Glutathione peroxidase in yeast. Presence of the enzyme and induction by oxidative conditions

Vol. 147, No. 3, 1987

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Pages 1200-1205

September 30, 1987

GLUTATHIONE PEROXIDASE E N Z Y M E AND I N D U C T I O N

IN YEAST. P R E S E N C E OF THE BY O X I D A T I V E C O N D I T I O N S

*

Oaliazzo

Francesca

Department and

**

, Alma

of

Biology,

" Center

**Institute

of

Tot

Molecular

of T e c h n o l o g y University

.-

Schiesser,

and Giuseppe

Vergata Biology

University, of

and A g r i c u l t u r a l

of Tuscia,

Rotilio

Viterbo

CNR,

Rome,

Rome,

Microbiology,

Italy

Received August 12, 1987

SUbIMARY. The p r e s e n c e of g l u t a t h i o n e p e r o x i d a s e a c t i v i t y is r e p o r t e d for the first time for a w i l d type s t r a i n of S a c c h a romyces cerevisiae. Both forms of enzyme, i.e. t~ah s p e c i f i cally active toward H 0 a l o n e and that d e c o m p o s i n g also orga2 2 nic p e r o x i d e s , w e r e f o u n d to be p r e s e n t The H 0 specific • ~ 2 2 form d i s a p p e a r e d when c e l l s were g r o w n in ehe a b s e n c e of oxygen, w h i l e the o t h e r form was m u c h i n c r e a s e d u n d e r the same c o n d i t i o n s • A d d i t i o n of c o p p e r to the c u l t u r e g r e a t l y i n c r e a sed b o t h forms. The r e s u l t s s h o w that g l u t a t h i o n e p e r o x i d a s e is to be included, as an i m p o r t a n t c o m p o n e n t that is also higly r e s p o n s i v e to o x i d a t i v e e n v i r o n m e n t s , in the e n z y m e defense s y s t e m of y e a s t a g a i n s t o x i d a t i v e damage. ® 1987 Academic Press, Inc.

Glutathione hydroperoxides

ROOH

Two

enzymes

and higher dent

GPX)

hydrogen

peroxidase

(GPX)

catalyzes

by GSH as r e d u c t a n t

according

+ 2 GSH

showing

Address

0006-291X/87

GPX

vertebrates• and

peroxide,

activity One

reacts

(selenium-independent

Biology, Italy.

> ROH

with

the

other

GPX)

were

contains both

has

GSSG

+

to the eq.

+ H20

selenium

not

negligible

$1.50 1200

of

i):

i)

in

mammals

(selenium-depen-

hydroperoxides containing activity

correspondence to: Francesca Galiazzo~ Tor V e r g a t a U n i v e r s i t y , Via O. R a i m o n d o

Copyright © 1987 by Academic Press, Inc. All rights of reproduction in any form reserved.

reduction

identified

organic

one,

the

and

selenium with

H202

Department of i, 00173 Rome,

Vol. 147, No. 3, 1987

(i,2). mes

Selenium-independent

of

zing

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

the

GSH-transferase

the c o n j u g a t i o n

have

been

sues

(1,2),

GPX

activity

but

more

recently

it

and

sycamore

cells

nach

was

the

other

wer

organisms,

involved

side,

in

derivatives catalase shown,

of

their

cing for

the

H202),

The

which

is

components.

revisiae

and

on

time the

in

A

be

exsist

In

cells

the

of

of

present type copper

studied,

excess the

of

and been

GPX

respect

with

was

redu-

detected

Saccharomyces

additions

to

in the p r e s e n c e

of

transition

with

cell,

to

the cells

intermediates

study

strain

oxygen

cells,

inside

of

in

enzymes

has

(9)

on

in lo-

dismutase

enzymes

flux

spi-

that,

reduced

redox-active

capability

a wild

was

in

of

maize,

other

eucaryotic

source

reaction

of

the

that

and b a c t e r i a

the p r e s e n c e

these

or

concerning

recalled

of

tis-

reported

superoxide

of

animal

yeast

partially

as

(8)

(4)

extracts

yeast,

widespread

effect

its a c t i v i t y

available

by the m e t a b o l i c

the

may

is

document

of

peroxidases

several

should

such

procaryotic

co~]tain

from

in

expression

to c o n t r o l

first

it

reports

metal-sequestering

cell

medium

and

reduction

the

(5).

compounds

glutathione

plants,

isoenz] .... cataly-

studies

detected

in p a r t i c u l a r

substrates.

ions,

was

detoxification

either

subjected

to

the (0~

be

metal

and

Previous

from higher

numerous

(6,7). in

oxygen

absent

purified

some

of e n z y m e s

hydrophobic

information

and m i c r o o r g a n i s m s .

to

a class

(3). W h i l e

and

little

identical

family,

center

demonstrated very

is

of GSH w i t h

ing an e l e c t r o p h i l i c

plants

GPX

the

ce-

culture

and absen-

ce of oxygen. The

results

portant

obtained

antioxidative

suggest role

that

also

this

in lower

enzyme

may have

eukaryotic

an im-

organisms.

M A T E R I A L S A N D M E T H O D S . B o v i n e s e r u m albumin, c u m e n e h y d r o p e r o xide, Tween 80, e r g o s t e r o l and c y c l o e x i m i d e were purchased f r o m Sigma. S o d i u m azyde a n d h y d r o g e n p e r o x i d e were o b t a i n e d from Merck. Glutathione (reduced), glutathione reductase and N A D P H w e r e from B o e h r i n g e r . Zymolyase (100000 U/g) was f r o m S e i g a k u K o g y o a n d y e a s t e x t r a c t f r o m Difco. C h l o r a m p h e n i col was o b t a i n e d f r o m Fluka. A w i l d type s t r a i n (D273-10B) of S a c c h a r o m y c e s c e r e v i s i a e was g r o w n a e r o b i c a l l y or a n a e r o b i c a l l y in a ~ e d i u m c o n t a i n i n g 0.5% glucose, i% y e a s t e x t r a c t ,

1201

Vol. 147, No. 3, 1987

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

o

o

~

i~ N H ~ C l , 0 . 0 3 ~ ~gSO 4, 0 . 0 9 % K 2 H P 0 4 , 0 . 2 2 % K H 2 P O . U p o n a n a e robic ~ conditions t~e medium was supplemente~ with ig/l Tween 80 and 30mg/l ergosterol. Increasing amounts of CuSO were added to the culture medium to r e a c h various fina~ concentrations in the range between 0.4 - imM. The copper concentration of the c u l t u r e m e d i u m as s u c h w a s 4 pM. Grown cells were harvested at the e n d of the logarithmic phase of g r o w t h . Before exposure to air, a n a e r o b i c cultures were incubated in the presence of 2g/l chloramphenicol and 100mg/l cycloeximide to prevent aerobic growth and de n o v o p r o t e i n synthesis. Cell free extracts were prepared as previously described (i0). Yeast cells were converted to s p h e r o p l a s t s by i n c u b a t i o n in 50 m M p o t a s s i u m phosphate buffer (pH 7.7) containing i.i M s o r b i t o l and 1.5 mg of zymolyase /g w e t w e i g h t of c e l l s . S o l u b l e e x t r a c t s obtained after sonication of the spheroplasts were extensively d i a l y z e d to r e m o v e f r e e c o p p e r a n d w a s a s s a y e d for s e l e n i u m -dependent and selenium-independent GPX activities (Ii). S p e c i f i c a c t i v i t y was e s p r e s s e d as ~ m o l e s of N A D P H o x i d i z e d x mln x mg proteln. Protein was determined by the m e t h o d of B r a d f o r d (12).

RESULTS The activity both

could

types

is

one

in

human

than

results

in

of

hundred

reported be

detected

in

glutathione fold

erythrocytes rat

in t a b l e

hepatocytes

1 show

aerobic

peroxidase,

less

in

(13)

yeast and

(14).

a

yeast

significant

cultures

although than,

one

When

that

for

thousand

cells

the

were

for value

instance, fold

grown

less under

TABLE I Effect of oxygen on yeast GPX activity

Growth condition

GPX (seleniumdependent)

GPX (seleniumindependent)

Aerobic* Anaerobic**

0.33 ± 0.08 0.12

3.52 ± 0.17 28.43

Activities are expressed as pmoles/min/mg protein (x 103 ) * The values reported are means ± S.D. of at least three exper~ ments. ** The results were obtained from a single set of experiments.

1202

Vol. 147, No. 3, 1987

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

TABLE Effect of copper

on GPX activity

II

in the presence

and in the absence

Aerobiosis* GPX (seleniumdependent)

Cu2+](x

of oxygen

Anaerobiosis**

GPX (seleniumindependent)

GPX (seleniumdependent)

GPX (seleniumindependent)

104M)

0.04 1.00 2.50 5.00 i0.00

0.33 0.58 0.80 1.15 1.20

-+ -+ -+ -+ -+

0.08 0.17 0.20 0.06 0.03

3.52 4.25 4.71 7.23 10.50

_+ ± ± _~ -+

0.17 0.25 0.22 0.38 0.80

0.12 0.91 3.00

28.3 36.1 63.4

ctivities are expressed as ~ m o l e s / m i n / m g protein (x 103 ) The values reported are means ± S.D. of at least three experiments. * Results were obtained from a single set of experiments.

strictly GPX of

was

and

2

conditions

detectable,

activity

Table of

anaerobic

was

reports

high

while

higher the

three

fold

the

of

increase type

in

A

pendent"

type

absence enzyme to

conditions.

of

of

oxygen

activity

a lesser

was

the

copper.

was

as

Also

increased,

the

overall

presence

"selenium-dependent"

and

activity of

induced the

under

in

oxygen.

fold

increase

activity

of

type

four

GPX

large

yeast An

the

of

well.

presence

growing

for

"selenium-independent" aerobic

in

of

"selenium-dependent"

"selenium-independent"

than

effects

concentration

no

were

the by

measured

"selenium-decopper

in

the

"selenium-independent"

these

conditions

although

extent.

DISCUSSION

The

data

presence

of

of

in

yeast

available GPX

spite

in of

the GSH

up

to

date,

(5)

seem

ant±oxidative

enzyme

being

in

present

1203

yeast

to

exclude

defense at

the

system

relatively

Vol. 147, No. 3, 1987

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

high concentrations strain

of

yeast

hydroperoxide

metabolism

is

pendent"

patterns

present

to the

gously

to

other

the

in

it

is

important

of u s i n g nal

the

be

sive

to

First red

fact

that

all,

when

cells

for

the

substrates, activity

to

and

and

the

can

addition the

the c o p p e r

This

indicates

compounds, an

effect that

to

increase

was

metal

also ion

sis

direct

faction.

Work

metals

may

similar

whether per

other

effect"

enzymes

is

related

have

representative to b i o l o g i c a l

of

was

not

which

it

should

be

absence not

greater

of oxygen. as a re-

biosynthe-

to e s t a b l i s h

and if the

general

H202

remarked

only

the e n z y m e

On

thiols

increased was

effects

as

is like-

intracellular

in the

the

peroxidase.

is in p r o g r e s s

oxygen

disappea-

which

a more

in v i e w

metabolism.

GPX,

to s t i m u l a t e

functio-

GSH-transferase

of

is a c t i n g

but

capable

peroxides

resulting

of

dox c o m p o u n d , in a m o r e

is l i k e l y

the

of

However

seen

This

type

rate

The

activity

to the culture,

with

type.

the

this

by

are

that

to be r e s p o n -

organic

explained

of C u S O

"Se-dependent"

that

be

with

view,

oxygen

anaerobiosis.

4 autoxidation

produced

to

each

However,

evident

found

peroxidase

measurable

of

that

even m o r e was

analo-

from

enzymes.

point

related

in

associated

reducing

generation, for

this is

increase

other

grown

activity

the c o n t r a r y , ly

stimuli

which,

of p e r o x i d e s .

of y e a s t

corres-

it is i m p o s s i b l e

organisms

is

H202-specific

are

that

finding

of

"Se-inde-

this

forms

purified

scavenger

GPX a c t i v i t y

the

the

and

distinguished

phylogenetic

among

this

physiological

of

case

of

on

pathway

a n d the e n z y m e

If

Se as c o f a c t o r ,

data

G S H as e n z y m a t i c

yeast

of two e n z y m e

the

included

in

are

for a w i l d

peroxidatic

specificity.

system,

of

from

significance

of the

presence

here

"Se-dependent"

substrate

absence

the

the

or not h a v i n g

say

reported

operative

both

mammalian

to

could

that

is

as

of

actual

by h a v i n g

yeast

The r e s u l t s

demonstrate

activity

ponds

(15).

"cop-

induction

of

activities.

A C K N O W L E D G E M E N T S . We are g r a t e f u l to Dr. L u i g i A l l i e v i (Depar~ m e n t of F o o d S c i e n c e T e c h n o l o g y and M i c r o b i o l o g y of M i l a n Uni-

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Vol. 147, No. 3, 1987

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

versity), for p r o v i d i n g a s s i s t a n c e in the p r e p a r a t i o n of anae robic e x p e r i m e n t . We w o u l d like to t h a n k Ms. P a t r i z i a C i v i t a reale for t e c h n i c a l assistance. This w o r k was s u p p o r t e d in part by " P r o g e t t o S t r a t e g i c o B i o t e c n o l o g i e " of CNR.

REFERENCES

i) 2)

3) 4) 5) 6) 7) 8) 9) i0 ii 12 13

14)

15)

M a n n e r v i c k , B. (1985) M e t h o d s Enzymol. ll3, 490-495. Wendel, A. (1980) in E n z y m a t i c Basis of D e t o x i f i c a t i o n , (Jacoby W.B., ed.) vol. i, pp. 333-348 Academic Press, N e w York. Prohaska, J.R. and Ganther, H.E. (1977) Biochem. Biophys. Res. Commun. 76, 437-445. Smith, J. and Shrift, A. (1979) Comp. Biochem. Physiol. 63B, 39-44. Overbaugh, J.M. and Fall, R. (1985) Plant. Physiol. 77, 437-442. Goscin, S.A. and Fridovich, I. (1972) Biochim. Biopsy. A c t a 289, 276-283. Seah, T.C.M., Bhatti, A.R. and Kaplan, J.G. (1973) Can. J. Biochem. 51, 1551-1555. Hassan, H.M. and Fridovich, I. (1977) J. Biol. Chem. 252, 7667-7672. Lee, F.J. and Hassan, M. (1985) J. Free Rad. Biol. Med. i, 319-325. Faye, G., Kujava, C. and Fukuharah, H. (1974) J. Molec. Biol. 88, 185-203. Lawrence, A.R. and Burk, R.F. (1976) Biochem. Biophys. Res. Comm. 71, 350-357. Bradford, M.M. (1976) Anal. Biochem. 72, 248-254. Rotilio, G.r C i r i o l o M.R. and Mavelli, I. (1986) in Free Radicals and A r t h r i c D i s e a s e s (Swaak, A.J.G. and Koster, J.F., Eds.) pp. 185-189 Eurage. Mavelli, I., Ciriolo, M.R., Dini, L. and Rotilio, G. (1986) in S u p e r o x i d e and Superoxide Dismutase in Chemistry, B i o l o g y and M e d i c i n e (Rotilio, G. ed.) pp. 425-428 Elsevier Science Publishers. Jaspers, C.J., Gigot, D. and Pennickx, M.J. (1985) P h y t o c h e m i s t r y 24, 703-707.

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