ARCHIVES

OF

BIOCHEMISTRY

AND

BIOPHYSICS

Rat Liver Glutathione

FRED

Department

H. STULTS,

ofFood

Science

(1977)

Peroxidase: Purification Multiple Forms’

JOHN

and

183, 490-497

W. FORSTROM, AL L. TAPPEL

Technology, Received

University April

DANNY

of California,

and Study of

T. Y. CHIU,

AND

Davis,

95616

California

14, 1977

The subcellular distributions of glutathione peroxidase and ‘-‘Se in rat liver were determined. Approximately 75% of the enzyme and 58% of the ‘“Se were contained in the cytosolic fraction. Rat liver cytosol glutathione peroxidase was purified 1029-fold from homogenate to yield a sample with a specific activity of 278 Fmol of NADPH oxidized/ min/mg of protein. The purified enzyme was subjected to disc-gel and sodium dodecyl sulfate-disc-gel electrophoresis, which confirmed the purity of the enzyme as well as the existence of multiple electrophoretic forms. Glutathione peroxidase existed as a large aggregate after homogenization, and means of dissociating the aggregate were investigated. The enzyme was isolated as a neutrally charged protein and became negatively charged upon storage, a phenomenon that was independent of the aggregation.

Glutathione peroxidase (glutathione:hydrogen peroxide oxidoreductase, EC 1.11.1.9) was first reported by Mills (1, 2) to catalyze the breakdown of hydrogen peroxide in bovine erythrocytes using glutathione as the hydrogen donor. Since that time, glutathione peroxidase has been studied in a number of animal tissues, but mostly in the erythrocyte (3, 4). A major breakthrough came when Rotruck et al. (5) discovered that the enzyme cochromatographed with 7Se. Flohe et al. (6) established that bovine erythrocyte glutathione peroxidase is a selenoprotein. The enzyme has been postulated to protect erythrocytes from damage by H,O, (71, and the finding by O’Brien and Little (8) that glutathione peroxidase will reduce lipid hydroperoxides led to the hypothesis that this enzyme may protect tissue against oxidative damage due to lipid peroxidation (9, 10). The ’ This investigation was supported by United States Public Health Service Research Grant AM 06424 from the National Institute of Arthritis, Metabolism, and Digestive Diseases and Grant ES00628-05Al from the National Institute of Environmental Health Sciences, Department of Health, Education, and Welfare and the Environmental Protection Agency.

importance of selenium as a nutrient has been acknowledged for some time, but the actual site of utilization has been a mystery. Glutathione peroxidase appears to be a primary site of selenium action. The liver, being a major site of detoxification and the first target of ingested oxidants, is a very important tissue in the study of the role of glutathione peroxidase in protection from lipid peroxidation. Rat liver glutathione peroxidase has been partially purified by other laboratories (11, 12), but little work has been done on its characterization. This paper will show that 7”Se-labeled selenite injected into rats prior to sacrifice is largely recovered coincident with glutathione peroxidase. In addition, it will be shown that there is more than one form of rat liver cytosolic glutathione peroxidase and that the in vivo form of the enzyme may be a large aggregate. MATERIALS

AND

METHODS

Materials. Sephadex G-100, G-25, and G-150 were obtained from Pharmacia Fine Chemicals; DEAE’agarose gel was from Bio-Rad Laboratories; reduced Z Abbreviations GSH, glutathione;

used: SDS,

DEAE, sodium

diethylaminoethyl; dodecyl sulfate.

490 Copyright All rights

0 1977 by Academic Press, Inc. of reproduction in any form reserved.

ISSN

0003-9861

RAT

LIVER

GLLJTATHIONE

glutathione, glutathione reductase, and NADPH were from Sigma Chemical Co.; cumene hydroperoxide was from Bio-Polymers, Inc.; and H2”Se0,, (404 &i/mg) was from New England Nuclear. Subcellular distribution of “Se and glutathione peroxidase. Two male Sprague-Dawley rats (300 g) were each injected subcutaneously with 50 PCi of ‘“Se-labeled selenite 3 days prior to sacrifice. The rats were decapitated and the livers were removed and homogenized 1:lO (wet weight:volume) in 0.25 M sucrose with a glass and Teflon homogenizer. The subcellular fractions were separated by differential centrifugation in a Spinco Model L preparative ultracentrifuge according to the method of de Duve et al. (13). Purification of glutathione peroxidase. Buffer pH was measured at 2o”C, while most of the purification procedure was done at 4°C. Thirteen male SpragueDawley rats (250 g) were decapitated and the livers were removed and placed in 10 mM potassium phosphate buffer (pH 7.0) isotonic in KCl. Three of the rats had received subcutaneous injections of 50 PCi of ‘“Se-labeled selenite 3 days prior to sacrifice. The livers were minced and homogenized 1:8 in the same buffer for 30 s in a Waring blendor. The homogenate was filtered through cheesecloth and centrifuged at 13,000 g for 15 min and the supernatant was then centrifuged at 27,000g for 30 min. The supernatant portion was made 5 mM in GSH and acidified to pH 5.3 with HCl to precipitate non-glutathione peroxidase protein. After storage at 4°C for 1 h, the sample was centrifuged at 14,500g for 20 min. The supernatant was readjusted to pH 7.0, heated to 50°C for 45 min, and centrifuged at 14,500 g for 20 min. The supernatant was made 50% in acetone at -20°C and centrifuged at 14,500 g for 20 min. The acetone precipitate was redissolved in 5 mM Tris buffer (pH 7.0) and centrifuged at 27,000g for 10 min. The supernatant was passed through a Sephadex G-25 column (2 x 30 cm, in two batches) to remove residual acetone and glutathione. The fractions with glutathione peroxidase activity were pooled and passed through a DEAE-agarose column (2.5 x 40 cm, 5 mM Tris buffer, pH 7.0) in two batches. The enzymatically active fractions were pooled and concentrated using a PM-30 membrane in an Amicon ultrafiltration cell. The concentrated sample was rechromatographed on a DEAE-agarose column (2.5 x 40 cm, 5 mM Tris buffer, pH 7.0) and the enzymatically active fractions were collected, pooled, and concentrated as described above. The enzyme concentrate was then passed through a Sephadex G-100 column (2.5 x 90 cm, 10 mM Tris buffer, pH 7.6) and the enzymatitally active fractions were pooled and concentrated as described above. This concentrated sample was passed through a Sephadex G-25 column (1.5 x 2 cm, 5 mM Tris buffer, pH 7.0) to exchange buffers, and the enzymatically active fractions were pooled and applied to a DEAE-agarose column (1.5 x 40 cm. 5

PEROXIDASE

491

mM Tris buffer, pH 7.01. The enzyme was eluted with a 200-ml linear salt gradient (0.0-0.2 M NaCl in 5 mM Tris buffer, pH 7.0) and was again concentrated as described above. The sample was then passed through a Sephadex G-150 column (1.5 x 90 cm, 10 rnM Tris buffer, pH 7.6). Assay for glutathionc peroxidase. Glutathione peroxidase was assayed by a modification .of the method of Paglia and Valentine (14). A 20-~1 sample was incubated for 5 min at 37°C with 0.25 mM GSH, 0.12 mM NADPH, 1 unit (1 pmol of NADPH oxidizedimin) per milliliter of glutathione reductase. 0.1 mM EDTA, and 50 mM Tris buffer (pH 7.3 at 37°C) in a final volume of 1.65 ml. The assay mixture was made 0.2 mM in cumene hydroperoxide to start the reaction and the absorbance at 340 nm was monitored for the rate of disappearance of NADPH in a thermostated Beckman DB spectrophotometer fitted with a Sargent recorder. The activity of glutathione peroxidase is reported as micromoles of NADPH oxidized per minute. Protein determination. Protein determinations were done by the Miller (15) method using bovine serum albumin as a standard. Elution of proteins from columns was followed by monitoring absorption at 280 nm. Gamma scintillation counting. “Se was detected in a Packard Model 3002 Tri-Carb scintillation spectrometer and Packard Model 3041 flow detector modified with a Bicron LSC-Gamma vial. This instrumentation provided an efficiency of approximately 16%. Electrophoresis. Purified glutathione peroxidase obtained after Sephadex G-150 chromatography was subjected to disc-gel electrophoresis according to the procedures of Ornstein (16) and Davis (17). Samples of 40 and 80 wg were applied to 7.5% gels that were stacked at pH 8.3 and electrophoresed at pH 9.5. SDS-disc-gel electrophoresis was performed according to the method of Wu and Bruening (18). Samples of the enzyme following Sephadex G-150 chromatography were applied to 8% SDS-disc-gels and electrophoresed at pH 9.7. Coomassic blue stain was used to detect protein in the gels, which were scanned at 540 nm in a Gilford Model 230 spectrophotometer fitted with a linear transport. The gels were then frozen, sliced, and counted for YSc radioactivity. An unstained gel was frozen. sliced. and assayed for enzymatic activity. Studies of the glutathionr pwoxidase aggregate,. Samples of freshly prepared rat liver cytosol as well as samples of the enzyme at various stages of purity were chromatographed on a Sephadex G-150 column (1.5 x 90 cm) equilibrated with 10 mM Tris buffer (pH 7.6). The fractions were assayed for enzymatic activity and the approximate molecular weight of protein in each peak of activity was determined. Treatments used to attempt disaggregation of the

492

STULTS

ET

large molecular weight glutathione peroxidase included: 0.2% Triton X-100, 10% propylene glycol, isotonic saline, 5 mM dithioerythritol, 5 mM glutathione (reduced), 10 FM selenite, 30 pg/ml of cumene hydroperoxide, and freeze-thaw four times in dry ice-acetone. Multiple-charge forms of glutathione peroxidase. Samples of rat liver supernatant were precipitated with 50% acetone at -20°C and immediately centrifuged at 27,000g. Portions of the precipitated protein were resolubilized in Tris buffers at various pH values and each was chromatographed on a Sephadex G-25 column (1.5 x 20 cm) and then on a DEAEagarose column (1.5 x 40 cm) with a 200.ml linear salt gradient (0.0-0.2 M NaCl).

20% of the injected the liver tissue. Purification

Distribution and 75Se

of Glutathione

The subcellular distribution of glutathione peroxidase in rat liver is shown in Table I. The cytosolic fraction contained approximately 75% of the glutathione peroxidase and 58% of the ‘“Se. The purity of the subcellular fractions was not determined. We assume that the glutathione peroxidase activity in the nuclear and microsomal fractions was due to cross contamination with mitochondria, which are known to contain glutathione peroxidase. The ratio of radioactivity to enzymatic activity was not unusually different throughout all of the fractions except the microsomes; in this fraction, most of the Y3e did not appear to be associated with glutathione peroxidase. Approximately TABLE SUBCELLULAR Fraction

Total

kin

pro-

(mg)

DISTRIBUTION Total

tein (%I

ity

Homogenate Nuclear Heavy mitochondrial Light mitochondrial Microsomal cytoso1

peroxidase

Total activ(units”)

of Glutathione

Peroxidase

Yield of activitg (%)

PEROXIDASE

Total 9e (cpm x 103

AND INJECTED “Se Yield of Relative ‘“Se “Se (%“r) (cpm)“/units” of activity

Percentage, activity/percentage

kin

3540 532

100 15

539 60

100 11

5800 842

100 15

2.0 2.6

0.73

639

18

96

18

523

9

1.0

1.00

705

20

36

7

464

8

2.3

0.35

503 1041

14 29

9 402

2 75

425 3352

7 58

8.7 1.5

0.14 2.59

a Units = micromoles b Values are normalized

of NADPH to data

oxidized obtained

in

I

OF GLUTATHIONE GSH

pro-

‘“Se was recovered

The data from the sequential purification steps of rat liver glutathione peroxidase are shown in Table II. The acid and heat precipitation steps are beneficial because they precipitate a large amount of non-glutathione peroxidase protein. The acetone precipitation serves to partially purify as well as concentrate the sample for application onto chromatography columns. The first two DEAE-agarose columns were eluted with buffer at pH 7.0, which resulted in retardation of the enzyme by the gel and elution after the major nonbound proiein peak. Fractionation on the Sephadex G-100 column produced two peaks of enzymatic activity: one in the void volume and one later at an elution volume that corresponded to a molecular weight of approximately 80,000 (Fig. 1). This multiple-form phenomenon is an important point that will be discussed later. The lower molecular weight enzymatically active fractions were pooled for further purification. During the third column fractionation on DEAE-agarose the enzyme was bound to the gel and it was eluted only with a salt gradient (Fig. 2). The binding of the enzyme to the third DEAE-agarose column, while it was only retarded on the first two DEAE-agarose columns, suggests an increase in negative charge on the

RESULTS

Subcellular Peroxidase

AL

per minute per milligram for heavy mitochondria.

of protein.

pro-

RAT

LIVER

GLUTATHIONE TABLE

PURIFICATION Fraction

a Units

11

OF GLUTATHIONE PEROXIDASE Total pro- Total activ--R;;w;;y tein (mg) ity (units”) ’

Recovery of activity (9)

Purificat1on (nfold)

Total activity/total “Se

-

-

-

68

85

1.6

0.73 0.92

51

71

2.2

1.01

3,780

34

55

3.5

1.18

950

3,750

22

54

14

1.85

1,000

90

1,580

11

23

65

1.58

21

700

61

1,280

7.4

18.6

78

1.84

67

290

8.7

580

3.1

8.4

246

2.00

49

116

4.4

216

1.2

3.1

180

1.86

278

40

0.28

79

0.4

1.1

Specific activity (uniW/mg of protein)

Total ‘“Se kpm >

0.27 0.44

9,380 6,350

25,500 13,200

6,880 5,870

0.60

4,800

4,800

4,860

0.94

3,200

4,040

3.9

2,030

18

Homogenate 27,000g supernatant Acid (pH 5.3) supernatant Heated (50°C) supernatant Acetone (O-50% resuspended pellet DEAE-agarose I (Nos. 36-50) DEAE-agarose II (Nos. 22-65) Sephadex G-100 (Nos. 37-43) DEAE-agarose III (Nos. 73-81) Sephadex G-150 (Nos. 40-44) = micromoles

493

PEROXIDASE

of NADPH

(0)

10-Y

oxidized

per minute

per milligram

1,029

1.98

of protein.

FRACTION NUMBER

FIG. 1. Purification of rat liver cytosol glutathione peroxidase by chromatography on Sephadex G100. After elution from DEAE-agarose column II, the active fractions were pooled, concentrated, and applied to a column (2.5 x 90 cm) of Sephadex G-100 equilibrated with 10 mM Tris buffer (pH 7.6). The 5ml fractions were assayed for protein (0) by absorbance at 280 nm, enzymatic activity (W) using 10 ~1 of sample, and We (A).

enzyme. It is important to note that this step decreased the enzyme specific activity, even though the fractionation pattern would suggest good purification. Following the DEAE-agarose chromatography,

FIG. 2. Purification of rat liver cytosol glutathione peroxidase by chromatography on DEAE-agarose column III. The pooled and concentrated sample from the Sephadex G-100 column was applied to the column (1.5 x 40 cm) and washed with 150 ml of 5 rnM Tris buffer (pH 7.0). The enzyme was eluted with a ZOO-ml linear salt gradient (5 mM Tris, pH 7.015 mM Tris, 0.2 M NaCl, pH 7.0) from fraction 60120. The 3-ml fractions were assayed for protein (0) by absorbance at 280 nm, enzymatic activity (W using 10 ~1 of sample, and ‘“Se (A).

the gel was analyzed for 7”Se and found to be radioactive. This 75Se-labeled protein was eluted from the gel with Tris buffer (pH 9.5) and found to have a molecular weight of approximately 80,000 by gel Cltration chromatography. This protein was

494

STULTS

enzymatically inactive and was assumed to be denatured glutathione peroxidase. The denaturation of the enzyme and its binding to the column during the DEAEagarose column chromatography may explain the loss of specific activity. The final Sephadex G-150 gel filtration resulted in nearly symmetrical and concurrent peaks of enzyme activity, radioactivity, and protein (Fig. 3). The pool of active fractions, 40-44, with a specific activity of 278 pmol of NADPH oxidized/min/mg of protein, was concentrated and subjected to electrophoresis. After the Sephadex G-150 chromatography there was a 1.1% recovery of initial homogenate enzymatic activity and a 1029-fold purification over the activity of the homogenate. The ratio of total enzymatic activity to total labeled selenium remained constant in all steps subsequent to the acetone precipitation step, which would suggest that the only seleno-protein present after that step was glutathione peroxidase.

ET AL.

GEL SLICE

NUMBER

FIG. 4. Disc-gel electrophoresis of the purified glutathione peroxidase with a specific activity of 278 Fmol of NADPH oxidizediminimg of protein. An 8%, polyacrylamide disc-gel was sliced into l-mm slices and each slice was eluted overnight in buffer. The eluates were assayed for enzymatic activity (ml and ‘“Se (A,). An identical gel was’ stained with Coomassie blue and scanned at 540 nm (0). The protein migrated toward the anode, on the right end in the figure.

incidence of protein, enzyme activity, and YSe suggests that the purified protein was Disc-Gel Electrophoresis pure glutathione peroxidase but that it The results of the disc-gel electrophore- consisted of multiple forms. This fact was sis of the sample with a specific activity of further substantiated by the SDS-disc-gel 278 pmol of NADPH oxidized/min/mg of electrophoresis, which resulted in a single protein are shown in Fig. 4. Even though coincident band of protein and YSe radiothe protein was not homogeneous, the co- activity. The subunit molecular weight was approximately 19,000, as shown by comparison with three standard proteins 04 OE7 -15 7- i of known molecular weight. These data, ;plus the results of gel filtration studies, 06-12 "0 support the existence of a tetramer with a z molecular weight of approximately 76,000, s I L consisting of four identical subunits. 0482 Studies of the Glutathione Peroxidase Aggregate OL FRACTION

NUMBER

FIG. 3. Purification of rat liver cytosol glutathione peroxidase on Sephadex G-150. The fractions with the highest specific activity from DEAE-agarose column III were pooled, concentrated, and applied to the Sephadex G-150 column (1.5 x 90 cm, 10 rn~ ‘Iris buffer, pH 7.6). The eluted 1.5ml fractions were assayed for protein (0) by absorption at 280 nm, enzymatic activity (ml using 5 ~1 of sample, and ‘“Se (A).

Figure 1 shows that during purification there existed two forms of glutathione peroxidase that differed in size. Figure 5A is a Sephadex G-100 column elution pattern of a sample of freshly prepared, untreated, rat liver cytosol. This pattern shows that all of the activity eluted with the void volume protein. This would suggest a molecular weight far greater than the 80,000 reported for the purified enzyme (19). Figures 5A-5C are sequential elution patterns from a single Sephadex G-100 column of

RAT

LIVER

GLUTATHIONE

FRACTION NUMBER

5. Chromatography of rat liver glutathione peroxidase on a Sephadex G-100 column (2.5 x 90 cm) at three subsequent times after preparation of the homogenate. (A) Elution pattern 5 h after sacrifice. (B) A second aliquot of the same preparation chromatographed after 29 h. (Ci A third aliquot chromatographed after 53 h. All samples were stored and chromatographed at 4°C. The 5-ml fractions were assayed for protein (0) by absorbance at 280 nm, enzymatic activity (ml using 20 ~1 of sample, and ‘“Se (A). FIG.

three aliquots of the same rat liver cytosolic sample over a S-day period. The first sample was chromatographed 5 h after sacrifice of the animals and the subsequent samples were chromatographed at 24-h intervals. All aliquots were stored at 4°C prior to chromatography. The enzymatic activity cochromatographed with the radioactivity during each elution, and the enzyme was shown to slowly disaggregate to a molecular weight of approximately 80,000 during storage. Of the several techniques used in an attempt to dissociate the aggregate of large molecular weight glutathione peroxidase, treatments with the following agents were ineffective: 0.2% Triton X-100, 10% propylene glycol, isotonic saline, 5 mM dithioerythritol, 5 mM glutathione (reduced), 10 mM selenite, and 30 pg/ml of cumene hydroperoxide. A freeze-thaw treatment repeated four times did cause a partial disaggregation, and storage at 4°C caused a substantial, but variable, dissociation of the aggregate. Study ofDifferent Charge Forms thione Peroxidase

of Gluta-

Figure 6A is the elution pattern of a sample of fresh, untreated rat liver cytosol chromatographed on DEAE-agarose at pH 8.0. There were three peaks of glutathione

495

PEROXIDASE

peroxidase activity (peaks I, II, and III) and each had a corresponding coincident peak of 7%e. When the constituent fractions of peak II were pooled and rechromatographed, both peak II and peak III were seen. When the constituent fractions of peak III were pooled and rechromatographed, the enzyme form was unchanged and only a peak corresponding to peak III was seen. Figure 6B shows the chromatogram of a similar sample of fresh rat liver cytosol that was prepared in and eluted with Tris buffer (pH 7.0). At this lower pH the absence of peak III is notable. An aliquot of the fresh cytosolic fraction that was stored for several days at 4°C chromatographed almost totally as peak III. These chromatograms illustrate that there was a change in the enzyme to a more negatively charged from. The proteins in both peak II and peak III had molecular weights of ap-

FRACTION NUMBER

FIG. 6. Multiple forms of glutathione peroxidase on DEAE-agarose column chromatography. A sample of fresh rat liver cytosol precipitated with 506 acetone was resolubilized in 5 rnM Tris buffer and passed through a Sephadex G-25 column. The sample was then applied to a DEAE-agarose column (1.5 x 20 cm) and the column was washed with 50 ml of buffer. A linear gradient (0.0-0.2 M NaCl) was used to elute the bound enzyme from the column. The 3-ml fractions were assayed for protein (0) by absorbance at 2&J nm and enzymatic activity (W using 20 ~1 of sample. (A) Elution at pH 8.0. (B) Elution at pH 7.0.

496

STULTS

proximately 80,000, as determined by gel filtration chromatography. DISCUSSION

The results of the subcellular distribution study of glutathione peroxidase correspond well with the report of Green and O’Brien (20) and the report of Flohb and Schlegel (21). Additional information was obtained by injecting rats with Ye-labeled selenite. From the ratio of radioactivity to glutathione peroxidase activity, it was determined that the microsomal fraction is the only fraction with a large amount of selenium that does not correspond with glutathione peroxidase. However, the purification shown in Table II reveals that at least 40% of the 75Sein the rat liver cytosol is in glutathione peroxidase. In this study, only 7% of the injected Y?e in rat liver was present in the microsomal fraction and 58% in the cytosol, at least 40% of which was in glutathione peroxidase. While keeping in mind the fact that the proteins into which selenium was incorporated in this study included only those that had a significant synthesis in the 3-day incorporation period, it is reasonable to propose that the seleno-protein glutathione peroxidase is a major site at which selenium is functional within the rat liver cell. The purification of rat liver glutathione peroxidase by the scheme described in this paper yielded an enzyme sample with a specific activity of 278 pmol of NADPH oxidizedlminlmg of protein. This purification in whole or in part has been repeated many times with similar final specific activities. Disc-gel elect,rophoresis of the purified enzyme sample resulted in a nonhomogeneous protein. The coincidence of protein, enzymatic activity, and 7”Seis evidence that all of the protein in the purified fraction was glutathione peroxidase, but the nonhomogeneity indicates the existence of proteins with different electrophoretic mobilities. Rat liver glutathione peroxidase appears to be quite unusual in several ways. First, it appears to be very stable to conditions that often denature proteins. In this purification procedure the enzyme sample

ET AL.

was heated to 50°C and acidified to pH 5.3. Second, the strong aggregation of this enzyme in an untreated cytosol preparation, presumably with other proteins, even in the presence of high concentrations of reductants, suggests the possibility of a large amount of hydrophobic character. Prohaska and Ganther (22) also have observed that crude cytosolic glutathione peroxidase from rat liver, testes, and brain appears to have a molecular weight much greater than purified enzyme unless it is treated with detergent and stored prior to chromatography. Third, the charge on the protein appears to become more negative with storage or with an increase in pH. This characteristic was utilized in the purification of the enzyme by subjecting it to DEAE-agarose chromatography before and after the increase in negative charge. This increase in negative charge appeared to be independent of the aggregation phenomenon. Both the neutrally and negatively charged protein peaks (Fig. 6A, peaks II and III) had molecular weights of approximately 80,000. The increase in the rate of change from the neutral form to the negatively charged form as the buffer pH was increased from 7.0 to 8.0 suggests the possibility that a disulfide interchange may be involved. While other workers have shown multiple-form phenomena associated with glutathione peroxidase, there are several important differences between their findings and those of this paper. Awasthi et al. (23) illustrated the existence of isozymes in the purified human red blood cell enzyme but did little to characterize them. In this paper we have shown that there are charge isomers of rat liver glutathione peroxidase and that one form can be favored over another by alterations in preparation. Lawrence and Burk (24) have reported two different enzymes from rat liver cytosol that differ in size, one being heat sensitive and having a molecular weight of 39,000. The multiple forms we report here are an aggregate with a very high molecular weight and two different charged forms of the enzyme, both of which have molecular weights of approximately 80,000. The multiplicity of forms of glutathione

RAT

LIVER

GLUTATHIONE

peroxidase in vitro may or may not represent true in uiuo forms. The hypothesis that glutathione peroxidase protects membranes from degradation due to lipid peroxidation requires that the cytosolic enzyme come into contact with the peroxide substrates in the membrane. If the aggregate is bound together by hydrophobic interactions, this would suggest much hydrophobic character to the enzyme and would support the enzyme’s ability to interact with hydroperoxides at the hydrophobic membrane surface. Study of the multiple forms may give information about the characteristics of the enzyme and the role it plays in protecting the cell from peroxidative damage. REFERENCES 1. MILLS, G. C. (1957) J. Biol. Chem. 229, 189-197. 2. MILLS, G. C. (1959) J. Biol. Chem. 234, 502-506. 3. SCHNEIDER, F., AND FLOH~, L. (1967) Hoppe Seyler’s Z. Physiol. Chem. 348, 540-552. 4. FLOH~, L., EISELE, B., AND WENDEL, A. (1971) Hoppe Seyler’s Z. Physiol. Chem. 352,151-158. 5. ROTRUCK, J. T., POPE, A. L., GANTHER, H. E., SWANSON, A. B., HAFEMAN, D. G., AND HOEKSTRA, W. G. (1973) Science 179, 588-590. 6. FLOH~, L., G~NZLER, W. A., AND SCHOCK, H. H. (1973) FEBS Lett. 32, 132. 7. COHEN, G., AND HOCHSTEIN, P. (1963) Biochrmistry 2, 1420-1428.

PEROXIDASE

497

P. J., AND LITTLE, C. (1969) Canad. J. 8. O’BRIEN, Biochem. 47, 493-499. 9. CHRISTOPHERSON, B. 0. (1968) Biochem. Biophys. Acta 164, 35-46. 10. CHOW, C. K., AND TAPPEL, A. L. (1972) Lipids 7, 518-524. 11. NAKAMURA, W., HOSODA, S., AND HAYASHI, K. (1974) Biochim. Biophys. Acta 358, 251-256. 12. LITTLE, C., AND O’BRIEN, P. J. (1968) Biochem. Biophys. Res. Commun. 31, 145-150. 13. DE DUVE, C., PRESSMAN, B. C., GIANNETTO, R., WATTIAUX, R., AND APPELMANS, F. (1955) Biothem. J. 60. 604. 14. PAGLIA, D. E., AND VALENTINE, W. N. (1967) J. Lab. Clin. Med. 70, 158-169. 15. MILLER, G. C. (1958)Anal. Chem. 31, 964. 16. ORNSTEIN, L. (1964)Ann. N. Y. Acad. Sci. 121, 321-349. 17. DAVIS, B. J. (1964)Ann. N. Y. Acad. Sci. 121, 404-427. 18. Wu, G.-J., AND BRUENING, G. (1971) Virology 46, 596-612. 19. CHIU, D. T. Y., FLETCHER, B., STULTS, F. H., ZAKOWSKI, J., AND TAPPEL, A. L. (1975) Fed. Proc. (abstr.) 34, 925. 20. GREEN, R.’ C., AND O’BRIEN, P. J. (1970) Biochlm. Biophys. Acta 197, 31-39. 21. FLOH%, L., AND SCHLEGEL, W. (1971) Hoppe Seyler’s Z. Physiol. Chem. 352, 1401-1410. 22. PROHASKA, J. R., AND GANTHER, H. E. (1976) J. Neurochem. 27, 1379-1387. 23. AWASHI, Y. C., BEUTLER, E., AND SRIVASTA, S. K. (1975) J. Biol. Chem. 250, 5144-5149. 24. LAWRENCE, R. A., AND BURK, R. F. (1976) Biothem. Biophys. Res. Commun. 71, 952-958.

Rat liver glutathione peroxidase: purification and study of multiple forms.

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Rat Liver Glutathione FRED Department H. STULTS, ofFood Science (1977) Peroxidase: Purification Mu...
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