Rabbit liver glutathione reductase. Purification and properties

ARCHIVES

OF BIOCHEMISTRY

AND

BIOPHYSICS

Vol. 198, No. 1, November, pp. 241-246, 19’79

Rabbit Liver Glutathione

Reductase. GIULIANA

Laboratory

of Biochemistry,

University

Purification

and Properties]

ZANETTI of

Milano,

Via

Celoria

2, 201.93 Milano,

Italy

Received May 8, 19’79; revised July 23, 1979 Hepatic glutathione reductase can be obtained in relatively good amounts from rabbit by a procedure which is fairly simple and sufficiently rapid. The purified flavoprotein shows absorbance ratios at 274,379, and 463 nm of 8.2:0.92:1.0, respectively; the FAD fluorescence is nearly completely quenched by the protein. Gradient ultracentrifugation and sodium dodecyl sulfate gel electrophoresis indicate that the enzyme is a dimer, consisting of subunits of about 56,000 molecular weight; flavin content suggests one FAD per chain. Gel filtration under a variety of conditions, on the other hand, yields a molecular weight in the range 56,000-6’7,000. It is proposed that rabbit liver glutathione reductase can be active also as monomer. Kinetic parameters ofthe enzyme have been determined under optimal conditions. The rabbit liver glutathione reductase is, at physiological pH, absolutely specific for NADPH.

The ubiquitous enzyme glutathione reductase (reduced NAD(P):oxidized glutathione oxidoreductase, EC 1.6.4.2.) belongs to the pyridine nucleotide-disulfide oxidoreductase group of flavoproteins (1). Its metabolic role and importance reflects that of its product, namely GSH,2 since the reaction is practically irreversible under most physiological conditions. Glutathione reductase has been purified to homogeneity from several sources (2-6). In spite of the remarkable role of glutathione metabolism in liver, few workers (7- 12) have been concerned with the hepatic enzyme and only recently has it been completely purified from rat liver (4). In the present paper we describe an isolation procedure to obtain homogeneous glutathione reductase from rabbit liver in relatively good amounts. In addition we determined several properties of the purified protein. ’ The research project was supported in part by grants from the Consiglio Nazionale delle Ricerche of Italy. 2 Abbreviations used: GSH, glutathione; GSSG, oxidized glutathione; p-HMB, p-hydroxymercuribenzoate; SDS, sodium dodecyl sulfate. 241

EXPERIMENTAL

PROCEDURES

Materials NADPH, GSSG, GSH, FAD, p-HMB, and all the protein markers were from Sigma. Affi-Gel blue and hydroxylapatite were obtained from Bio-Rad and Sephadex G-200 and G-100 from Pharmacia.

Methods Protein determination. Protein concentration was determined by the biuret method (13) up to the third purification step; thereafter it was calculated from the absorbance at 280 nm (14) or estimated by the method of Lowry et al. (15). Enzyme assay. The activity was measured at 25°C following the decrease in absorbance at 340 nm in l-ml final volume mixture containing 50 mM potassium phosphate, pH 7, 1 mM EDTA, 0.1 mM NADPH, and 0.5 mM GSSG. The oxidation of 1 pmol of NADPHimin under these conditions is used as 1 unit of glutathione reductase activity. Estimation of molecular weight. Sodium dodecyl sulfate gel electrophoresis was carried out either on slabs or on rods according to Weber and Osborn (16). Gel filtration was performed on Sephadex G-200 or G-100 column (2 x 56 cm; 2.2 x 95 cm) under different medium conditions. The G-200 column was equilibrated in 0.1 M potassium phosphate, pH 6.8, containing 1 mM EDTA (25 mM P-mercaptoethanol); the G-100 column was in 50 mM Tris-HCI, pH 7.5, containing 50 mM NaCl. Density gradient centrifugation was carried out

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242

GUILIANA

ZANETTI

according to Martin and Ames (17). One-tenth milliliter of the protein mixture was layered on top of the preformed glycerol linear gradient (5-20% v/v) in 50 mM potassium phosphate, pH 7, containing 0.1 mM EDTA. Centrifugation was run at 1°C in the 3 x 6 ml swingout bucket rotor at 40,000 rpm for 15 h. Fluorescence measurements were made in a PerkinElmer spectrofluorimeter Model MPF 2A and absorption spectra in a Cary Model 118 spectrophotometer. RESULTS

Purifiation Red&use

Step 3: (NH,)$O, fractionation. The fraction precipitated between 45 and 75% of ammonium sulfate saturation was collected. The precipitate was redissolved in a minimum volume of 10 mM buffer and dialyzed extensively against the same medium. These three steps were repeated on another lot of livers and the dialyzed fractions combined. Step 4: P-cellulose chromatography. The dialyzed solution, clarified by centrifugation, was applied to a Whatman Pll phosphocellulose column (28 x 3.4 cm) previously equilibrated with 10 mM buffer. The column was then washed with 1.5-2 vol of the same buffer. Elution was effected by a linear gradient of potassium phosphate, 10 to 280 mM, pH 6.8 (total volume, 800 ml), containing 1 InM EDTA. The best fractions were pooled and dialyzed extensively against 0.05 M buffer. Step 5: Affi-Gel blue chromatography. The solution, after centrifugation, was chromatographed on a column (15 x 1.4 cm) of Affi-Gel blue, which had been previously equilibrated with 0.05 M buffer. Following 1.5-2 vol of washing, elution was performed with 45 ml of 1 mM NADPH (dissolved in 0.05 M potassium phosphate, pH 6.8). The column was left to equilibrate with the nucleotide solution for 30 min, prior to collecting fractions. Immediately after activity determination, the fractions were diluted with an equal volume of cold distilled water, pooled, and adsorbed on a hydroxylapatite

AND DISCUSSION

Scheme of Glutathione

Unless otherwise stated, all operations were performed at 0-4°C and the buffer used was potassium phosphate, pH 6.8, containing 1 mM EDTA. Step 1: Homogenate. Livers were obtained from Red Burgundy rabbits (average weight 2 kg) and usually were stored frozen at -20°C. Four livers (about 300 g) were cut into small pieces and washed carefully with cold distilled water. After adding 3 vol of 50 InM buffer containing 6 PM FAD, the livers were homogenized in a Vir Tis blender. The homogenate was centrifuged at 20,OOOg for 30 min and the pellets were discarded. Step 2: Heat treatment. The supernatant was rapidly brought to 60°C using a large thermostatic bath held at 72°C. After 5 min at 6O”C, the solution was immediately cooled to 4°C and centrifuged as described above. TABLE

I

PURIFICATIONSCHEME OFGLUTATHIONE REDUCTASE FROM RABBIT LIVER

Fraction Homogenate Heat treatment Ammonium sulfate fractionation Two fractions combined Phosphocellulose chromatography Af6-gel blue chromatography G-200 gel filtration

Volume (ml)

Total protein b-47)

Total activity (units)

700 590

17,500 5,220

1,050 987

0.06 0.19

1 3.2

100 94

78 185

1,616 3,150

808 1,575

0.50 0.50

8.3 8.3

77 75

Specific activity (units/mg)

Purification factor

Recovery vd

60

72.8

1,092

15

250

52

10 3

6.5 3.6

819 650

126 180

2,100 3,000

39 31

RABBIT

LIVER

GLUTATHIONE

column (4.5 x 1.4 em) which was equilibrated with 0.01 M potassium phosphate buffer, pH 6.8. After washing, the enzyme was eluted with 0.25 M potassium phosphate buffer, pH 6.8, in a narrow peak. This step is necessary to free the enzyme from the NADPH to avoid inactivation and it results in a concentration of the enzyme solution. Step 6: Sephadex G-2OOgelfiltration. The fractions were pooled and if necessary concentrated by ultrafiltration with a Millipore molecular separator (or on a Diaflo PM 10 membrane) before gel filtration on a column of Sephadex G-200 (2.5 x 95 cm), equilibrated with 0.1 M buffer. The best fractions were collected and concentrated by a Millipore molecular separator. The experimental data of the purification procedure are summarized in Table I: The most powerful steps are the phosphocellulose and the affinity columns. The enzyme thus obtained, has a specific activity of 180 prnol NADPHemin-‘Vmg protein determined by the method of Lowry et al. Using the extinction coefficient at 280 nm (E$$ = 18.6 (2)) the value of specific activity would be 253, thus comparing fairly well with the value of 250 obtained for the gluA

4

REDUCTASE

Mobility

FIG. 1. SDS-polyacrylamide gel electrophoresis of glutathione reductase from rabbit liver. (A) Electrophoretic pattern in the absence (1) or in the presence of the protein markers (2, 3). (B) Calibration curve for the estimation of the molecular weight. a, bovine serum albumin; b, catalase; c, ovalbumin; d, yeast alcohol dehydrogenase; e, a-chymotrypsinogen A; f, myoglobin.

243

FIG. 2. Visible and ultraviolet spectra of oxidized glutathione reductase from rabbit liver. Spectra were recorded in 0.1 M potassium phosphate (pH 6.81, containing 1 mM EDTA, at 25°C.

tathione reductase from rat liver (4). The enzyme seems to be homogenous, showing only one band on SDS-polyacrylamide gel electrophoresis (Fig. 1A). The total purification was more than 3000-fold with a yield of 31%. In the oxidized form the glutathione reductase from rabbit liver displayed a typical flavoprotein absorption spectrum with peaks at 274, 379, 463 nm, minima at 322 and 407 nm, and a shoulder at 490 nm (Fig. 2). The ratio Az,,:A 463, a characteristic index of purity for flavoproteins, was 7.3. The flavin was released from the protein by boiling the enzyme for 3 min and identified as FAD by measuring its fluorescence before and after adding phosphodiesterase (18). The flavin fluorescence is nearly completely quenched in the enzyme, being only 3-4% of an equimolar solution of FAD. Molecular

Relative

PURIFICATION

Weight Analysis

It was apparent during the last step of the purification scheme (gel filtration on G-200) that rabbit liver glutathione reductase was behaving like a molecule of M, about 67,000. The gel filtration was repeated several times with different enzyme preparations in the presence and absence of 5 mM P-mercaptoethanol; no significant differences in the Ve/Vo values were found. The enzyme at an early stage of purification was also filtered on a calibrated Sephadex G-100 column obtaining a major peak and a shoulder corresponding, respectively, to M, of 56,000 and to M, of 98,000 (Fig. 3). The values of molecular weight obtained by gel filtration are in fair agreement with the value of 56,000 determined by SDS-gel

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FIG. 3. Gel filtration of rabbit liver glutathione reductase. (A) Elution pattern from Sephadex G-100. In the absence of GSSG, NADPH oxidation was completely abolished. (B) Calibration curves obtained on Sephadex G-200 and G-100 respectively. a, -y-globulins; b, bovine serum albumin; c, ovalbumin; d, myoglobin.

electrophoresis (Fig. 1B). However, a determination of molecular weight by ultracentrifugation in a glycerol density gradient (17) gave a value of 108,000. Thus we are lead to the tentative conclusion that the rabbit liver glutathione reductase exist as a dimer as well as an active monomer, depending on the conditions used. Also the erythrocyte enzyme from rabbit has been reported to be a monomer (19) as well as the glutathione reductases from rat liver (10, 12) from Rhodospirillum rubrum (20), from pea seedlings (21), and from spinach leaves (G. Zanetti, unpublished). However, at least for the human erythrocytes enzyme, there is some

PI-I

FIG. 4. pH activity curves of rabbit liver glutathione reductase. The buffers used were: 0.1 M sodium acetate (pH range 4.5-5.8); 40 mM potassium phosphate (pH range 5.8-7.8); 0.1 M Tris-HCl (pH range 7.29.0). The Z was maintained constant = 0.1 M by the addition of KCl. 0 - - - A, NADPH activity; 0 A, NADH activity (NADH was 0.1 mM in the assay).

evidence (22) that elements of the active site are contributed by each subunit. Kinetic

Properties

The influence on the glutathione reductase activity of several parameters of the reaction medium has been studied. Figure 4 describes most of the data obtained. The activity of this enzyme is dependent on the ionic strength of the medium with a maximum at Z = 0.1 M (160% of the control in 10 mM phosphate buffer pH 7.1); nevertheless at Z = 1 M the activity is still 25% of the control. Thus in the experiments of Fig. 4 the activity has been measured at constant Z = 0.1 M; in these conditions the different buffers used did not show any differences. The pH optimum is 6.9, although the activity remains over 90% in a wide range of pH (kO.6 pH unit). In the same figure is reported the activity measured with NADH as the pyridinenucleotide substrate: The maximal activity is only 2.6% of that with NADPH at the same concentration (0.1 mM) and the pH optimum is shifted to much lower value (pH 5-5.5). Thus at pH ‘7, the specificity of the hepatic glutathione reductase for the pyridine nucleotide coenzyme is virtually absolute. The kinetic parameters were determined under the optimal conditions thus far established. The K, for NADPH is 8 PM and the K, for GSSG, 58 Z.&M. The maximal velocity is 357 units *mg protein-’ or 23,000 units * pmol flavin-‘, based on an cd63value of 11.3 mM-’ cm-’ (2). Whereas the Michaelis constants are quite similar to those published

RABBIT TABLE

LIVER

GLUTATHIONE

II

INHIBITION OF RABBIT LIVER GLUTATHIONE REDUCTASE” Activity (%)

Conditions 0.05% SDS 4 M guanidine-HCI 8 M Urea 1.25 PM p-HMB 1.25 pM p-HMB, 1.25 @M p-HMB, 1 mM NADPH 0.25 mM NADPH

0.1 mM NADPH 10 FM GSH

65 21 98 51 0 93 47 75

a The enzyme was preincubated with the effecters for 5 min in 50 mM potassium phosphate buffer, pH 7, containing 1 mM EDTA at 25°C. In the experiments with p-HMB the concentrations reported are those in the assay medium.

for rat liver glutathione reductase (4), the turnover number is substantially higher. Stability of the Putijied Reductase

Glutathione

The enzyme is fairly stable. It has been kept frozen at -2o”C, with several freezethawing cycles, for more than 2 years without loss of activity. Treatment at 75°C for 1 h destroys only 25% of the activity. The effect of several compounds which inhibit the glutathione reductase activity are reported in Table II. The enzyme seems quite stable to the action of urea. It is well known that the glutathione reductases from other sources (1) have a disulfide in the active center which participates in catalysis. The rabbit liver enzyme is no exception since mercurials are powerful inhibitors at very low concentrations if present in the assay medium. A short preincubation with NADPH at the concentration of p-HMB which gives 50% inactivation brings about complete inactivation. This inactivation is reversible by GSH or other sulfhydryl compounds and if the enzyme has not been completely inhibited, the assay solution by itself will restore the enzyme activity. It has been reported (23, 24) that preincubation with NADPH can inhibit the cata-

REDUCTASE

PURIFICATION

245

lytic activity of erythrocyte glutathione reductase. Table II shows that also the hepatic enzyme is inactivated by this treatment, although only at high concentration of NADPH. This inhibition is fairly slow (about 5 min to reach 90% of maximal inactivation); it is concentration dependent and is increased at higher pH. There is no protection or reversal by adding 0.5 mM GSH or 1 mM NADP+. Thus, the mechanism of inhibition seems to differ from that proposed by Worthington and Rosemeyer (24). Whether this inhibition has any physiological implications in the case of liver glutathione reductase should await further studies. REFERENCES 1. WILLIAMS, C. H., JR. (1976) in The Enzymes (Boyer, P. D., ed.), 3rd ed., Vol. 13, pp. 89173, Academic Press, New York. 2. MASSEY, V., AND WILLIAMS, C. H., JR. (1965) J. Biol. Chem. 240, 4470-4480. D. J., AND ROSEMEYER, M. A. 3. WORTHINGTON, (1974) Eur. J. Biochem. 48, 167-177. 4. CARLBERG, I., ANDMANNERVIK, B. (1975)J. Biol. Chm. 250, 5475-5480. 5. PIGIET, V. P., AND CONLEY, R. R. (1977) J. Biol. Chem. 252, 6367-6372. 6. HALLIWELL, B., AND FOYER, C. H. (1978) Plunta 139, 9-17. 7. MANN, P. J. G. (1932) Biochm. J. 26, 785-790. 8. RALL, T. W., AND LEHNINGER, A. L. (1952) J. Bid. Chem. 194, 119-130. 9. MIZE, C. E., AND LANGDON, R. G. (1962) J. Biol. Chem. 237, 1589-1595. 10. MIZE, C. E., THOMPSON, T. E., AND LANGDON, R. G. (1962) J. Biol. Chem. 237, 1596-1600. 11. BUZARD, J. A., AND KOPKO, F. (1963) J. Biol. Chm. 238, 464-468. 12. ONDARZA, R. N., ESCAMILLA, E., GUTIERREZ, J., AND DE LA CHICA, G. (1974) Biochim. Biop&s. Acta 341, 162-171. 13. GORNALL, A. G., BARDAWILL, C. J., AND DAVID, M. M. (1949) J. Biol. Chem. 177, 751-760. 14. KALCKAR, H. M. (1947) J. Biol. Chem. 167, 461475. 15. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J. (1951) J. Biol. Chem. 193, 265-271. 16. WEBER, K., AND OSBORN, M. (1969) J. Bid. Chem. 224, 4406-4412. 17. MARTIN, R. G., AND AMES, R. (1961) J. Biol. Chm. 236, 1372- 1379. 18. BESSEY, 0. A., LOWRY, 0. H., AND LOVE, R. H. (1949) J. Biol. Chem. 180, 755-769.

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19. RAY, L. E., AND PRESCOTT, J. M. (1975) PTOC. Sot. Exp. Bill. Med. 148, 402-409. 20. BOLL, M. (1969) Arch. Mikrobiol. 66, 3’74-389. 21. MAPSON, L. W., AND ISHERWOOD, F. A. (1963) Biochem. J. 86, 173-191. 22. SCHULZ, G. E., SCHIRMER, R. H., SACHSEN-

ZANE’ITI HEIMER, W., AND PAI, E. F. (19’78) Nature (London), 273, 120-124. 23. ICEN, A. L. (1967) Stand. J. Clin. Lab. Invest. Suppl. 96, l-67. 24. WORTHINGTON, D. J., AND ROSEMEYER, M. A. (1976) Eur. J. Biochem. 67, 231-238.