molecular weight is slightly higher than has been reported for the yeast (Mavis and Stellwagen, 1968) or animal (Carlberg and Mannervik, 1975; Worthington and Rosemeyer, 1976) enzymes, the spinach enzyme resembles the other glutathione reductases in being a dimer with subunits of similar size. When assayed in the forward direction using N A D P H and G S S G as substrates, the spinach enzyme has a p H optimum of 8.5-9.0. In contrast, glutathione reductases from animal tissues show p H optima in the range 7-8 (Massey and Williams, 1965; Carlberg and Mannervik, 1975, Worthington and Rosemeyer, 1976). However, the spinach enzyme is largely located in the stroma of the chloroplast, which has a p H of 8 or more in the illuminated leaf (Halli-

16

B. Halliwell and C.H. Foyer: Properties and Function of Glutathione Rednctase

well, 1978). Hence the enzyme is adapted to the environment in which it works. There was little activity when NADPH was replaced by NADH, and the pH optimum for NADH-dependent GSSG reduction is lower than that for NADPH-linked reduction. A similar observation has been made with glutathione reductases from other organisms (Carlberg and Mannervik, 1975, Worthington and Rosemeyer, 1976). Lipoic acid or cystine cannot replace GSSG as a substrate for the spinach enzyme: this is consistent with the observation that chloroplast extracts in the presence of N A D P H can reduce GSSG but not lipoate or cystine (Halliwell et al., 1977). The enzyme was inhibited by compounds that react with -SH groups and by heavy-metal ions such as Zn 2+ , which may also be reacting with -SH groups. It seems that at least some of the essential -SH groups on the enzyme are located at its active site, since GSSG protected the enzyme considerably against inhibition by Zn 2+ or thiol reagents. The Km of spinach-leaf glutathione reductase for N A D P H is comparable to the values reported for other glutathione reductases, but its Km for GSSG (ca. 200 gM) is greater than has been observed previously, the maximum so far reported being 65 gM (Mapson and Isherwood, 1963 ; Massey and Williams, 1965; Staal et al., 1969; Carlberg and Mannervik, 1975; Worthington and Rosemeyer, 1976). When isolated chloroplasts are transferred to the dark, their internal concentration of N A D P H does not fall to zero, largely because the enzyme glucose-6phosphate dehydrogenase becomes active in the dark (Anderson, 1975). The concentration of NADPH in spinach chloroplasts in the dark has been quoted as 15 nmol/mg chlorophyll (Lendzian and Bassham, 1975). If we assume the osmotic volume of spinach chloroplasts to be 21 gl/mg chlorophyll, an internal concentration of 710 IaM for N A D P H may be calculated, which is more than sufficient to saturate the glutathione reductase. This enzyme, unlike many Calvin cycle enzymes, is equally active in chloroplasts exposed to light or dark (Table 3). Its equilibrium position greatly favours GSSG reduction and high activities of the enzyme are present in chloroplasts (Foyer and Halliwell, 1976). The enzyme will be halfmaximally active at a GSSG concentration of 0.2 mM, which is small in relation to the total glutathione concentration inside chloroplasts (3.5ram Foyer and Halliwell, 1976). Hence one would expect chloroplast GSH/GSSG ratios to remain high in both light and dark, which is in agreement with our experimental results. Hence changes in the ratio of GSH to GSSG cannot be involved in the rapid light-induced activiation of chloroplast enzymes, since the ratio remains con-

stant, and very large, for at least the first 10 rain of darkness. Also, GSH could not replace dithiol compounds in activating NADP+-glyceraldehyde-3phosphate dehydrogenase in chloroplasts and neither GSH nor GSSG affected the activity of alkaline fructose diphosphatase. Wolosiuk and Buchanan (1977) reported that GSSG inhibited fructose diphosphatase assayed in the presence of reduced ferredoxin and "protein factors". However, to achieve a 47% inhibition of enzyme activity they added 25 lamol of GSSG to a 1.5 ml reaction mixture, which gives a final concentration of 16.7 raM. Since the concentration of total glutathione in chloroplasts was reported by them to be 1 mM (compared with 3.5 mM reported by Foyer and Halliwell, 1976) it may be seen that a concentration of 16.7 mM GSSG could never be achieved. In any case, over 90% of chloroplast glutathione is GSH, which has no effect on fructose diphosphatase. It is likely that the function of GSH and glutathione reductase in chloroplasts is to stabilise Calvin cycle enzymes and probably also to help remove H z O 2 and superoxide. It may be argued that the reason why glucose-6-phosphate dehydrogenase activity is enhanced in chloroplasts kept in the dark (Anderson, 1975) is to provided some N A D P H for the activity of glutathione reductase, so maintaining the GSH/ GSSG ratio and preventing inactivation of chloroplast enzymes. C.H.F. thanks the Science Research Council for a Research Studentship. We are grateful to the Rank Prize Funds and to the Central Research Fund of the University of London for financial support.

References Anderson, L.E. : Light modulation of the activity of carbon metabolism enzymes. In: Proceedings of the Third International Congress on Photosynthesis. pp. 1393-1405. Avron, M. ed. Amsterdam: Elsevier 1975 Anderson, L.E., Avron, M : Light modulation of enzyme activity in chloroplasts. Plant Physiol. 57, 209-213 (1976) Andrews, P. : Estimation of the molecular weights of proteins by Sephadex gel-filtration. Biochem. J. 91,222 228 (1964) Arnon, D.I. : Copper enzymes in isolated chloroplasts. Polyphenol oxidase in Beta vulgaris Plant Physiol. 24, 1 15 (1949) Bradbeer, J.W.: The activities of the photosynthetic carbon cycle enzymes of greening bean leaves. New Phytol. 68, 233-245 (1969) Brodelius, P., Larrson, P., Mosbach, K,: The synthesis of three AMP analogues and their application as general ligands in biospecific affinity chromatography. Eur. J. Biochem. 47, 81-89 (1974) Buchanan, B.B., Schurmann, P., Kalberer, P.P.: Ferredoxin-activated fructose diphosphatase of spinach chloroplasts. J. Biol. Chem. 246, 5452-5459 (1971)

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Mannervik, B., Jacobsson, K. Boggaram, V. : Purification of glutathione reductase from erythrocytes by the use of affinity chromatography on T5'ADP-Sepharose 4. EBS Lett. 66, 221 224 (I976) Mapson, L.W., Isherwood, F. : Glutathione reductase from germinated peas Biochem. J. 86, 173-191 (1963) Massey, V., Williams, C.H. : On the reaction mechanism of yeast glutathione reductase. J. Biol. Chem. 240, 4470M480 (1965) Mavis, R.D., Stellwagen, E.: Purification and subunit structure of glutathione reductase from Baker's Yeast. J. Biol. Chem. 243, 809-814 (1968) Moroff, G., Brandt, K.G.: Yeast glutathione reductase: studies of the kinetics and stability of the enzyme as a function of salt concentration. Biochim. Biophys. Acta 410, 21-31 (1975) Ornstein, L.: Disc electrophoresis, I.: Background and theory. Ann. N.Y. Acad. Sci. 121, 321 349 (1964) Owens, C.W.I., Belcher, R.V.: A colorimetric micro-method for thedetermination ofglutathione. Biochem. J. 94, 705 711 (1965) Schaedle, M., Bassham, J.A.: Chloroplast glutathione reductase. Plant Physiol. 59, 1011 1012 (1977) Schnarrenberger, C., Oeser, A., Tolbert, N.E.: Two isoenzymes each of gIucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase in spinach leaves. Arch. Biochem. Biophys 154, 438M48 (1973) Staal, G.E.J., Visser, J., Veeger, C.: The reaction mechanism of glutathione reductase from human erythrocytes. Biochim. Biophys Acta 185, 39-48 (1969) Tietze, F. : Enzymic method for quantitative determination of nanogram amounts of total and oxidised glutathione. Anal. Biochem. 27, 502-522 (I969) Walker, D.A.: Chloroplasts (and grana): aqueous-including high carbon fixation ability. Methods Enzymol. 23A, 2l 1-220 (1971) Weber, K., Osborn, M.: The reliability of molecular weight determination by dodecyl-sulphate-polyacrylamide gel electrophoresis. J. Biol. Chem. 244, 4406 4412 (1969) Wolsiuk, R.A., Buchanan, B.B. : Thioredoxin and glutathione regulate photosynthesis in chloroplasts. Nature 266, 565-567 (1977) Worthington, D.J. Rosemeyer, M.A.: Human glutathione reductase: purification of the crystalline enzyme from erythrocytes. Eur. J. Biochem. 48, 167 177 (1974) Worthington, D.J., Rosemeyer, M.A.: Glutathione reductase from human erythrocytes : Catalytic properties and aggregation. Eur. J. Biochem. 67, 231 238 (1976)

Received 29 July; accepted 14 November 1977