Membrane-bound glutathione peroxidase-like activity in mitochondria

Vol.

96, No.

September

1, 1980

8lOCHEMlCAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

Pages 85-91

16, 1980

MEMBRANE-BOUND GLUTATHIONE Aspandiar

PEROXIDASE-LIKE

G. Ratki*l

ACTIVITY

and Charles

IN MITOCHONDRIA

E. Myers+

*Radiobiology Division, Tufts-New England Medical Center Hospital, 171 Harrison Avenue, Boston, Massachusetts 02111, and +Clinical Pharmacology Branch, National Cancer Institute Bethesda, Maryland 20205 Received

July

21,198O

SUMMARY

A membrane-bound glutathione peroxidase-like activity has been detected in liver and cardiac mitochondrial membrane. This enzyme activity differs from the cytosol and mitochondrial matrix selenium-dependent glutathione peroxidase in that it is membrane bound, sensitive to sonication and tritonX-100, and is unaffected by prolonged feeding of a selenium-free diet. This mitochondrial membrane-bound enzyme activity differs from the glutathione-stransferases which exhibit glutathione peroxidase activity in that it is capable of utilizing both cumene hydroperoxide and hydrogen peroxide as substrates. Digitonin fractionation studies indicate that this enzyme is not located in either inner or outer mitochondrial membrane but rather within inter-membrane space. This newly described membrane-bound enzyme activity may play an important role in the maintenance of cardiac mitochondrial integrity in that mitochondrial matrix does not contain glutathione peroxidase. INTRODUCTION As part

of our

investigation

rubicin

cardiac

toxicity

H202 in

cardiac

mitochondria.

mitochondrial Cardiac

H202/min/mg to result oxidation, for lipid

in

turn, in

peroxidation

of glutathione

and "high

swelling amplitude"

(11).

peroxidation the

and

is

H202 are

to

lipids

function

and has

of liver swelling

Detoxification

of

of

4 (3,4).

0.3-0.6

nmol

and H202 is known (7,8).

mitochondria are

state

produce

of superoxide

of

byproducts

in

membrane

in doxo-

detoxification

greatest

estimated

production

mitochondrial

amplitude"

anion

been

of mitochondrial

peroxidase for

This

of lipid

re-examined

production

have

fact,

affects

"high

we have

their

(3,5,6).

in peroxidation

role

Superoxide

and in

protein

example,

1Requests Institute,

(1,2),

metabolism

mitochondria,

of the

lessened superoxide

been

Lipid

per-

implicated, (9,lO).

by the and

reprints should be addressed to: A. G. Ratki, National Building 10, Room 6N119, Bethesda, Maryland 20205.

Both addition hydrogen Cancer

0006-291X/80/170085-07$01.00/0 85

Copyright 0 1980 by Academic Press, Inc. All rights of reproduction in any form reserved.

Vol.

96, No.

peroxide

by mitochondria

Mitochondria (13), are

BIOCHEMICAL

1, 1980

have

and all

has thus

been

shown

to

selenium-dependent

contained

in hepatic

of cardiac

examined,

especially

with

In the present

study,

matrix.

both the

the

described

reported

a glutathione

with

catalase

These

enzymes

Peroxide

detox-

with

activity.

mitochondria

lack

in

some properties

mitochondrial activity

which

glutathione

peroxidase

the selenium-

liver

peroxidase

selenium-dependent

glutathione-S-transferases

(12),

(14-16).

peroxidase

cardiac

space

interest.

have not been as comprehensively

previously

detected

considerable

(12,13-16).

to glutathione

inter-membrane

previously

matrix

COMMUNICATIONS

dismutase

peroxidase

we show that

we have

of

super-oxide

mitochondria

regard

RESEARCH

a subject

contain

peroxidase

Instead,

the mitochondrial

been

mitochondrial

mechanisms

glutathione

BIOPHYSICAL

glutathione

ification

dependent

AND

within

differ

from

peroxidase

and

activity.

METHODS AND MATERIAL CDFl male mice were obtained from Dublin Laboratories, Dublin, VA, and kept on selenium-containing or selenium-free diet as described earlier and liver subcellular fractions and mitochondrial sonication (2). Cardiac and triton-X-100 treatment were done as described in legends to tables. Glutathione peroxidase activity was determined according to Lawrence and Burk (17) with some modifications as described in the legend to Table I. Protein was determined by the method of Lowry et al (18). For most of the glutathione peroxidase assays, physiologic pH was used in order to ensure that the ratio of activities in the various fractions were measured under a reasonable approximation of in vivo conditions. In the digitonin experiments, in order to gain greater sensitivity, the membrane-bound enzyme was assayed at its pH optimum of 8.0. were

RESULTS AND DISCUSSION Of the one,

the

hydrogen

enzymes

known

selenium-dependent peroxide

and

ary

hand,

selenium

cumene

tathione mals results

peroxidase

utilize For

(Table

these

exhibit

as

peroxide

reasons,

animals

substrates

thatmitochondria

to

and are not

utilize

both The

activity,

affected

tissue

on

by diet-

distribution

peroxides are

only

(17,18-21).

peroxidase

in cardiac both

activity,

able

the subcellular

using

86

is

glutathione

I) was measured

mice reveal

peroxidase

peroxidase,

hydrogen

and in selenium-deficient from control

glutathione

hydroperoxide

which

cannot

(23,24).

exhibit glutathione

glutathione-S-transferases the other

to

of glu-

from control as substrates.

the richest

intracellu-

aniThe

Vol.

96, No.

1, 1980

Table

BIOCHEMICAL

I.

AND

BIOPHYSICAL

COMMUNICATIONS

SUBCELLULAR DISTRIBUTION OF GLIJTATHIONE PEROXIDASE ACTIVITY IN HEART MUSCLE WITH DIFFERENT SUBSTRATES IN CONTROL AND SELENIUM-DEFICIENT CDFl MICE CONTROL

Cell

RESEARCH

Hydrogen Peroxide As Substrate (nmoles/min/mg

Fraction

SELENIUM-DEFICIENT Cumene Hydrogen HydroPeroxide Peroxide As As Substrate Substrate (nmoles/min/mg protein)

Cumene HydroPeroxide As Substrate protein)

Homogenate

5.45

7.00

5.78

9.70

Pellet

5.31

6.96

7.51

8.38

Mitochondria

10.30

9.48

Microsome

0

0

1.22

1.86

4.33

5.11

1.28

2.85

Soluble

11.25

10.80

Subcellular fractionation was done by preparing 10% heart homogep ate in 250 mM sucrose, 1 mM Tris, 10 mM EDTA, pH 7.2, in a Brinkmann model PCU-2-110 Polytron 3 times for 5 set at 5.5 settings with cooling in between cycles. After sedimentation of the nuclear fraction (700 x G for 10 min), the supernatant was centrifuged at 10,000 x G for 15 mln to sediment mitochondria. The post-mitochondrial supernatant was centrifuged at 105,000 x G for 60 min to sediment microsomes. Nuclear, mitochondrial, and microsomal pellets were resuspended in the initial buffer. Glutathione peroxidase activity was determined by the decrease in absorbance at 340 w by incubating enzyme in 100 mM Tris, 3 mM EDTA, pH 7.0, 1.2 units glutathione reductase, 0.25 mM GSH, 3.0 mM NaN3, 0.25 mM NADPH, and 0.04 ml4 H202 or 0.075 mM cumene hydroperoxide in 1 ml final volume. Blank reactions with the enzyme source replaced by distilled water were subThe activity is expressed as nmoles NADPH tracted from each assay. oxidized/ min/mg protein using the extinction coefficient for NADPH Succinate-cytochromecreductase activity of 6.22 x lo3 mol-l cm-l. and rotenone-insensitive NADH-cytochrome 2 reductase activity were determined as mitochondrial and microsomal marker enzymes, respectively.

lar

source

twice

that

(Table

I,

tion is

of

glutathione

of the homogenate columns

glutathione less

(Table

than I,

peroxidase

activity

sults

suggest

which

is

more

3 and 4) did

30% of that 1). showed

I,

In

contrast,

a slight

to

with

effect

mitochondrial

peroxidase

effects

87

of

on the

with

selenium

deficiency

soluble

frac-

where

activity

hydrogen

peroxide

glutathione selenium

activity

Selenium

dependent,

mice when assayed

with

a specific

1 and 2).

be selenium

increase

the

heart

expected

of glutathione to

the

columns

the

from control

the presence resistant

have known

peroxidase,

column

cific

(Table

in

peroxidase

deficiency. activity deficiency

spe-

These

re-

in mitochondria than

is

the

Vol.

96, No.

1, 1980

previously

BIOCHEMICAL

described

to utilize

both

Previous

cytosolic cumene

studies

(15,24).

in liver

sonication

(14)

chondrial glutathione the

into

of mitochondria

activity.

selenium-deficient suggest bound

that

but less

Because for

liver

these

we have

X-100

treatment

results

(Table

supernatant.

In contrast,

also

found

selenium-deficient

for

post-sonication

is

free

of glutamembrane

Triton-X-100 complete

treatment loss

of

090%)

of mitochondria

from

These results

enzyme but have

deficiency

so sharply

and

the

matrix

those

with

is

a membranesensitive

to both confirm

to

remains

most

mice.

in the

selenium-deficient

after

sonication Furthermore,

of the matrix

88

activity

the

in the triton-

from

and liver

post-treatment

post-treatment

mice,

However,

is

sonication

in

activity

pellet

both peroxidase

little

enzyme.

reported

and

glutathione in activity

matrix

sonication that

increase

from

previously

of the enzyme activity

by the

control

mice,

most

Our results

release

of this

from

the

reported

in enzyme activity.

mitochondria

B).

in the membrane

of mitochondria

mito-

mitochondrial

that

treatment

by

the

mitochondrial

in nearly

the matrix

where

mitochondria

dependency

disrupt

paralleled

contains

by selenium

liver II

as reflected

liver

lack

contrast

subjected

mitochondria

selenium

which

drop

mitochondria

be liberated

Triton-X-100

(14-16,24)

treatment

of

should

mitochondrial

cardiac

that

existence

so to sonication.

triton-X-100

ernate

it

of

able

substrate.

the

the

of which

A shows

resulted

unaffected

mitochondria

matrix,

mice

mitochondria is

II

enzyme

mice gave a similar

enzyme which

triton-X-100

Table

activity.

cardiac

liver

50% of the enzyme activity.

control

peroxidase

from

behavior

to see if

The pellet,

about

from

the

reason,

a matrix

in

still

as

only

located

both

is

peroxide

reported

liberated

(15,16,24),

enzyme.

possesses

glutathione

been

was assessed

which

peroxidase

fragments,

has

this

mitochondrial

supernatant thione

For

peroxidase

liver

peroxidase

and triton-X-100

membrane.

have

COMMUNICATIONS

but which

hydrogen

mitochondria

enzyme

RESEARCH

peroxidase and

glutathione

This

BIOPHYSICAL

glutathione

hydroperoxide

of a seleniunrdependent matrix

AND

sup-

confirming

the

some enzyme activity and triton-X-100

in

liver is

lost

treatment

mitochondria but

is

29.3%

from of the

Vol.

96, No.

BIOCHEMICAL

1, 1980

Table

II.

AND

EFFECTS OF SONICATION LIVER MITOCHONDRIAL

BIOPHYSICAL

RESEARCH

AND TRITON-X-100 ON CARDIAC AND GLUTATHIONE PEROXIDASE ACTIVITY SONICATION* (nnoles/min/mg

A.

COMMUNICATIONS

TRITON-X-100' protein)

Heart

Intact

mitochondria Post-treatment Post-treatment

Se-deficient intact Post-treatment Post-treatment B.

11.06 0 5.63

supernatant pellet

12.65 0 0.99

mitochondria supernatant pellet

14.41 0.21 2.64

Liver

Intact

mitochondrial Post-treatment Post-treatment

25.45 74.60 24.55

supernatant pellet

Se-deficient intact Post-treatment Post-treatment

25.45 61.15 9.76

mitochondria supernatant pellet

13.99 2.84 4.10

*Mitochondria, 2 to 6 mg/ml protein, were sonicated 10 set x 3 by Branson Sonifier Cell Disruptor 185 with microtip, setting control output on 6 and meter reading 42-44 watts/second, and were sedimented at 105,000 x G for 60 min to separate the mitochondrial membrane pellet, which was resuspended in 250 mM sucrose, 1 mM Tris, 10 mM EDTA, pH 7.2. +To the mitochondrial suspension, 1 to 2 mg/ml, in 250 mM sucrose, 1 mM Tris, 10 mM EDTA, pH 7.2 was added triton-X-100 in 50 mM Tris, 1.5 ml4 EDTA, pH 7.0 to final concentration 0.5% and incubated at 0' for 10 min. The suspension was sedimented at 105,000 x G for 60 min to separate mitochondrial membrane pellet which was resuspended in 250 mM sucrose, 1 mM Tris, 10 mM EDTA, pH 7.2. 110% liver homogenate in 250 mM sucrose, 1 mM Tris, 10 ml4 EDTA, pH 7.2 was centrifuged at 600 x G for 10 min to sediment nuclei. The post-nuclei supernatant was centrifuged at 8,500 x G for 12 min to sediment mitochondria, suspended in 250 mM sucrose, 1 mM Tris, 10 mM EDTA, pH 7.2 to 4-6 mg/ml.

original

mitochondrial

persists.

These

membrane-bound

glutathione

results

consist becane

the

of

treatment contents

the

a standard

the

as

the

localizing

activity

well

as

located outer

membrane

the

previously

the

pellet

possess

a

described

in the matrix.

and

membrane yields

and matrix.

enzyme activity

in

may also

mitochondrial space

89

found

mitochondria

inter-membrane

mitochondrial

one for

liver

peroxidase

disrupts of

inner

that

peroxidase

glutathione

Digitonin releases

suggest

glutathione

selenium-dependent

peroxidase

within

and

mitoplasts This

thus which

technique

the mitochondrial

has

Vol.

96, No.

1, 1980

Table

BIOCHEMICAL

III.

AND

BIOPHYSICAL

RESEARCH

EFFECT OF DIGITONIN TREAlMENT ON CARDIAC GLDTATHIONE PEROXIDASE ACTIVITY

MITOCHONDRIAL

SPECIFIC (nmoles/min/mg Intact

1.59

membrane

Inter-membrane Inner

26.3

space

membrane

1.61

1.89

Matrix

Mitochondria

were

isolated

Digitonin fractionation except that buffer and 250 mM sucro.se, athione peroxidase Table I in Tris-EDTA,

membrane.

Table

III As

inter-membrane

space.

found located

It

remains

truly

pool cytosol

selenium

which

turns

complex

analysis

in

we have

in liver

independent

matrix enzyme

Cardiac while

localized

in

to the

of

addition

to

the

glutathione

peroxidase

a glutathione

peroxidase

mitochondria

both

cardiac

detoxification

thought;

detected

to

types

are

lacking

of glutathione

per-

mitochondria. this

or whether slowly

applied

is clearly

previously

enzyme,

to be seen whether

this

in legend

the mttochondrial

membrane.

present

more

such

and selenium-dependent

matrix,

or mitochondrial

and characterize

than

matrix

over

of

suggest

mitochondrial

are

activity

as described

the enzyme activity

results

selenium-dependent

oxidase

I.

out as previously described (25) Tris HCl, pH 7.4, with 10 mM EDTA proteinwas 11.1 mg/ml. Glut-

results

catalase

the

Table

was determined

the

mitochondrial

within

in the

is

identified

in

pH 8.0.

these

may be more

in the

activity

can be seen,

In conclusion,

previously

as described

was carried used was 1 ti and mitochondrial

shows

mitochondria.

peroxides

ACTIVITY protein)

82.5

mitochondria

Outer

COMMUNICATIONS

than

membrane-bound it

is simply

glutathione dependent

selenium

the

pool

enzyme.

We are currently

further

in

order

to

peroxidase upon a selenium supports

which

attempting answer

this

the

to purify question.

REFERENCES 1.

Myers, Young,

C-E.,

McGuire,

R.C.

(1977)

W-R., Science

Liss,

R.H.,

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165-167.

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Ifrim,

I.,

Grotzinger,

K.,

and

Vol.

96, No.

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1, 1980

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

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