Activation of microsomal glutathione S-transferase activity by sulfhydryl reagents

Vol.

87, No.

April

3, 1979

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS Pages

13, 1979

ACTIVATION

OF

MICROSOMAL

Ralf Department

GLUTATHIONE

Morgenstern.

of

Joseph

Biochemistry,

February

7,

W.

ACTIVITY

DePierre,

BY SULFHYDRYL

and Lars

Ernster

University

of

Arrhenius Laboratory, 91 Stockholm, Sweden

S-106

Received

S-TRANSFERASE REAGENTS

657-663

Stockholm,

1979

SUMMARY Rat liver microsomes exhibit glutathione S-transferase activity with 1-chloro-2.4-dinitrobenzene as the second substrate. This activity can be stimulated 8-fold by treatment of the microsomes with N-ethylmaleimide and 4-fold with iodoacetamide. The c&responding glutathione S-transferase activity of the supernatant fraction is not affected by such treatment. These findings suggest that rat liver microsomes contain glutathione S-transferase distinct from those found in the cytoplasmi and that the microsomal transferase can be activated by modification of microsomal sulfhydryl group(s).

INTRODUCTION Glutathione the

biotransformation

compounds have in

S-transferases

and,

broad

both

microsomal

view

date

4).

see

our

efforts

the

microsomal

be

stimulated

activity

greatly

whereas

the

by

treatment.

corresponding

Abbreviations: IAA, iodoacetamide;

is

CDNB,

by

to

the

has

due

to

towards

role

These

been

the

that

the

enzymes one

(for

the

microsomes

activity

of

the

supernatant

most

a recent

microsomal

we

discovered

(CDNB) with

sulfhydryl

fraction

NEM,

re-

glutathione

I-chloro-2,4-dinitrobenzene of

of

(2,3),

contamination,

treatment

another

occurrence

reported enzymes

cytoplasmic

1-chloro-2,4-dinitrobenzene; DTNB, 5,5'-dithiobis-(2-nitrobenzoic

in active

resemble though

cytoplasmic

demonstrate

(I).

substrates Even

activity on

not

most

important

pharmacologically agents

hydrophobic.

concentrated

activity

that

but and

an

including alkylating

S-transferase

have In

S-transferase

such

potential

electrophilic

play

xenobiotics,

specificities;

glutathione to

many

especially,

substrate

being

studies

of

(E.C.2.5.1.18)

can reagents;

is

not

affected

N-ethylmaleimide; acid).

UUU6-291x/79/070657-07$Ol.C0/0

657

Copyright @ 1979 by Academic Press. Inc. All rights of reproduction in my form reserved

Vol. 87, No. 3, 1979

MATERIALS

BIOCHEMICAL

AND METHODS

Glutathione, N-ethylmaleimide from the Sigma Chemical and purchased from common

tained grade

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

(NEM), and iodoacetamide Co. ALL other chemicals were commercial sources.

(IAA) were of reagent

ob-

Liver microsomes and the supernatant fraction from 180-200 g male Sprague-Dawley rats that had been starved overnight were prepared as described previously (51, with the minor modification that the microsomes were washed twice with 0.15 M Tris-CL, pH 8, in order to remove contaminating cytoplasm (additional washes had no effect on the microsomal glutathione S-transferase.activity). ALI experiments were performed on material prepared the same day. Treatment with NEM and IAA was carried out as follows: 1.5-2.2 mg protein of the microsomal or supernatant fractions was incubated at room temperature in 90 (or 80) mM sodium phosphate, pH 7.5-25 (or 12.5) mM sucrose in a total volume of 1 ml and with IAA (or NEH) at the concentrations indicated in the tables. Before treatment with NEM the supernatant fraction was passed through a Sephadex G-25 M column equilibrated with 0.25 M sucrose in order to remove low molecular weight thiols (chiefly glutathione). ALiquots were withdrawn at the times indicated and assayed for glutathione Stransferase activity with CDNB as described by Habig et al. (6). except that 3 mM glutathione was used in the assay in order to saturate the microsomal enzyme. The aliquots taken were small enough so that the concentration of NEM (or IAA) in the assay medium was never more than l/60 (or 116) of the glutathione concentration. Determination 5.5'-dithiobis-(2-nitrobentoic Protein albumin

serum

of

fast-reacting

sulfhydryl (DTNB)

acid)

was determined as the standard.

by

the

method

groups

was

carried

al.

(8)

out

with

(7). of

Lowry

et

with

bovine

RESULTS Treatment creases

the

fold

in

al

activation

Low

50

is

more

No change

I).

was

assured

the

activation

nearly

groupsl

protein.

These

in

the

reactive in

activation towards

the

liver

microsomes

When by

lower

by

NEM

microsomaL

for remains

measured

with

10

of min.

suggest

microsomal

that

glutathione

microsomal

glutathione

sulfhydryl S-transferase

658

With

was the

pH 7.5

NEM were

in-

about

8-

used,

maxim-

concentrations and

microsomal

DTNB,

at

CDNB

6-7-foldi

The with

observations

NEM than

assayed

about

activation.

of

1 mM NEM

concentrations

incubation

as

with

activity

half-maximal

sulfhydryl

nmoles/mg involved

rat

S-transferase

(Table

uM.

gives

reacting

fresh

glutathione

1 min.

as

still

of

IO

content found

to

be

fast-

about

40

group(s)

S-transferase

activity

PM NEM of

sulfhydryl

groups

as

activity in was

general. found

in

Vol.

87,

No.

BIOCHEMICAL

3, 1979

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Table

Effect of microsomesa

NE11 treatment

on

I

Glutathione-S-transferase

activity

mf4

Incubation

time

0

96.3

-+ 1.1

112

+ 0.6

1.16

0.005

10

137

_+ 3

1.94

0.01

10

321

t

4

3.33

0.05

10

591

_+ 16

6.14

0.1

10

646

_+ 26

6.71

1

1

733

_+ 44

7.77

1

10

664

_* 16

7.04

microsomal

Quadruplicate

of

determinations

in

added

before

Originallyr

between

the

thione

S-transferases

natant

fraction

min.

were

with

of

addition

known

of

rat

liver

NEM

for

the

to

be

of

with

the

microsomal

when

an

NEM

(not

rats shown

and are the

excess

of

gluta-

shown). in

attempt

catalyzing inhibitor

the

mM IAA

(another

distinguish

of

used

here

CDNB

(Table

sulfhydryl

S-transferase

S-trans-

cytoplasmic

treatment

periods

to

glutathione of

with

glutathione

three

values

a IO-fotd

assayed

659

of mean.

performed

time

10

the

However.

short

activity

enhances

NEM or

(9).

S-transferase microsomes

of

proteins

is

each

The

error

were

soluble

on

calculated.

of

experiments and

NEM

performed

were standard

absence the

microsomal

activity.

Treatment

the

these

ferase

glutathione

protein/ml

of these quadruplicates these averages t the

incubated was

20

_+0.8

10

controls

for

94.3

0.001

mg of

liver

treated/control

10

averages means

its

minb

0

b

thione

nmollmg

min.

1

a 1.5

rat

Activity

Conditions: NEM.

in

glutathe

did

supernot

affect

II). reagent) activity

about

Vol. 87, No. 3, 1979

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

II

Table Effect supernatant

of

Condi NEM,

IAA

NEM and fractiona

tions

on

glutathione

S-transferase

activity

min.

nmol/mg

1017

_+ 1

0

IO

1006

_* 28

1

1

1044

_+ 33

1

10

993

_+ 13

0.1

10

1077

_+ 32

0.01

10

966

_+ 18

1

1333

_+ 44

nt4

0

0

60

1297

_+ 10

10

60

1410

_+ 17

1

60

1383

_+ 49

aIAA treatment centration (not with DTNB uas NEM treatment Thiol concentration bLiver soluble Quadruplicate ages of these

means

4-fold

of

l

1).

The

of

to

mM IAA

use

reach

(see

S-transferase

performed with 1.5 mg of soluble protein/ml. precipitable by 5 % TCA (Trichloroacetic acid)) 60-80 PM. was performed uith 1.6-1.7 mg of desalted soluble (not precipitable by 5 % TCA) was not more protein -SH is around 100 nmol/mg protein (11). determinations were performed on each of two rats quadruplicates were calculated. The values shown

averages

required

10

uas

these

(Fig.

activation with

b

min.

1

time

the

Activity time

0

IAA,

of

liver

:

Incubation

mM

treatment of rat

also in

the

lower

maximat Table

standard

III).

is

seen

of

the

concentrations

activation the

manner. (Table

IAA

decreases

and are

both the

supernatant very

protein/ml. than 4 DM. averthe

mean.

of

and

When

a corresponding

activity

error

Thiol conas measured

prolongs extent

fraction

little

change

of is

in

the this

treated

glutathione

II).

DISCUSSION Microsomal substrate

is

glutathione enhanced

many-fold

S-transferase by

activity

treatment

660

with

with both

CDNB NEM and

as

the

IAA.

second The

ac-

Vol.

87,

No.

3, 1979

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

-

I 30

I 60

I 90

TIMElm

Fig.

1.

Effect of IAA on glutathione S-transferase activity microsomes (2.2 mg protein/ml). Measurements were ruplicate on material from 1 rat and performed as the methods section. l = 10 mM. I= 1 mM. A= 0.5 IAA.

Table Effect of microsomesa

IAA

treatment

on

III

Gtutathione-S-transferase

activity

Conditions: IAA.

mM

in

Incubation

time

min.

nmollmg

1

minb

64.8

_+ 2.6

71.3

_+ 3.5

90

0.5

90

207

_+ 6.5

2.90

1

90

246

f

7.0

3.45

10

60

274

% 12

3.96

b

mg of

see

tivation

microsomal

footnote

thus

whether

which

catalyzes

tein.

e.g.

b to

protein/ml table

apparently

this

inhibitor.

I.

results

sulfhydryl the

an

liver

treated/control

0

a 2.2

rat

Activity

0

but

in rat liver done in quaddescribed in mM, o = 0 mM

from

group(s)

glutathione

attack

is

on

actually

S-transferase

remains

to

be

661

microsomal

sulfhydryl

localized activity

established.

on or

In

on

the

group(s);

the some

case

protein other

of

pro-

fructose

Vol.

87,

I,6

No.

3, 1979

BIOCHEMICAL

diphosphatase

[E.C.3.1.3.11

modification

of

In

either

transferase

groups

on

case

the

possibility

is

group

form,

the

S-transferase

this

increases

thione

in

strates philic

is, It

may

vivo ----

hydryl

it

been

has

cyclic

AMP

enzyme

is

react

with

can

extentr

greatly

S-transferase

question

the

whereas

after

substrates be

expected

after

system

have

the

microsomal

involving the

an

liver

maximal

activation

for

the

gluta-

to

dissolve

ex-

become

conversion of

even

non-activated

total

xenobiotics

only

S-

the

to

subelectro-

endoplasmic

greater

role

to

play

glutathione

and Levels

activated

to

that

its

own

groups

group

the

diurnally,

possibility

substrates, both

involved.

between

varies

Another

S-transferase

sulfhydryl formation

occursl

(11~1.8).

sulfhydryl

the

disulfide

glutathione

by

glutathione

and

is which

enzymically

that

electroto

a certain

of

different

activities a different can

only

responses to

sulfhydryl

enzyme(s) be

answered

of

in by

soluble

and

reagents the

probably

microsomal

isolation

of

microsomal

fraction. the

microsomal

glutathione

reflect Of

the course.

We would suggestions

like

to and

thank

Drs.

T.

Bartfai

advice.

662

and

B.

Mannervik

enzyme(s).

for

prethis

ACKNOWLEDGEMENTS

helpful

is

the

being and,

sulf-

non-enzymically. The

sence

of

demonstrated

by

tic.

of

addition

proteins

% of

hydrophobic

may

by

figures.

activation

of

a mechanism

5-10

would

P-450

activity

groups

microsomal

many

occur

glutathione

in

most

and

cytochrome

these

that by

affected

phi

be

occurs

Indeed,

the

microsomal by

Since

S-transferases

by

indicated

%.

since

glutathione

the

30

to

that

only

CDNBi

hydrophobic and

intermediates

than

about

by

calculated

for with

shown

microsomal

vivo ----

be

COMMUNICATIONS

(10).

that

--in

accounts

are

the

reticulum,

--in

to

membranes;

for

regulated can

been

itself

arises

It

RESEARCH

has

protein

activity

S-transferases

tensively

being

fraction

glutathione figure

the

modification.

microsomal

BIOPHYSICAL

1 activation

-SH

activity

sulfhydryl

AND

their

Vol.

This

87,

No.

study

"Polycyclic

BIOCHEMICAL

3, 1979

uas

supported

Hydrocarbon

by

AND

National

Metabolism

BIOPHYSICAL

Cancer in

the

Institute Respiratory

RESEARCH

Contract

COMMUNICATIONS

No.

NO1

33363,

Tract".

REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10.

11. 12.

Grover. P.L. (1977) in Drug Metabolism - Microbe Man, Symp. (Parke. D-V., Smith, R-L.. eds.) pp. 105-122. Taylor & Francis, London. Booth. I., Boyland, E. and Sims, P. (1961) Biochem. J. 79, 516-524. Kraus, P. (1978) in Conjugation Reactions in Druq Biotransformation. CA. Aitio, Ed.) p. 503, Elsevier/North Holland B;omedical Press, Amsterdam. Jakoby, W-B. (1978) Advances in Enzymology (Meister. A., ed.) Vol. 46, pp. 383-414. Ernster, L. Siekevitz, P. and Palade, G-E. (1962) J. Cell Biol. 15. 541-562. Habig. W.H., Pabst, M-1. and Jakoby. W-B. (1974) J. Biol. Chem. 249, 7130-7139. ELLman. G.L. (1959) Archiv. Biochem. Biophys. 82. 70-77. Lowry, O.H.. Rosebrough. N.J.. Farr, A.L. and Randall, R.J. (1951) J. Biol. Chem. 193, 265-275. Askelbf, P., Guthenberg, C., Jakobson, I. and Mannervik, 8. (1975) Biochem. J. 147, 513-522. Pontremoli, S. and Horecker, B.L. (1970) in Current Topics in Cellular Regulation (Horeckerr B-L.. Stadtman, E-R., eds.) Vol. 2, pp. 173-199. Academic Press. New York and London. Isaacs, 1. and Binkley, F. (1977) Biochim. Biophys. Acta 497, 192-204. Isaacst J-T. and Binkley, F. (1977) Biochim. Biophys. Acta 498. 29-38.

663