BIOCHEMICAL

Vol.71,No.4,1976

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

GLUTATHIONE PEROXIDASE ACTIVITY IN SELENIUM-DEFICIENT Richard

A. Lawrence

Department of Internal Health Science Center Veterans Administration

Received

May 18,

RAT LIVER'

and Raymond F. Burk

Medicine, at Dallas Hospital,

University and Dallas,

of Texas Texas

75235

1976

SUMMARY: Glutathione peroxidase activity in the liver supernatant from rats me-deficient diet for 2 weeks was 8% of control when measured with H 0 but 42% of control when assayed with cumene hydroperoxide. Two peaks of g fZu athione peroxidase activity were present in the Sephadex G-150 gel filtration chromatogram of rat liver supernatant when 1.5 mM cumene hydroperoxide was used as substrate. Only the first peak was detected when 0.25 mM H 0 was used as substrate. The first peak was absent from chromatograms of Se- ii e f* 1cient rat liver supernatants; but the second peak, which eluted at a position corresponding to M.W. = 39,000, appeared unchanged. The second peak thus represents a second glutathione peroxidase activity which catalyzes the destruction of organic hydroperoxides but has little activity toward H202 and which persists in severe selenium deficiency. INTRODUCTION:

11.1.9) it

Glutathione

was discovered

(2).

This

sources

in various

intake

activity

most

investigators,

tion

of a variety

other

substrates

tial

glutathione

%upported (R.A.L.)

Se which

from

H202 (lo),

toward Since

glutathione

of organic such

grants

Copyright 0 I976 by Academic Press, Inc. All rights of reproduction in any form reserved.

tightly

bound

rat

the substrate

(ll),

952

form

from

(5,7,8)

liver

several

and its

on dietary

Se

has no glutathione

used for

its

assay

by

can catalyze

the

some workers

have used

With

has been reported

ROl ES-01017

and characterized

is dependent

Se-deficient

hydroperoxides

activity

purified

in homogeneous

peroxidase

EC 1.

H202 oxidoreductase,

species

as cumene hydroperoxide.

peroxidase

by U.S.P.H.S.

is

several

It has been shown that

(9).

peroxidase

been obtained

It contains tissues

(glutathione:

(1) who partially

by Mills

enzyme has since

(3,4,5,6).

activity

peroxidase

(R.F.B.)

this

substrate

destruc-

substan-

in Se-deficient

and T32 AM-07100

rat

BIOCHEMICAL

Vol. 71, No. 4, 1976

liver.

(12).

Using

dase activities

cumene hydroperoxide

that

were

14 months.

This

glutathione

peroxidase

finding

indicated which

activity

is

Se status,

a non-Se-dependent source

of error.

to resist

stress

from

activity,

which

fed

the

possible

not

be Se-dependent.

glutathione

studies

agents

should

not be measured rat

liver

the glutathione

peroxidase

Since

of the into

of the

for

glutathione or functional could

be an

ability

of an animal

account

such an

H202.

x 9 supernatants

activity

diet

of an additional

activity

with

peroxi-

a Se-deficient

nutritional

take

by assays

105,000

glutathione

existence

peroxidase

In addition,

oxidative

might

cumene hydroperoxide

in rats

used as a means of assessing

important

we have fractionated

might

RESEARCH COMMUNICATIONS

we have observed

30% of control

peroxidase

measured

AND BIOPHYSICAL

For these

on Sephadex fractions

reasons G-150

using

and

both

and H202 as substrates.

METHODS: Weanling male Holtzman rats were fed a Torula yeast-based Se-defimet (13) or the same diet supplemented with 0.5 mg Se as sodium selenite per Kg (controls). The rats were exsanguinated under ether anesthesia. The livers were perfused with 1.14 M NaCl and homogenized in 0.25 M sucrose. The supernatant fraction was prepared by centrifugation at 105,000 x 9 for 1 hour. Enzyme activity was measured by a modification of the coupled assay procedure of Paglia and Valentine (14). The reaction mixture consisted of 50 mM potassium phosphate buffer (pH 7), 1 mM EDTA, 1 mM NaN3, 0.2 mM NADPH, 1 E.U./ml GSSG-reductase, 1 mM GSH, 1.5 mM cumene hydroperoxlde or 0.25 mM H202 in a total volume of 1 ml. All ingredients except enzyme source and peroxide were combined at the beginning of each day. Enzyme source (0.1 ml) was added to 0.8 ml of the above mixture and allowed to incubate 5 min. at room temperature before initiation of the reaction by the addition of 0.1 ml peroxide solution. Absorbance at 340 nm was recorded for 5 min. and the activity was calculated from the slope of these lines as pmoles NADPH oxidized per minute. Blank reactions with enzyme source replaced by distilled water were subtracted from each assay. Protein was measured by the method of Lowry et al. (15). RESULTS AND DISCUSSION:

Table

liver

rats

supernatants

The activity

from

measured

with

1 shows glutathione fed Se-deficient

by 2 weeks

but

had decreased

much less,

to 42% of control.

Figure rats

fed

peroxidase

1 shows

the

gel

experimental

activity

and control

H202 had decreased

8% of control

the activity

filtration diets

appeared

in

3 months.

the chromatogram

953

in the

measured

chromatograms for

peroxidase

activity diets

Se-deficient

with

of liver Two peaks

in

for

2 weeks. rats

to

cumene hydroperoxide

supernatants

from

of glutathione

of the supernatant

from

the

Vol.71,No.4,

BIOCHEMICAL

1976

TABLE I: Effect on Rat Liver

AND BIOPHYSICALRESEARCH

of Dietary Glutathione

Selenium Supplementalion Peroxidase Activity

GSH-Peroxidase Dietary

COMMUNICATIONS

Activity*

f S.E.

Cumene.OOH

Se

0 wm

28 ?r 7.5

314 f 18

0.5

354 f 65

748 i 81

ppm

'Rats were fed the respective diets from weaning for 2 weeks before killing and processing as described in the text. 'Results are expressed min. per mg protein.

as pmoles NADPH oxidized per There were 4 rats per group.

A. CONTROL zo-

40

50

FRACTION

Fig.

1

Se-adequate the second cates

60

70

80

go-

NO. (-3mVFroctd

Sephadex G-150 gel filtration of rat liver supernatant. Gel filtration was performed on a 5 x 22.5 cm column of Sephadex G-150 eluted with 0.05 M tris buffer (pH 8.6) containing 1 n&l EDTA and 0.1 M .K2HPO4 at a flow rate of 60 ml/hr. The sample was applied in a 10 ml volume.

rat

(Fig.

1A) when cumene hydroperoxide

peak was not detected

the presence

of 2 glutathione

was used as substrate

when H202 was used as substrate. peroxidases

954

capable

of destroying

This

but indi-

cumene

BIOCHEMICAL

Vol.71,No.4,1976

hydroperoxid'e,

only

The chromatogram activity

but

1 of which

of the

AND BIOPHYSICAL

destroys

Se-deficient

Se-dependent

glutathione

peroxidase.

on the basis

of its

assay

conditions.

1B) showed

Peak I thus

Peak II

weight,

these (Fig.

II activity.

of peak

molecular

H202 under supernatant

persistence

RESEARCH COMMUNICATIONS

no peak I

appears

to be the

seems to be a different

substrate

specificity,

enzyme

and persistence

in Se deficiency. Our assay

procedure

measures

the oxidized

glutathione

peroxidase

reaction

by coupling

glutathione

reductase.

There

was a possibility,

catalyzed GSH.

the oxidation

We have also

of GSH remaining catalyzes Table

the

of NADPH directly

been able as described

oxidation

Time (min.)

it

to the oxidation therefore,

rather

to measure

this

by Hafeman,

formed

than

activity

et al.

in the of NADPH via

that

through

II

peak

oxidation

by direct

of

measurement

(10) indicating

that

peak

of GSH.

II shows results

TABLE II:

glutathione

Effect

of a heat

of Heating

denaturation

at 60' Peak I.

% of Control

experiment.

Peak II

C on GSH-Px Activity' Peak II % of Control

0

loo2

1002

10

50

0

20

20

0

30

10

0

'Aliquots of the combined fractions having peak I and peak II activity from Sephadex G-150 chromatography were heated in a thermostated water bath at 60" C. At the times indicated 0.1 ml samples were withdrawn, added to 0.8 ml ice cold reaction mixture, then allowed to incubate at room temperature for 5 minutes and assayed for GSH-Px activity as described in the text. 'Contained 0.2 enzyme units expressed as change in O.D. at 340 nm per minute for peak I and 0.38 enzyme units for peak II per ml.

955

II

BIOCHEMICAL

Vol. 71, No. 4, 1976

activity

is

I activity

is

provides the

completely still

further

protein

39,000

evidence

II

2.

after

of peak

peak II

of the

peak I enzyme,

at 60" C but

at 60"

This

C.

peak

at a volume G-150

peak

experiment

I and peak

by other

peroxidase

weight

II and of

has been found

II

enzyme could

.6r

I

as shown for

to contain

weight

I

column

the

(3,4,5,6).

about

be a dimer

I

to M.W. =

determined

investigators

enzyme has a molecular

the peak

corresponding superfine

the molecular

peroxidase

glutathione

and the

10 minutes

between

eluted

one-half

glutathione

(3,4,5,6)

30 minutes

on the Sephadex

is about

Se-dependent

for

II.

enzyme activity

This

Se-dependent

by heating

of a difference

when chromatographed

in Fig.

the

detectable

nature

The peak

destroyed

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Since 4 subunits

one-half

that

of the peak

I

1

K (1” .4 -

.3 -

I

4.2 Log

Fig.

2

H202.

that

retains

activity

M.W

no activity

in

does not dissociate

to give

peak

activity

toward

We have rechromatographed

and have observed

II

I

4.6

Molecular weight determination of glutathione peroxidase peak II. Rat liver supernatant was chromatographed on a 2.6 x 29.4 cm column of Sephadex G-150 superfine eluted with the same buffer as Fig. 1 at a flow rate of 6 mljhr. The sample was applied in a 1.5 ml volume. This column was calibrated using cytochrome c (M.W. = 12,384), achymotrypsin (M.W. = 22,500) and ovalbumin (W.W. = 45,000).

enzyme subunits toward

I

4.4

is

not diminished

peak

the peak II II

under

by severe

956

organic

hydroperoxides

I fractions region,

these

on Sephadex indicating

conditions.

Se deficiency,

but

it

that Also,

probably

not

G-150 peak since does

I peak

not

BIOCHEMICAL

Vol. 71, No. 4, 1976

contain

Se as does the

has not yet suggests turally

these

related,

would

differences

The onset

mentation

liver

diets

By increasing

II enzyme which

such

as the

sheep which

peak II

rat

which

trations

peroxidase

detected

I.

still

give

peroxidase at these

peak II

in

by Table should

glutathione

of the

separation

from

could

time

be struc-

of the

tissues

observed

(16).

substrate

where

both

using

results

indicate,

enzymes

of H202. so assay

peak

II

have much liver

the

present

assay

at the usual

G.C. G.C.

(1957) J. Biol. (1959) J. Biol. and FTbhq. 34B;l~;0-~52.and

::

%%$~h;m:

5.

J. Biol' Chem' 250 ch,m:,-%ithK'H.E.,

activity

specific must

for

51;4-ii49. and Hoeks tra,

957

W.G.

(1974)

the

be done after

m. 229, 189-197. Chem. z, 502-506. (1967 ) Hoppe-Seyler's Z. Sr ivastava, S.K. (1975)

l3-, 1825-1829.

concen-

cannot

REFERENCES: Mills, Mills,

of

as is

peak I enzyme.

::

in

activity.

enzyme activity

No substrate

of its

species

of peak I or Se-dependent

estimate the

this

in Se and

peak II for

are

H202 as substrate

since

for

in the

a problem

of

acids.

In addition,

than

low

by supple-

amino

necrosis

poses

diets

The existence

liver

I activity

peak

fed

can be delayed

I enzyme activity

peak

a reliable

activity,

in rats

provide

dietary

peak II enzyme

Assays

occurs

acids,

preliminary

has higher

concentrations

the

importance

in Se deficiencv.

develop

than

enzyme has been found

should

of the sulfur-containing they

may, as our

The existence

demonstrated

do not

they

or methionine

decreased

enzyme activity

to the

glutathione

effect

is not

E deficiency

contrast

which amino

the GSH concentration

peak

at this

them.

cysteine

this

the

II enzyme

peak

available

Even if

from

necrosis,

with

enzyme may explain

higher

detract

E and sulfur-containing

of these

vitamin

not

RESEARCH COMMUNICATIONS

of purified

The evidence enzymes.

between

of dietary

in Se, vitamin

Se analysis

however.

are 2 different

this

physiological

this

peak I enzyme.

been performed,

that

AND BIOPHYSICAL

Biochem.

be

Vol.71,No.4,

6.

7. 8. 9. 10.

11. 12. 13. 14.

1976

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Nakamura, W., Hosoda, S., and Hayashi , K. (1974) Biochem. Bio h s Acta 358, 251-261. !%$$$ m,Pope, A.L., Ganther, H .E., Swanson , A.B., Hafeand Hoekstra, W.G. (1973) Science 179-, 588-590. Flo;e,'L:: Gunzler, W.A., and Schock, H.H.973) FEBS Lett.

32. 132-134.

z&her, H.E., Hafeman, D.G., Lawrence, R.A., Serfass, R.E., and Hoekstra, W.G. (1976) in Trace Elements in Human Health and Disease (Prasad, A.S., Edmpress,cZemicPress,N.'f. HafemG., Sunde, R.A., and Hoekstra, W.G. (1974) J. Nutr. 104. 580-587. i?lGle, C., and O'Brien, P.J. (1968) Biochem. Biophys. Res. Commun. 31, 145-150. Chow, (:.c, and Tappel, A.L. (1974) J. Nutr. 104, 444-451. Burk, R.F., and Masters, B.S.S. (1975r -~ m. mchem. Biophys. l70, 124-131. Paglia, R.E., and Valentine, W.N. (1967) J- -Lab. -Clin. Med.

70, 158-169. 15. i&&y, O.H., Rosebrough, N.J., Farr, A.L., (1951) J. Biol. Chem. 193, 265-275. 16. Schwarz, K.v65)ed. &. 24, 58-67.

958

and Randall,

R.J.