Effect of S-adenosylhomocysteine on sulfhydryl xenobiotic transmethylases in rat liver

Vol.

128,

No.

2, 1985

BIOCHEMICAL

AND

RESEARCH COMMUNICATIONS

BIOPHYSICAL

Pages

April 30, 1985

Sulfhydryl

Effect of S-Adenosylhomocysteine Xenobiotic Transmethylases

965-971

on in Rat Liver

Loo and John T. Smith

George

Department of Nutrition and Food Sciences College of Home Economics and Agricultural Experiment Station The University of Tennessee, Knoxville, TN 37996-1900 Received

March

19,

1985

SUMMARY Rat liver cytosolic thiopurine methyltransferase and microsomal thiol methyltransferase were each found to be subject to control by the absolute molar ratio of S-adenosylmethionine to S-adenosylhomocysteine using cell-free enzyme preparations. As this ratio was lowered, inhibition of both sulfhydryl xenobiotic transmethylases occurred. On the other hand, when the ratio was decreased in vivo by the administration of D,L-homocysteine thiolactone to animals, tFs=ration was accompanied by an inhibition of only thiopurine activity. Thiol methyltransferase activity was not methyltransferase significantly affected after drug treatment, which would suggest that there is a 0 1985 compartmentalization of S-adenosylhomocysteine in the intact hepatocyte. Academic

Press,

Inc.

Transmethylation including

xenobiotic

catalyze

the

is

thiol

S-methylation

enzymic

in the

counterpart.

(81,

the

thiopurine

Abbreviations

(2,5)

drug,

been

the

as the

can also

S-methylate

SAM, S-adenosylmethionine;

965

more recently

other

subcellular

to be separated

(6,7),

of agent, it

some aryl

its

include the

(4).

is frcm

methyltransferase

metabolism

Recently,

thiol

methyltransferase

captopril

antineoplastic

(5).

in

disulfiram

also

to

(EC 2.1.1.67).

although

reported

(l),

in

(l),

thiol

known

compounds:

thiopurine

for

and

2-thiouracil

methyltransferase

used:

(9),

such

enzyme

and appears

substrates

involved

sulfhydryl

contrast,

cytosol

processes, are

methyltransferase

also

In

dithiothreitol

substances,

antithyroid

has

biochemical

methyltransferases

thiopurine

and 2,3-dimercaptopropanol

is

important

foreign

as a microsomal

Exogenous

methyltransferase pharmacological

and

(2,3,4).

localized

Z-mercaptoethanol thiols

various

activity

fractions

apparently

of

reported

methyltransferase

many

Two similar

(EC 2.1.1.9)

was first

particulate

in

metabolism.

methyltransferase The former

involved

alkane

Thiopurine some

important

6-thiopurine, was thiols

and

reported

that

(lo),

which

SAH, S-adenosylhomocysteine. 0006-291X/85 $1.50 Copyright 0 1985 by Academic Press, Inc. All rights of reproduction in any form reserved.

Vol. 128, No. 2, 1985

interestingly

is

a catalytic

Nevertheless,

(11). thiopurine

it

donor.

of

methyltransferase enzyme

(3)

preparations.

are

xenobiotic

the

cell

has

shared

asserted

distinct

is

the

reaction,

and

thiopurine such,

it

shown

to

thiol

thiol

methyltransferase

methyltransferase

by thiol

methyltransferase

achieved

using

SAH, is

a potent

inhibitor

of

methyltransferase

(2)

using

SAM as

plausible

that

metaholism

intracellular

control

some

and

(2).

either

and the

of the

by

that

enzymes

is

tra'nsmethylases

by modulation

been

also

transmethylation,

As

sulfhydryl intact

been

methyltransferase,

The product

ratio

has

xenobiotic

thiopurine

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

feature

methyltransferase

Sulfhydryl or

BIOCHEMICAL

the

methyl both

thiol

cell-free

SAH may

regulate

of their

substrates

SAM:SAH ratio. other

group

the in

Altering

methyltransferases

this

--in

vivo

adenosine

and

(12,13,14). Recently,

it

was demonstrated

D,L-homocysteine levels

of

thiolactone

SAH --in

D,L-homocysteine in

the

of

in

order

of protein to

more

liver

SAM:SAH ratio

thiol

present

of D,L-homocysteine

study

methyltransferase

was altered

effect

This

--in

vitro

of

a marked

shown

increase

later

elevation to

was accompanied

the

--in vivo of

the

was conducted

to

compare

with

thiopurine that

obtained

(15), (16).

sulfhydryl the

methyltansferase --in vivo

to

by a decrease

--in vitro

regulation

and

tissue

(EC 2.1.1.6)

as measured methylation

in

be attributed

catechol-O-methyltransferase

and phospholipid define

co-administration

produce

(EC 2.1.1.8)

clearly the

transmethylases, rat

mouse brain

the to

an

(17).

methyltransferase

by inhibition

able

(15,16),

thiolactone

activities

histamine-N-

vivo

is

that

via

and

and also Therefore, xenobiotic

activities when

of the

administration

thiolactone. MATERIALS

AND METHODS

Materials: [I4C-methyl]-S-adenosyl-L-methionine (sp. act. 49 mCi/mmole) was obta'ned from ICN Chemical and Radioisotope Division, Irvine, CA, as was the a d enosine (52 mCi/mmole) needed for the synthesis and purification of [8-I+ All other chemicals were purchased from the S-C8-1 Cladenosylhomocysteine (18). Sigma Chemical Co., St. Louis, MO. Male Sprague-Oawley rats (350-425 g bwt) were used in Animals and Treatment: Animals all experiments and maintained on standard chow and water, ad libitum. receiving D,L-homocysteine thiolactone were injected intraperitoneally (500 or After 1000 mg/2ml 0.9% NaCl/kg bwt) and sacrificed 40 minutes later. One decapitation, livers were rapidly excised and sectioned into two pieces. piece was immediately placed in ice-cold 5mM potassium phosphate buffer, pH 7.5, to be stored at -80" C for future enzyme analysis. The other piece was quickly placed in ice-cold 10% TCA and a 1:5 (w/v) homogenate prepared to prevent degradation of SAM (19) during quantitative analyses of SAM and SAH (see below). Tissue levels of SAM la4nd SAH were Quantitative Analyses of SAM and SAH: with [ C-methyl]-Sthe ether-extracted TCA supernatant, determined in

Vol. 128,No.

2, 1985

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

adenosyl-L-methionine and S-[8- 14C]adenosylhomocysteine as internal standards, using a coupled radiochromatographic-spectrophotometric procedure (18). :;;yT,t\;say:: For the assay of the sulfhydryl xenobiotic transmethylases, a tissue homogenate was prepared in ice-cold 5 mM potassium phosphate buffer, pH 7.5. Following centrifugation at 10,000 x g for 10 minutes at 4' C, the supernatant was next centrifuged at 100,000 x g for 60 minutes. The specific activities of thiol methyltransferase in the microsomal fraction and thiopurine methyltransferase in the "soluble" fraction were assayed using the radiochemical procedures of Rorchardt and Cheng (3) and Walker et al. (20), respectively. Substrate concentrations for SAM and v-mercaptoethanol in the thiol methyltransferase assay were 1.1 and 9.6 mM, respectively. For the assay of thiopurine methyltransferase, 23 UM SAM and 9.7 mM 6-thiopurine were used. Protein was determined by the method of Ohnishi and Barr (21) with bovine serum albumin as the standard. Evaluation of statistical differences were performed using ANOVA Statistics: and Duncan's new multiple range test (22).

RESIJLTS Decreasing enzyme

the

SAM:SAH ratio

incubation

mixture

resulted

methyltransferase

activity,

the

of SAM was 10,

concentration

effect

of the

the (Figure

of

shown

thiolactone both

a

100,

or

120

1.

This

A somewhat

SAM and

of SAH in the

inhibition

methyltransferase

PM

level

ratio-related

or 1000 PM.

on thiopurine

40,

by

the

SAM and SAH --in

of

SAM:SAH ratio 0.67

there

vivo.

in the molar

inhibition

only

mg/kg

by about

Table

However, drug

the

of

occurred similar

thiol whether

regulatory

was also appropriate

observed level

was no significant

administration

of

increase

in the

in

of

SAH

with

500 or This

the

in

the

activity

of

on the

1000

group

mg/kg,

IJnder

the

of thiol

of

relative

as reflected

was associated

to 0.91

thiopurine

activity

levels

Specifically,

control

respectively.

effect

tissue greater

in turn

methyltransferase. 3.19

D,L-homocysteine

was a substantially

of SAM to SAH.

from

35 and 63X,

there

treatment

ratio

bwt),

1,

a dose-related

thiopurine

was lowered

(1000

in

produced

of SAH following

by a decline

reduced

data

to animals

elevation

and

in

the

2). As

the

4,

by increasing

as can be seen from Figure

SAM:SAH ratio

presence

--in vitro

with

when (500 mg/kg

methyltransferase

the bwt) was

same conditions,

however,

methyltransferase.

DISCUSSIoN It

is

well

SAM-dependent thiopurine absolute both

established

that

SAH is

methyltransferases methyltransferase

molar

of these

ratio sulfhydryl

of

(23), (2).

SAM to xenobiotic

a potent

including However,

SAH may ultimately transmethylases 967

inhibitor

thiol our

--in

vitro

of

methyltransferase results

indicate

regulate

the

in

rat

liver

(3) that

activities (Figures

many and the of 1 and

Vol. 128, No. 2, 1985

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

100 90 80 f

0’

0

0

2

1

’

4

6

a

8

SAM : SAH

8

10

1

8

12

1

14

16

0

01 0

1 2

2

Ratio

1 4

6

8

1 12

10

SAM : SAH

1 14

1 16

Ratio

Figure 1 - Regulation of rat liver microsomal TMT in vitro by the SAM:SAH ratio. The livers fran three animals were pooled zmobtain the enzyme The assay procedure was perfoned with preparation used in these experiments. three different concentrations of SAM: 10 uM (@--O), 100 uM (O---Q), and The concentrations of SAH was appropriately adjusted in 1000 d (cl--a). each case to obtain the desired SAM:SAH ratio in the reaction mixures. Each point represents the average of three duplicate determinations. of rat liver cytosolic TPMT in vitro by the SAM:SAH Figure 2 - Regulation ratio. The livers from three animals were pooled %jobtain the enzyme preparation used in these experiments. The assay procedure was performed with three different concentrations of SAM: 4 uM (O---O), 40 uM (@--a), and 120 uM (o--a). The concentrations of SAJi was appropriately adjusted in Each each case to obtain the desired SAM:SAH ratio in the reaction mixures. point represents the average of three duplicate determinations.

TABLE

l-

Effect

Treatment

of levels

D,L-homocysteine of SAM and SAM

thiolactone administration and the activities of

SAH SAH

98 i

4a

tissue

TMT

cpm 32 *

liver

TPMT

SAM: SAH

nmol/g Saline (2 ml/kg

on rat and TllT

TPMT

x 10m3/mg

Pro

2a

3.19

*

0.33a

2.5

f

O-la

17.5

f

2.1a

bwt)

O,L-homocysteine thiolactone (500 mg/2 ml saline/kg but) D,L-homocysteine thiolactone (1000 mg/2 ml saline/kg but)

223

k 45b

253

f

31b

0.91

*

0.20b

1.6

t

0.2b

17.8

t

3.4a

360

t

560

t

62c

0.67

t

0.09b

0.9

t

0.3c

15.7

t

2.9”

35c

Animals were injected intraperitoneally represent the average f SEM for 5 rats. column are not significantly different

and sacrificed Data sharing (p>O.O5).

968

40 minutes later. a common superscript

Values in a

BIOCHEMICAL

Vol. 128, No. 2, 1985

This

21.

is

in

accord

with

AND BIOPHYSICAL

research

demonstrating

modulate

the

activity

of

2.1.1.17)

using

cell-free

enzyme preparations

that

altering

the

could

alter

thiol

the

proceeds

due

the

3.3.1.1),

a

secondarily

ratio,

however, is

conditions.

not This

of SAH to

once

this

drug

treatment.

to

achieved

purine

interact

deeply

l),

with

other the

has

been

words,

the

SAH-binding This

embedded

within

there

may be competition

also

limit

the

would the

for

accessibility

especially

of

synthesis

of

SAH

hydrolase of

in

SAH and

thiol

the

of

within these

SAM:SAH

thiopurine

the

by the

endoplasmic experimental

rather

impermeable

endoplasmic in

the

reticulum, cytosol

have been

if

thiol

it

methyltransferases the

the

level

due

to

methyltransferase

In addition,

to

after

available

methyltransferase

be true

of

Somewhat

same

SAH may not

bilayer.

SAH

the

cytosol.

synthesized

on

SAH among the

as

fall

such as the

generated

membrane

part

elevation

the

under

rapidly

site

precursory

the most

inhibition

localized

(28),

of

for

the

be explained

membranes

nucleoside

of

in

affected may perhaps

intact

an

which

may be due to inhibition

the

methyltransferase

observation

In

for situated

significantly

fully

Thus,

the

--in vitro.

S-adenosyl-L-homocysteine

because

also

activities

this

membrane

The latter

accounts

thiol

compartmentalization. is

--in vivo.

the

as measured

shown of

factors

control

adenosine,

(27).

Furthermore,

(Table

unexpectedly,

nature

enzyme

likely

methyltransferase

of

have

that

thiolactone,

endogenous

can (EC

activity

conceivable

hepatocyte

of

ratio

studies

the

methyltransferase

activity

cytosolic

most

is

may similarly

rat

presence

reactions.

this

reticulum

the

catalytic

of SAM, is

transmethylation

vivo

it

SAM:SAH

Other

regulate

D,L-homocysteine

permeates

to

can

Thus,

--in

of

In the

the

methyltransferase

(24,25).

vivo

and thiopurine

presumably

L-homocysteine.

(EC.

ratio

administration

compound

--in

(15,26).

SAM:SAH

methyltransferase Upon

ratio

as well

that

phosphatidylethanolamine

SAM:SAH

methyltransferases

RESEARCH COMMUNICATIONS

is

known

that

(2g),

which

could

site

on

thiol

liver

tissue

binding

methyltransferase. It

has been demonstrated

inhibit

(261,

the which

transferase. identical such vesicles

activity shares

of

microsomal

the

same

The endoplasmic to the

as

that

SAH-binding

sites

has

indicated

subcellular contains

of the

phosphatidylethanolamine %9

as

SAH-binding

SAM-dependent

can

methyltransferase

localization

methyltransferase. that

of SAH in

phosphatidylethanolamine

reticulum

phosphatidylethanolamine (31)

raising

thiol

sites

methyl-

which

methyltranferases Work

with

may be (30),

microsomal

methyltransferase

is

Vol. 128,No.

situated

2, 1985

to the

in essence Hence,

external

making

although

has

not

BlOCHEMlCALANDBlOPHYSlCALRESEARCHCOMMUNlCATIONS

this

the

in

SAH

may

be

positioned

luminal

side

the

the

the

endoplasmic

semi-sealed methyltransferase

thiopurine

(32),

it

sulfhydryl

However,

reticulum

This

has

xenobiotic the our

enzymic

SAH generated

the

microsomal

turn

after

could

implied

the from

drug

other

argument

have different treatement

endoplasmic

separated

SAH

can

l),

it

from

inhibit is

formation the

thiol

possible

of

binding

thiol

of

that

changes

to

"leaky"

or

SAH to

thiol

enzyme --in vitro.

associated the

the

morphological

the

allow

microsome

that

essentially

induced

activities

entity

of

cytosol.

the

speculate

(Figure

in

the

within

though

may have

membrane,

SAH in

interior

microsome,

even

inhibit

seems to support

methyltransferase

the

of the

transmethylase

same data

been

to

to

the

resulting

in

and therefore

Finally,

represent

fraction

microsomes.

in

preparations

microsomal

reticulum

accessible

reasonable

Thus,

microsomal

endoplasmic

methyltransferase

is

cytosol. using

of

thiol

it

methyltransferase isolation

of

delineated,

to the

of the

enzyme more

topography

methyltransferase i.e.

side

particular

the

been

reticulum,

(cytosolic)

research examined with that

in

the thiol

subcellular inhibited

(11)

that

the

present

microsomal

the

cytosolic,

two work

fraction.

methyltransferase localizations

only

the

and

(2),

since but

not

enzyme.

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