Eur. J. Biochem. 98, 487-499 (1979)

Mouse-Liver Glutathione Reductase Purification, Kinetics, and Regulation Juan LOPEZ-BAREA and Chi-Yu LEE Laboratory of Environmental Mutagenesis, National Institute of 1:nvironmental Health Sciences, Research Triangle Park, North Carolina (Received September 29, 1978/March 26, 1979)

Glutathione reductase from the liver of DBA/2J mice was purified to homogeneity by means of ammonium sulfate fractionation and two subsequent affinity chromatography steps using 8-(6-aminohexyl)-amino-2’-phospho-adenosinediphosphoribose and N6-(6-aminohexyl)-adenosine 2‘, 5’-bisphosphate-Sepharose columns. A facile procedure for the synthesis of 8-(6-aminohexyl)amino-2’-phospho-adenosine diphosphoribose is also presented. The purified enzyme exhibits a specific activity of 158 U/mg and an A280/A460 of 6.8. It was shown to be a dimer of M , 105000 with a Stokes radius of 4.18 nm and an isoelectric point of 6.46. Amino acid composition revealed some similarity between the mouse and the human enzyme. Antibodies against mouse glutathione reductase were raised in rabbits and exhibited high specificity. The catalytic properties of mouse liver glutathione reductase have been studied under a variety of experimental conditions. As with the same enzyme from other sources, the kinetic data are consistent with a ‘branched’ mechanism. The enzyme was stabilized against thermal inactivation at 80°C by GSSG and less markedly by NADP’ and GSH, but not by NADPH or FAD. Incubation of mouse glutathione reductase in the presence of NADPH or NADH, but not NADP’ or NAD’, produced an almost complete inactivation. The inactivation by NADPH was time, pH and concentration dependent. Oxidized glutathione protected the enzyme against inactivation, which could also be reversed by GSSG or other electron acceptors. The enzyme remained in the inactive state even after eliminating the excess NADPH. The inactive enzyme showed the same molecular weight as the active glutathione reductase. The spectral properties of the inactive enzyme have also been studied. It is proposed that auto-inactivation of glutathione reductase by NADPH and the protection as well as reactivation by GSSG play in vivo an important regulatory role.

Glutathione is a thiol-containing tripeptide which, in its reduced state, participates in several functions of vital importance to the cell. It maintains in the proper redox state the thiol groups of soluble and structural proteins, and participates in the detoxification of hydroxyperoxides and a-oxoaldehydes [ 11. Reduced glutathione also detoxifies a variety of potentially harmful electrophilic compounds which are later excreted as mercapturic acids [2]. Glutathione is reduced from its oxidized disulfide form by glutathione Abbreviation. br’NADP’, C-8 bromo-substituted analog of NADP’. Enzymes. Ferredoxine-NADP’ reductase (EC 1.6.7.1); fructose 1,6-bisphosphatase (EC 3.1.3.1 1); glucose-6-phosphate dehydrogenase (EC 1.1.1.49); glutathione reductase (EC 1.6.4.2); glycogen synthase (EC 2.4.1.11); hexokinase (EC 2.7.1.1); nitrate reductase (EC 1.6.6.1); nitrite reductase (EC 1.6.6.4); 6-phosphogluconate dehydrogenase (EC 1.1.1.44).

reductase, which employs NADPH generated by the hexose monophosphate pathway [3]. The properties of glutathione reductase have been recently reviewed [4]. The enzyme from yeast and human erythrocytes has been most extensively studied. It has also been purified from sea urchin eggs [5] and rat liver [6,7]. In the mouse two electrophoretic alleles have been described for the gene encoding for glutathione reductase, GR-1A and GR-1B [S]. This then provides an animal model system for the study of the biochemical differences between the enzyme variants. Although a ping-pong mechanism was first postulated for glutathione reductase [9,10], the competitive inhibition by NADP’ and the properties of the reverse reaction led to the formulation of a ‘branched’ mechanism in which the enzyme could function by both the ping-pong and the sequential patterns, depending on the GSSG concentration [7, l l - 141. Sep-

488

arate binding sites for GSSG and NADPH have been demonstrated by an X-ray crystallographic study of human erythrocyte glutathione reductase [15]. The nitrate, nitrite and NADP' reductases from bacteria, fungus and higher plants are inactivated upon reduction by their own electron donors. The inactive enzymes can then be reactivated by oxidation [16- 181. Several previous reports seemed to indicate that glutathione reductase could have similar properties [6,10,14]. The role of glutathione and glutathione reductase in the hexose monophosphate pathway [19 - 211, gluconeogenesis [22] and glycogen biosynthesis [23] prompted us to investigate a possible regulatory mechanism for glutathione reductase. This paper reports the purification of glutathione reductase from strain DBA/2J of mouse (allele GR-1A) using a new affinity chromatography procedure, its molecular characterization and kinetic properties. The inactivation of this enzyme by reduced coenzymes has been investigated in detail in an attempt to understand its possible regulatory role in vivo. MATERIALS AND METHODS

Mouse-Liver Glutathione Reductase

Beckman spectrophotometer (model 25) at a constant temperature of 25 "C. The reaction mixture contained in a final volume of 1 ml, 100 mM potassium phosphate buffer pH 7.0,2.5 mM GSSG, 0.2 mM NADPH and 0.01 -0.05 ml of the enzyme solution. One unit of enzyme activity is defined as the amount of enzyme which reduces one micromole of GSSG per minute under the described conditions. Specific activities are expressed as units of enzyme per milligram of protein. For the kinetic characterization of glutathione reductase, 0.1 M potassium phosphate buffer pH 7.0 was used to dissolve the enzyme, substrates and inhibitors in all the assays. NADPH concentrations were standardized by absorbance at 340 nm and that of GSSG by the decrease in absorbance at 340nm in the presence of excess NADPH and glutathione reductase. All the assays were initiated by the addition of NADPH. Protein Determination

The protein concentration was estimated by the biuret-phenol method [24] using bovine serum albumin as standard.

Chemicals

Liquid bromine, 2-mercaptoethanol, Dowex-1x8 resin (200 - 400 mesh), blue dextran, bovine serum albumin, lactic dehydrogenase, aldolase, ovalbumin, a-chymotrypsinogen A, ribonuclease A, and myoglobin were purchased from Sigma Chemical Co. Oxidized glutathione, cytochrome c, and Freund's complete adjuvant were purchased from Calbiochem. NADPH and NADP' were obtained from Boehringer. Urea, dodecylsulfate and HCl were purchased from Pierce. Acrylamide, bisacrylamide and bromophenol blue were obtained from Bio-Rad. Dithiothreitol was obtained from GIBCO, Coomassie blue G-250 from Serva, and the ampholines were purchased from LKB. Cyanogen-bromide-activated Sepharose and N6-(6aminohexy1)-adenosine 2', 5'- bisphosphate-Sepharose were obtained from Pharmacia Fine Chemicals. 1,6diaminohexane was purchased from Aldrich. All other chemicals purchased were of the highest purity and used without further purification. Animals

DBA/2J male mice, eight weeks old, were purchased from Jackson Laboratories. The animals were sacrificed by cervical dislocation, and the livers were removed and frozen at - 70 "C for use later in enzyme purification. Enzyme Assays

The glutathione reductase activity was followed by the decrease in absorbance at 340nm using a

Glutathione Reductase

The pure enzyme was stored at - 70°C at a concentration of 0.25 mg/ml in 0.1 M potassium phosphate buffer p H 7.0, containing 1 mM EDTA, 1 mM dithiothreitol, 20 pM FAD and 5 mM NADP'. Prior to any inactivation experiment the endogenous NADP' and FAD were removed by gel filtration in a Sephadex G-25 column (1.6 x 58 cm) equilibrated with 0.2 M Tris-HC1 buffer, pH 8.0. Preparation of 8- (6-aminohexyl)-amino-2'-phosphoadenosine diphosphoribose-Sepharose

The synthesis was carried out according to the following procedure. NADP' (1 g, 1.2 mmol) was dissolved in 10 ml of 1 M sodium acetate buffer pH 4.5. Liquid bromine (0.3 ml, 5.4 mmol) was then added dropwise to the rapidly stirring solution and the pH was maintained at 4.5 with 1 M NaOH. After 30 min of reaction at room temperature the unreacted bromine was extracted 3-times with 10 ml of carbon tetrachloride. 40ml of cold acetone was added to the aqueous phase which contained the br*NADP+ and then stored overnight at - 70°C [25]. The yellow precipitate was washed with 40 ml of cold acetone and redissolved in 5 ml of water. To the solution of br8NADP+ was added 1,6-diaminohexane (3 g, 25 mmol) and the solution was heated at 60°C. The progress of the coupling reaction was monitored spectrophotometrically and after 3.5 h the absorbance maximum shifted from 263 to 278 nm and the A278/

489

J. Lopez-Barea and C.-Y. Lee

I

I

-

A

1M NaOAc pH 4.5, 30 min I

NADP'

@

bra NADP'

@

Sepharose 4B CNBr

8 8-(6-Aminohexyl)-amino-2'-phosphoadenosine diphosphoribose - Sepharose

8 -( 6 -Aminohexyl) -amino -2'-phospho adenosine diphosphoribose

-

ac-H 0

II

+

OH

2-Hydroxynicotinaldehyde

Fig. 1. Scheme f o r the synthesis of 8-(6-aminohexyl)-amino-2'-phospho-adenosinediphosphoribose and gel bound ligand. R, represents ribose and P, phosphate

,4263 changed from 0.74 to 1.1. The resulting reaction mixture contained 8-(6-aminohexyl)-amino-2'-phospho-adenosine diphosphoribose and presumably 2-hydroxy-nicotinaldehyde [26]. The reaction mixture was diluted 100-fold to 1000 ml and loaded into a Dowex-1x8 column (260 ml bed) which had been washed with 1 M ammonium carbonate and equilibrated with water. The column was then washed with 5 vol. of water and the elution was made with a 0.1 M ammonium carbonate gradient (1 1 each). The fractions with A,,, at 278 nm were pooled and lyophilized [271. Thin-layer chromatography was performed to follow the synthesis using Eastman silica gel plates with a fluorescent indicator. A solvent system containing 0.1 M phosphate pH 6.8/ ammonium sulfate/propan-1-01 (100/60/2) was used. The chromatograms were visualized by ultraviolet light. Ninhydrin was applied to monitor the extent of reaction. The RF values under these conditions were: NADP+, 0.56; 8-(6-aminohexyl)-amino-2'-phospho-adenosine diphosphoribose, 0.46; and br'NADP',

0.14. The molar absorption coefficient of br8NADP+ and 8 - (6 - aminohexyl) - amino - 2'- phospho - adenosine diphosphoribose were 20700 and 18000 at 263 and 278 nm respectively. The overall yield of the synthesis was 65%. Covalent attachment of the 8-(6-aminohexyl)amino-2'-phospho-adenosinediphosphoribose to Sepharose was achieved by the cyanogen bromide method [28]. Fig. 1 presents a general scheme for the synthesis of this affinity gel. Sucrose Density Gradient Centrifugation

The sedimentation coefficient was determined according to the method of Martin and Ames [29], using a SW 50.1 rotor at 49000 rev./min for 16 h at 4°C in a Beckman ultracentrifuge (model L2 65B). Linear gradients 5 - 20 % in sucrose were prepared in 100 mM potassium phosphate buffer pH 7.0, 1 M KCl, 1 mM EDTA, 1 mM dithiothreitol and 20 pM FAD. 50 pg of pure glutathione reductase were applied in 0.1-ml

Mouse-Liver Glutathione Reductase

490

samples. Rabbit muscle lactic dehydrogenase with a sedimentation coefficient of 7.2 S [30] was used as a marker. Gel Filtration

The Stokes radius of the native enzyme was determined following the procedure of Siege1 and Monty [31] using a column of Sephadex G-200 superfine (1.6 x 76 cm) equilibrated with 100 mM potassium phosphate buffer pH 7.0, 1 mM EDTA, 1 mM dithiothreitol and 20 pM FAD. Blue dextran, rabbit muscle aldolase (4.74 nm Stokes radius), bovine serum albumin (3.70 nm), ovalbumin (2.76 nm), a-chymotrypsinogen A (2.26 nm) and ribonuclease A (1.80 nm) were employed as standards. Polyacrylamide Gel Electrophoresis

Electrophoresis under denaturing conditions was performed according to the method of MacGuilliwray [32]. A separating gel consisting of 15% acrylamide, pH 8.9, and a stacking gel of 2.5 % acrylamide, pH 6.7 was used. Both gels contained 4 M urea and 0.1 % sodium dodecylsulfate. Bovine serum albumin (MI 67 000), ovalbumin ( M ,45 000), a-chymotrypsinogen A (MI 25 700), myoglobin ( M , 17200) and cytochrome c (13370) were used as markers. The samples were dialyzed overnight against 10 mM sodium phosphate buffer pH 6.8, 8 M urea, 1 % sodium dodecylsulfate and 1 % 2-mercaptoethanol, and boiled for 1 min prior to electrophoresis. The gels were run at 5 mA/ gel for 3 h, fixed overnight in 50% methanol/lO% acetic acid, and stained using the Coomassie blue G-250/perchloric acid method [33]. Analytical polyacrylamide disc gel electrophoresis was performed by the method of Davis [34] using 7.5% acrylamide in 0.037 M Tris-HC1 buffer pH 8.9 for the separating gel and 1.25 % acrylamide in 0.03 M phosphate buffer pH 6.9 for the stacking gel. The reservoir buffer was 0.04 M Tris-glycine pH 8.3. The gels were run at 2 mA/gel with 0.01 % bromophenol blue as a tracking dye. Protein was stained as above [33]. The gels were soaked in the enzymatic reaction mixture without GSSG for 1 h at 4 °C followed by the addition of 0.1 ml of 0.125 M GSSG. The zymograms were monitored by the disappearance of NADPH fluorescence under ultraviolet light.

Isoelectric Focusing

Isoelectric focusing of glutathione reductase was carried out in an LKB 8101 isoelectric focusing column (1 10 ml) using 2 % ampholines of pH 3.5- 10 at 800 V for 64 h. Fractions of 1.O ml were collected for enzyme assay and pH measurement.

Absorption Spectrum

Endogenous NADP' and FAD were removed from 80 U of pure glutathione reductase by gel filtration in a column of Sephadex G-25 ( 1 . 6 ~ 5 8cm) equilibrated with 0.2 M potassium phosphate buffer pH 7.0 and 1 mM EDTA. The absorption spectrum of the peak fraction with known protein concentration was recorded (Fig. 3). The spectrum of the oxidized and aerobically reduced glutathione reductase was recorded using an Aminco spectrophotometer (model DW-2a) using 0.02 A as full scale (Fig. 9). Amino Acid Analysis

250 pg of glutathione reductase were extensively dialysed against 0.1 M ammonium hydroxide and lyophilized. They were then dissolved in 2 ml of 6 M HC1 containing 0.02 % 2-mercaptoethanol and divided in two samples which were then sealed under vacuum and incubated for 24 and 48 h at 1 1 0 T . After the reaction, the hydrolysates were dried in a dessicator under vacuum in the presence of NaOH. The samples were then dissolved in 5 ml of 0.2 M sodium citrate buffer pH 3.25, mixed thoroughly, and centrifuged for 5 min at 12000 x g. 1-ml samples were then analyzed in a Jeolco JLC-6AH analyzer according to the method of Moore and Stein [35]. 8 nmol of amino acid was required for the full-scale deflection in the maximum sensitivity range. Tryptophan was determined spectrophotometrically after dissolving the protein in 6 M guanidine hydrochloride [36]. Immunological Procedures

Antibodies against glutathione reductase were prepared according to Harboe and Ingild [37]. Two New Zealand white rabbits were innoculated subcutaneously on days 1, 14 and 28 with 40 pg each of pure protein emulsified with Freund's complete adjuvant in a total volume of 2.7 ml. On days 42, 56 and 70 the protein was emulsified with incomplete adjuvant. Blood was taken from the marginal ear vein prior to the first injection and on days 49, 63 and 77. The blood was allowed to clot at room temperature for 30 min and then carefully rimmed. After two more hours at room temperature it was incubated overnight at 4°C. The serum was removed and centrifuged at 5000 x g for 20 min. The sera were kept frozen at - 20 "C in the presence of 0.01 % sodium azide. Immunoprecipitation was performed by the method of Ouchterlony [38] in 0.046 M potassium phosphate buffer pH 7.4, containing 0.15 M NaCl, 0.01 % sodium azide and 1 % agar noble. To estimate the antibody titer, 1 pg of pure enzyme was diluted to a final volume of 0.12 ml with the control serum and serial amounts of different immune sera. A control was prepared

49 1

J. Lopez-Barea and C.-Y. Lee

with the buffer instead of immune serum. The mixtures were incubated at 37°C for 1 h and kept overnight at 4°C. They were then centrifuged for 4 min at 3000 x g and the supernatant was assayed for residual enzyme activity. Thermal Inactivation 6 pg of glutathione reductase were dissolved in 0.05 M potassium phosphate buffer pH 7.0, containing known concentrations of substrates and nucleotides in a total volume of 100 p1. The tubes were then sealed with parafilm, heated at 80 "C for 10 min and immediately cooled down at 0 "C. The remaining enzymatic activity was assayed in duplicate 10-pl samples. Inactivation of Glutathione Reductase under Reducing Conditions Unless otherwise indicated, glutathione reductase was inactivated by incubation in 0.05 M Tris-HC1 buffer pH 8.0, in the presence of 0.3 mM reduced pyridine nucleotide for 3 h at 37 "C. All the inactivations were performed aerobically. RESULTS Purification of Glutathione Reductase In order to simplify the experimental conditions all the operations were performed at 4°C. All the buffers used were 0.02 M potassium phosphate pH 7.0, 1 mM EDTA, 1 mM dithiothreitol and 20 pM FAD [39], unless otherwise specified. Centrifugations at 25 000 x g for 30 min were employed whenever necessary. Preparation of Crude Extract. 300 g of liver were thawed and homogenized with a total of 1100 ml of buffer in six batches, for 6 min each, with a Polytron homogenizer (Brinkman Instruments). The homogenate was centrifuged and the supernatant was retained as the crude extract after carefully removing the overlaying lipids. Ammonium Suvate Fractionation. The crude extract was brought to 40 % saturation. After 30 min of continuous stirring the precipitate was removed by centrifugation. The resulting supernatant was brought to 80 % saturation, stirred and centrifuged as described above. The pellet was resuspended in 150 ml of the buffer and dialyzed for 18 h against 30 1 of the buffer with two changes. 8- (6-Aminohexyl)-amino-2'-phospho-adenosine Diphosphoribose-Sepharose Chromatography. The dialysate was cleared by centrifugation and loaded on a column of affinity gel (3.2 x 22.5 cm) equilibrated with extraction buffer. The column was washed with 20column volumes of the same buffer. A linear

gradient of 0-0.5 mM NADP' (800 ml each) in the same buffer was used to elute the enzyme which had been completely retained by the affinity gel. Glutathione reductase was eluted in a single sharp peak immediately after a dark-colored protein. Several other peaks of proteins free of glutathione reductase activity were also noticeable. The fractions with activity were pooled, concentrated by ultrafiltration, and dialyzed overnight against 2 1 of the same buffer. N6-(6-Aminohexyl)-adenosine 2 ',5 BisphosphateSepharose Chromatography. The dialysate was loaded on a column of the second affinity gel (1.3 x 14 cm) equilibrated with 0.05 M potassium phosphate buffer pH 7.0, 1 mM EDTA, 1 mM dithiothreitol and 20 pM FAD, and washed with the same buffer. The first peak eluted after the void volume contained all the darkcolored proteins which were coeluted with glutathione reductase in the previous step. On the contrary, the glutathione reductase was completely retained in this column. After washing with 0.1 M potassium phosphate buffer pH 7.0, containing 1 mM EDTA, 1 mM dithiothreitol and 20 pM FAD with 15 column volumes, the enzyme was eluted with a linear gradient of 0- 10 mM NADP' in the same buffer (100 ml each). Two subsequent peaks of protein were eluted and glutathione reductase activity was found in the second peak with high NADP' concentration (> 7 mM). At this stage the enzyme was homogenous as indicated by gel electrophoresis in both -nondenaturing (Fig. 2 D) and denaturing conditions. Both Fig.2 and Table 1 show the progress of the purification and point out the importance of both affinity chromatography treatments. The 8-(6-aminohexy1)-amino-2'- phospho-adenosine diphosphoriboseSepharose column produces a 35-fold purification, and the N6-(6-aminohexyl)-adenosine 2',5'-bisphosphate-Sepharose products an additional 22-fold to final purity. As presented in Table 1, about 5 mg of pure enzyme was obtained from 300 g of mouse liver after 4700-fold purification and 66 % recovery. The purification, which can be performed in 7 days, yields an enzyme with a specific activity of 158 U per mg of protein. In the presence of NADP' and FAD it can be stored frozen at - 70 "C for 8 months without loss of activity. The activity after ammonium sulfate precipitation and subsequent dialysis was consistently higher than the total activity in the crude extract. The most extreme case was a 217% recovery after ammonium sulfate fractionation and 241 % recovery after 8-(6-aminohexyl)-amino-2'-phospho-adenosine diphosphoribose-Sepharose chromatography obtained in one particular preparation. I-

Molecular Size and Isoelectric Point The sedimentation coefficient of the pure glutathione reductase was estimated to be 6.13 k 0.06 S

492

Mouse-Liver Glutathione Reductase

activity with a p l of 6.46 determinations.

in three independent measurements by sucrose density gradient centrifugation. No aggregates with higher S value were found even at low ionic strength, in the absence of thiols, or in the presence of 0.1 mM NADPH. The Stokes radius has been determined to be 4.18 nm. From this data and the sedimentation coefficient a native molecular weight of 105000 and a frictional ratio of 1.34 have been determined. The subunit molecular weight has been estimated to be 55 000, thus, the mouse glutathione reductase can be viewed as a dimer with a M , of 105000. After column preparative isoelectric focusing the pure glutathione reductase shows a single peak of

A

B

C

0.36 in seven independent

Amino Acid Composition The amino acid composition of mouse glutathione reductase is presented in Table 2. The number of residues have been calculated based on a subunit molecular weight of 52 500.

Spectral Properties The absorption spectrum of glutathione reductase is shown in Fig. 3. The protein has absorption peaks at 213, 365 and 460 nm with absorbance ratios A2731 Table 2. Amino acid composition of mouse glutathione reductase The results are calculated as relative molar ratios (residues/52000-Mr subunit) with respect to the total yield of all amino acids except threonine, serine, methionine, isoleucine, tyrosine, half-cysteine and tryptophan. n.d., not determined

D

Occurrence in glutathione reductase

Amino acid

-

~~

found

nearest integer

residues/subunit ___ ___

41.23 35.26" 32.00" 43.50 24.36 49.91 41.38 47.63 9.54 23.81 36.48 10.56 19.33 32.04 13.70 13.82 4.27" n.d.

Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Lysine Histidine Arginine Tryptophan Half-cysteine

Fig. 2. Polyacrylumide gel electrophoresis U I diJkrent stages of purification. (A) 1 mg of protein from crude extract. (B) 2 mg of protein after ammonium sulfate fractionation. (C) 150 pg of protein after 8-(6-aminohexyl)-amino-2'-phospho-adenosine diphosphoriboseSepharose and (D) 40 pg of enzyme after Nb-(6-aminohexy1)adenosine 2',5'-bisphosphate-Sepharose

a

~~~

41 35 32 43 24 50 41 48 10 24 36 11 19 32 14 14 4 n.d.

Determined by extrapolation to zero time. 48-h result. Determined spectrophotometrically.

Table 1. Purification of mouse glutathione reductase Purification step

Crude extract 40- 80 % (NH4)zS04 8-(6-Aminohexyl)-amino-2'-phospho-adenosine diphosphoribose-Sepharose N6-(6-Aminohexyl)-adenosine2', 5'-bisphosphate-Sepharose

Volume

Total activity

Total protein

Specific activity

Purification

ml

U

mg

Uimg

-fold

1020 275

1246 1495

36946 6783

0.034 0.220

1 6

100 120

98

1077

155

6.972

207

86

19

815

158.357

4695

66

5.1

Recovery

%

493

J. Lopez-Barea and C.-Y. Lee

A460, A28c/A46cand A365/A460 of 8.17, 6.81 and 1.17 respectively. The absorbance at 273 nm of a protein solution of 1 mg/ml has been determined to be 1.418. Immunological Properties

A single identical immunoprecipitation line was obtained when both pure glutathione reductase and a concentrated crude extract were tested against immune serum. No precipitation lines were produced against control serum. The antibody did not crossreact with yeast glutathione reductase. 1 pg of glutathione reductase was precipitated by 1.15 pl of the first serum, however, only 0.56 p1 of the second serum and 0.49 p1 of the third were required to produce the same precipitation. Kinetic Studies

The activity of glutathione reductase was assayed with NADPH or NADH as coenzyme in 0.1 M suc-

cinate, phosphate and Tris-HC1 buffers with pH ranging from 5.0 to 8.5. With NADPH as coenzyme glutathione reductase showed a pH optimum of 7.25 with a specific activity of 158 Ujmg, whereas with NADH it exhibited a pH optimum of 6.0 with a specific activity of only 19 U/mg. Steady-state kinetic studies of mouse glutathione reductase were performed with various concentration of NADPH and GSSG in the assay mixture. The results are presented in Fig.4. A series of parallel lines can be obtained in the plot of Ijv versus l/[NADPH]. A strong inhibition by high NADPH concentrations was found, especially at low GSSG concentrations (Fig. 4A). Similarly, parallel lines were also obtained in the plot of l / v versus l/[GSSG] (Fig.4B). It is again noticeable that at high NADPH and low GSSG concentrations a deviation from the general pattern of parallel lines was obtained. A V of 8295 pmol x min-' x pmol-' of flavin was determined. K, values for NADPH and GSSG were determined to be 5.9 and 107 pM respectively. An investigation of the product inhibition by NADP' was carried out to further probe the reaction mechanism of the mouse glutathione reductase. A linear competitive inhibition pattern of NADP' with respect to NADPH was observed. An inhibition constant of 45 pM was calculated for NADP'. Very high concentrations of GSH ( > 5 mM) were required to produce inhibition of the enzyme activity, thus making quantitation difficult. The inhibition of GSH versus GSSG seemed, however, to be non-competitive. Heat Stability

400 500 Wavelength (nm)

300

Fig. 3. Absorption spectrum o~'puri~edg1utathione reductase. Protein concentration, 193 pg/ml

Mouse glutathione reductase was stable at 37 "C for at least 8 h. Heat treatment at 75 "C for 10 min also failed to produce any appreciable inactivation.

70t

I 0

50 100 l / [ N A D P H I (mM-')

150

[NADPH] (JLM

B

+a

i

lo0 0

1

2

3

4

5

6

7

8

9

1

0

l/[GSSG] (mM-1)

Fig. 4. Effect of diJferent NADPH and GSSG concentrations on the steady-state kinetics of mouse glutathione reductase. The experimental conditions were as indicated in Materials and Methods. Enzyme 0.28 pg/assay. u, is expressed as pmol/min

Mouse-Liver Glutathione Reductase

494 GSSG

120

/

/

100

I

.2"

-

80

.-> ."

Table 3. Inactivation of mouse glutathione reductase under reducing conditions 2.5 pg of ghtdthione reductase were preincubated in the presence of 0.3 mM of each pyridine nucleotide, in a total volume of 200 pl, as described in Materials and Methods. The activity was then determined in 40-p1 aliquots. 100 "/,activity corresponds to 1.2 U/ml System of preincubation

Activity

Control NADPH NADH NADP' NAD'

100 5 6 102 72

w

"/, control

c

60 C m

.C ._

E"

$ 40

20

0 0

l 0

"

"

"

"

1

00

000 [GSSG]or [NADP'] or [GSH] (&M)

Fig. 5. Effect of different concentrations of substrates on the heat inrictivation of mouse glutathione reductase. (u 0-0, and V V ) Represent GSSG, NADP' and GSH concentrations respectively. 100 % activity represents 8.7 U/ml

GSSG, NADP', GSH and dichloroindophenol (but not NADPH or FAD) protected the enzyme against thermal inactivation at 80 "C. Fig. 5 shows the protective effect of different concentrations of GSSG, GSH and NADP'. It is noticeable that 50% protection was achieved at GSSG concentrations similar to its K,, while with NADP+ a much higher concentration than its Ki was required to obtain a similar protective effect. GSH seemed to provide little protection for the enzyme. The lack of protection by FAD could indicate that the enzyme was already saturated with FAD due to its presence along the purification procedure. It is interesting to note that NADPH, with a high affinity for the enzyme ( K , = 5.9 pM), not only failed to protect but inactivated the enzyme far beyond the heat-treated controls. Inactivation of Glutathione Reductase under Reducing Conditions

The effect of oxidized and reduced pyridine nucleotides on the glutathione reductase is shown in Table 3. Both reduced coenzymes NADPH and NADH, produced a dramatic inactivation of the enzyme at a concentration of 0.3 mM, while the oxidized pyridine nucleotides had essentially no effect on the enzyme. Fig. 6 presents the time course of glutathione reductase inactivation by NADPH at pH 7.0 and 8.0. NADPH modification [40] was also monitored by following the spectral changes of the coenzyme during the incubation period. The rate of glutathione reductase inactivation increased sharply with pH, the half life of the enzyme diminished from 14 min at pH 7.0

Time (rnin)

Fig. 6. Time course of mouse glutathione reductase inactivation by NADPH at different p H . Glutathione reductase activity in the presence of 0.3 mM NADPH in 0.1 M phosphate buffer pH 7.0 (-0) or in 0.1 M Tris-HCl buffer, pH 8.0 ( W B ) . Controls and t - 0 respecwithout NADPH at pH 7.0 and 8.0 (0-0 tively). Absorbance at 340 nm at pH 7.0 and 8.0 (A----A and A----A respectively). Each preincubation mixture contained 9 pg of enzyme in a final volume of 800 pl. 100% activity corresponds to 1.75 Uiml

to 4 min at pH 8.0 in the presence of NADPH. However, the stability of NADPH increased with pH; only 9.6% of the initial NADPH was converted to NADPH-X at the end of the incubation period at pH 8.0 (28 pM). In the absence of reduced coenzyme, glutathione reductase remained fully active at either pH. Inactivation of glutathione reductase was found to depend on NADPH concentration. 1.75 pg of enzyme in 200 pl of final volume (80 nM) was maximally inactivated by 7.5 pM NADPH, when incubated at 37 "C for 3 h. 50 % inactivation was produced at 1 pM NADPH, a concentration which is only sixfold higher than the subunit concentration of the enzyme. It should be mentioned that the apparent affinity of the glutathione reductase for the coenzyme in the

495

J. Lopez-Barea and C.-Y. Lee Table 4. Protection by dijferent substrates and cofactors ofglutathione reductase inactivation Mouse glutathione reductase, 1.2 pg, was preincubated for 7 h at 0 "C in 0.05 M Tris-HC1 buffer, pH 8.0 and 0.3 mM NADPH plus 1.0 mM substrates or cofactors when added, in a total volume of 100 pl. The activity was then determined in duplicate 40-pl samples. 100% activity corresponds to 2.1 U/ml. 2',5'-ADP, adenosine 2',5'-bisphosphate System of preincubation

Table 5. Effect of different oxidized compounds on the reactivation of glutathione reductuse Mouse glutathione reductase, 13 pg, was preincubated for 7 h at 0 "C in 0.05 M Tris-HCI buffer, pH 8.0 and 0.3 mM NADPH in a total volume of 1 ml. Then 2 mM of oxidized compounds were added, and after 8 h at 37°C the activities were determined in duplicate 4O-pl samples. A control was prepared without NADPH. 100% activity represent 2.1 U/ml. The activities are corrected for dilution

Activity System of preincubation

Activity

Control NADPH NADPH NADPH NADPH NADPH

100 3 9 65 48 34

% control Control NADPH NADPH NADPH NADPH

+ GSSG + GSH + 2',5'-ADP

100 9 81 19 23

inactivation process is higher than during the catalytic reaction ( K , = 5.9 pM). No appreciable changes in the extent of inactivation were observed with a longer incubation time, even at the lowest NADPH concentrations.

"/, control

I

100 e

I

+ NADP+ + GSSG + ferricyanide + dichloroindophenol

OCIC-.-*

+ = = F - L - - - o

Protection and Reversion of Glutathione Reductase Inactivation The protective effect of different substrates and cofactors against the inactivation of glutathione reductase by NADPH was studied. The results are presented in Table 4. Oxidized glutathione (1 mM) effectively prevented the inactivation, while reduced glutathione at the same concentration had only a slight protective effect. The effect of 2',5'-adenosine bisphosphate (the adenosine moiety of NADPH) could be due due to competition with the reduced coenzyme for the binding site. The inactivation of glutathione reductase by reduction and the protection by its oxidized substrate led us to investigate the effect of oxidized electron acceptors on the inactive enzyme. Table 5 presents the results of an experiment in which the enzyme inactivated by NADPH was incubated for 8 h at 37 "C in the presence of NADP', GSSG, ferricyanide and dichloroindophenol. Although the control with NADPH remained fully inactive, the enzyme partially recovered its activity after the addition of the oxidized compounds dichloroindophenol, ferricyanide and most extensively the oxidized substrate GSSG. NADP' failed to produce such an extensive reactivation even at the high concentration employed. Fig.7 shows the time course of protection and reactivation by oxidized glutathione. GSSG (2 mM) fully protected glutathione reductase against inactivation when present in the incubation mixture prior to the addition of NADPH. GSSG not only protected the enzyme against inactivation, but also reactivated in a short period of time the enzyme which had been

vO

30

60

90 120 Time (min)

150

180

Fig.1. Time course of protection and reactivation of mouse glutathione reductase inactivated by NADPH. 9 pg of enzyme were preor in incubated in 800 pl of final volume in the absence (+O) as) indicated in Materials and the presence of NADPH (H contained ) 2 mM Methods. The closed circle experiment (M GSSG prior to NADPH addition. The arrow indicates the addition of GSSG to an aliquot of the inactivated enzyme at a final concentration of 2 mM. The activities have been corrected for the dilution. 100% activity corresponds to 1.75 U/ml

inactivated by NADPH. 70% of the initial activity was recovered after 3 h. Properties of the Inactive Enzyme It became interesting at this point to know if the enzyme would remain inactive once inactivated, even in the absence of NADPH, or the continuous presence of the reduced coenzyme was necessary. As it is shown in Table 6 glutathione reductase remained inactive even after removal of the excess NADPH by gel filtration on a Sephadex G-25 column. Addition of GSSG was still necessary in order to reactivate the

Mouse-Liver Glutathione Reductase

496 Table 6. EfJect of dialysis on the glutathione reductase inactivated by NADPH 18 pg of protein was preincubated for 16 h at 0°C in 0.2 M TrisHCI buffer pH 8.0, in the presence or the absence of 0.3 mM NADPH. Both samples were then dialyzed through a Sephadex G-25 column equilibrated with the same buffer. One aliquot of the inactive enzyme was then incubated for 12 h at 37°C in the presence of 2 mM GSSG. 1000/, activity correspond to 2.9 U enzyme -~

Experiment

Treatment

Total activity

Control

none dialysis

100 114

Inactivated

none dialysis dialysis

2 4 70

"/,

+ GSSG

m 0

c .r 1.0 -0 c

c

0.8

0.6

enzyme. A permanent modification of glutathione reductase was thus the result of the inactivation by reduction. The molecular weight of the inactive enzyme was compared with that of the active glutathione reductase (Fig. 8). 50 pg of active glutathione reductase (top) or inactivated enzyme with 15 remaining activity (bottom) were filtered through a Sephacryl S-200 column (exclusion molecular weight 250 000). After the elution profile of the inactive enzyme was analyzed (shaded line), the fractions were supplemented with 2 mM GSSG and incubated at 37 "C. The elution process was again studied after 1, 4 and 7 h of reactivation. The successive profiles coincided with the initial profile of the inactive enzyme and with the profile of the active enzyme, ruling out the possibility of an aggregation upon inactivation. In order to study the spectral changes associated with the inactivation of glutathione reductase by NADPH, the spectra of an enzyme solution of 90 pg/ml were recorded at 25°C in the initial oxidized state and sequentially after NADPH addition. Aliquots of the incubated enzyme solution were drawn from the cuvette at different time intervals to follow the enzyme inactivation by activity assays. Although the inactivation required some time (half life = 20 min), the glutathione reductase spectrum shifted instantaneously to that of the two electron reduced state, EH2 [4], and remained unchanged throughout the inactivation process. The oxidized and half-reduced spectra in aerobic conditions are shown in Fig. 9. DISCUSSION Purification of glutathione reductase by conventional procedures was often complicated by the fact that several other proteins have similar charge and size

0.4

a2 0

20

25

30

35

40

45

50

Fraction number

Fig. 8. Molecular weight of the active and inuctivated glutathione reductase. Elution from a Sephacryl S-200 column (1.2 x 50 cm) equilibrated with 0.2 M Tris-HC1 buffer pH 8.0, of 50 pg of active glutathione reductase ( e - 0 ) . Elution pattern of 50 pg of inactivated enzyme immediately after elution (* -*) and ( a - - - O , A----A and @---0) after 1, 4 and 7 h of reactivation respectively in the presence of 2 mM GSSG. The arrows indicate the elution volume of cytochrome c and the void volume 0.014 I

"

/'

---___ ..__ -.__

'

--._._

400

4500,\0,5 Wavelength (nrn)

600

656

Fig. 9. Absorption spectra of mouse glutathione reductase. The spectra of an enzyme solution of 90 pg/ml were recorded (--) prior and (----) sequentially after aerobic addition of NADPH at a final concentration of 0.1 M

[5-7,10,41-441. The introduction of N6-(6-aminohexy1)-adenosine 2',5'-bisphosphate-Sepharose as a new gel for general ligand affinity chromatography [45] made possible a fast and more effective approach to the purification of glutathione reductase. However, this did not solve the problem due to the copurification of several NADP+-linked dehydrogenases with similar

497

J. Lopez-Barea and C.-Y. Lee

affinities for that gel [46]. Thus, another type of gel with a different affinity for the NADP' dehydrogenases would be advantageous. 8-(6-aminohexyl)-amino-2'-phospho-adenosinediphosphoribose-Sepharose is a NADP+ derivative in which the nicotinamide has been removed from the coenzyme due to the basic treatment during the coupling of br8NADP' with 1,6-diamine hexane [26]. The synthesis is relatively simple and has a high yield (65 %) (Fig. 1). This new affinity gel could be very useful in the purification of NADP'-dependent enzymes. In this study, mouse glutathione reductase was purified to homogeneity by a new procedure which combined both affinity column steps (Table 1 and Fig. 2). Although both gels are structurally related to NADP', they show different affinities for the NADP+linked dehydrogenases, thus making it possible to purify mouse glutathione reductase to homogeneity (Fig. 2). The new procedure in fast (7 days) and allows a high recovery (66%). A maximum specific activity of I58 U/mg has been found for the mouse enzyme (Table 1). This is close to that reported for the rat enzyme, 207 U/mg [7] and for the human enzyme, 165 U/mg [43] or 240 U/mg [44]. The differences could be due to different methods of protein determination. The absorption spectrum of the pure mouse glutathione reductase (Fig. 5 ) confirms that the enzyme is indeed a flavoprotein. The ratio A280/A460 of 6.8 is similar to those described for the human enzyme, 9 [43] and 6.7 [44]. Although the ratio for A365/A460 is higher than that reported for other flavoproteins [4], it is probably not due to contaminating proteins in our purified enzyme sample. Several biochemical and immunological evidences as well as A280/A460 clearly demonstrated the high purity of the prepared enzyme (Tables 1 and 2, and Fig. 2). Since the purification was carried out in the presence of FAD, higher activities were recovered during the initial stages of purification than in the crude extract (Table 1). This is probably due to the presence of some apoenzyme in the homogenate [47]. Mouse glutathione reductase has a molecular weight and subunit composition similar to those previously reported for the enzymes from other sources [4]. No aggregates corresponding to higher molecular weight were detected either by sucrose-density gradients or by gel filtration, even under those conditions favoring the aggregation of the human enzyme [14,48]. Four bands of protein with enzymatic activity have been described upon submitting the pure human glutathione reductase to thin layer polyacrylamide gel electrofocusing [49]. However, we found a single peak after column preparative isoelectric focusing of the mouse enzyme. The amino acid composition of mouse glutathione reductase (Table 2) appeared more similar to that of

the human enzyme than that from yeast or Eschevichiu coli [48]. It seems, however, that the mouse enzyme has a lower methionine content and higher amount of proline and phenylalanine than the human enzyme. The kinetic behavior of mouse glutathione reductase (Fig. 4) is consistent with the branched mechanisms previously proposed for the same enzyme from yeast [12,13], rat [7] and human erythrocytes [ l l , 141. The data presented in Fig. 4 indicate a strong substrate inhibition by NADPH, especially at low GSSG concentrations. The same phenomenon has been previously described in human [lo, 111 and rat [7] glutathione reductases. A linear competitive inhibition of NADP' with respect to NADPH has been found at 2 mM GSSG; a hyperbolic competitive inhibition was, however, found for the yeast [12,13] and rat enzymes [7], but only at low NADPH and GSSG concentrations. Similar to the human enzyme [44], mouse glutathione reductase is very stable at high temperature. The mouse enzyme is inactivated within 10 min at 80 "C; this inactivation is efficiently protected by GSSG, probably due to interaction with its binding site, but less markedly by NADP' and GSH (Fig. 5). The effect of NADPH on the heat inactivation of mouse glutathione reductase is in good agreement with a similar phenomenon reported for the human enzyme [lo]. Mouse glutathione reductase is inactivated when preincubated with NADPH or even NADH (Table 3). The enzyme can be protected by GSSG (Table 4) or reactivated by GSSG and other oxidizing compounds (Table 5 and Fig. 7). It could be argued that substrate inhibition by NAD(P)H or their decomposed products may account for the origin of inactivation. At neutral pH, NADH is converted by acid catalysis to NADH-X, which has no absorption at 340 nm but exhibits an increase in the region of 280300 nm [50]. The rate of conversion is accelerated by phosphate buffers. The same transformation occurs with NADPH [40] and NADPH-X has been demonstrated to inhibit several NADP+-linked dehydrogenases [40,49]. The inactivation of mouse glutathione reductase by NADPH cannot be explained, however, on the basis of inhibition by NADPH-X. First, a faster rate of inactivation was observed at pH 8.0 in Tris-HC1, at which NADPH is much more stable, than at pH 7.0 in phosphate buffer (Fig.4, cf. also [40]). Second, the conditions of incubation and assay of glutathione reductase activity would only introduce into the assay mixture a maximum of 1.1 pM NADPH-X (at pH 8.0) which is considerably lower than its Ki, 22 pM [49]. Neither can the inactivation of mouse glutathione reductase by NADPH be attributed either to substrate inhibition by NADPH. In fact, inhibition by excess NADPH should be instantaneous, while glutaI

498

thione reductase inactivation is time dependent (Fig. 6 and 7). The higher apparent affinity for NADPH during the inactivation process also seems to rule out the substrate inhibition explanation. The modification of glutathione reductase upon inactivation seems to be a permanent change of the enzyme itself, since no excess reduced coenzyme is necessary to maintain the inactive state (Table 6). Enzyme inactivation does not parallel the spectral changes observed upon aerobic NADPH addition (Fig, 9). Indeed, the two-electron reduced spectrum (considered to take part in the catalytic cycle, cf. [4]) appears immediately after NADPH is added, while the enzyme is still fully active, and remains unchanged through the timedependent inactivation process. The reactivation of glutathione reductase by GSSG (Table 5 and Fig. 7) and the enhanced inactivation at higher pH could suggest that the inactivation is associated with some other modification, such as an alteration of the disulfide present in the active site [4,51]. There have been some reports regarding the regulation of the glutathione reductase. This activity was shown to decrease significantly during cell division of Ehrlich ascites-tumor cells [52]and sea urchin eggs [53] and increase towards the subsequent mitosis. Inhibition by a lipoprotein was postulated to be the regulatory mechanism in sea urchin eggs [53,54]. Human hemolysate glutathione reductase was profoundly 'inhibited' during incubation with 6-phosphogluconate, although this compound did not directly inhibit the enzyme [20]. Moreover, patients with glucose-6-phosphate dehydrogenase deficiency showed a 24% increase in their red cell glutathione reductase which remained then unexplained [55]. On the other hand, the effect of NADPH on glutathione reductase activity has been previously described. k e n [lo] reported that NADPH enhanced the inactivation of the enzyme by heat, urea and thiol reagents. Worthington and Rosemeyer [14] have described a 50% decrease in activity upon 5 min preincubation of human erythrocyte glutathione reductase with 1 mM NADPH. The inactivation was directly protected by GSH and indirectly by GSSG, through its reduction to GSH. The authors found aggregation upon ultracentrifugation in the presence of NADPH (at a protein concentration of 5 mg/ml) and correlated the inactivation with intermolecular association through the thiols produced in the active site by NADPH. An intramolecular modification of the active site disulfide was alternatively suggested [14]. However, no aggregates have been found upon sucrose density gradient centrifugation of mouse glutathione reductase in the presence of NADPH. In addition, the molecular weight of the mouse enzyme which had been inactivated by NADPH was identical to that of the active enzyme (Fig.8). We have also found that GSSG protects the enzyme much more

Mouse-Liver Glutathione Reductase

effectively than GSH against inactivation (Table 4), and that GSSG can also reactivate the enzyme (Fig. 7). This seems to restrain the alteration produced by inactivation to the individual glutathione reductase molecule, possibly affecting the disulfide localized in its active site. Further investigation will be necessary to probe this hypothesis. Several other reductases have also been shown to be inactivated by reduction and reactivated by oxidation, both in vivo and in vitro [16- 181. The assimilatory nitrate reductase from bacteria, algae and higher plants was inactivated by its coenzyme, NADH, and protected by its oxidized substrate, NO;. The inactivated enzyme could be immediately reactivated by oxidation with ferricyanide [161. The pyridine-nucleotide-dependent nitrite reductase from bacteria [16] and fungus [17], and the ferredoxin-NADP' reductase from blue-green algae [181 also behaved similarly. The ratio of oxidized to reduced glutathione was found to have effect on several enzymes. This was the case of the red cell hexose monophosphate pathway [19] probably due to the fact that glutathione reductase regenerates NADP', the coenzyme of glucose6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase [56]. Alkaline rabbit liver fructose 1,6-bisphosphatase was activated by covalent modification of specific thiol groups, and the activation was reversed by GSH [22]. A similar disulfide exchange mechanism has been implicated in the regulation of the hexokinase [57]. Finally, rat liver glycogen synthase was, in contrast, inactivated by GSSG and reactivated by GSH [23]. The results presented in this paper indicate that the activity of glutathione reductase could be adjusted to the physiological needs of the cell. In the absence of oxidized glutathione the intracellular NADPH (40 - 50 pM) [56,58] would inactivate glutathione reductase, consequently diminishing the hexose monophosphate pathway metabolism. On the contrary, the inefficiency of the hexose monophosphate pathway such as that produced by glucose-6-phosphate dehydrogenase deficiency would result in higher glutathione reductase activities due to a lower (NADPH)/ (NADP') ratio. The presence of oxidized glutathione would protect against this inactivation. We have found that mouse glutathione reductase retained 70 % of its activity upon preincubation with 50 yM NADPH in the presence of 50 yM GSSG and 1 mM GSH; conditions which mimic the physiological GSSG/GSH ratio [59,60]. However, a lower concentration of GSSG than NADPH would produce a more pronounced inactivation. In the absence of an active glutathione reductase the concentration of GSSG would steadily build up at the expense of GSH, and the enzyme could be reactived as it has been shown in vitro (Fig. 7).

J. Lopez-Barea and C.-Y. Lee We thank Dr R. DiAugustine for his invaluable help with the amino acid analysis. We also thank C. 0. Shore and J. Lorrain for their technical help, and Dr L. Lazarus, Dr R. M. Philpot and D. Bronson for their editorial review of the manuscript.

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J. Lopez-Barea, Departamento de Bioquimica, Facultad de Cieneias, Universidad de Extremadura, Badajoz, Spain

C.-Y. Lee, Laboratory of Environmental Mutagenesis, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina, U.S.A. 27709