Immunochemical study on the participation of cytochrome b5 in drug oxidation reactions of mouse liver microsomes

Vol. 91, No. 1, 1979 November

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

14, 1979

Pages 207-213

IMMUNOCHEMICAL STUDY ON THE PARTICIPATION IN DRUG OXIDATION Mitsuhide

Noshiro,

Department

Nobuhiro

of Biology,

September

Harada,

Faculty

Higashi-ku, Received

OF CYTOCHROME b5

REACTIONS OF MOUSE LIVER MICROSOMES and Tsuneo

of Science,

Fukuoka

812,

Omura

Kyushu

University,

Japan

13, 1979

Summary: Highly purified divalent and monovalent antibodies against cytochrome b5, anti-b5 immunoglobulin G (IG) and anti-b5 Fab', were used in elucidating the role of this cytochrome in the drug-oxidizing enzyme system of mouse liver microsomes. Anti-& IG strongly inhibited not only NADHsupported but also NADPH-supported oxidation of 7-ethoxycoumarin and benzo(a)pyrene, but had no inhibitory action on the oxidation of aniline. Anti& Fab' also inhibited NADH-supported and NADPH-supported benzo(a)pyrene hydroxylation. These observations indicate an essential role of cytoch?ome &s in the transfer of electrons not only from NADH but also from NADPH to cytochrome P-450 in the microsomal oxidation of some drugs, but not of aniline. It

is

generally

monooxygenase also

plays

reactions

role

CoA desaturation microsomes.

that

(5,6)

However,

reactions

microsomes.

the second

the oxidation

of the process

&

to cytochrome

several

investigators

(7,8)

reported

purified

cytochrome

as into

oxidation

of aminopyrine

Lu and co-workers maximal cytochrome

rate

(9)

whereas

it

that

P-450

for

required

proposed

one cycle

cytochrome

incorporation

of b5

contrary,

of detergent-

inhibited

NADPH-supported experiments

bs was required

for

reactions

first

On the

of chlorobenzene

source‘

been made as

(1)

via

b5

oxidation

Reconstitution

cytochrome

oxidation was not

required

membrane

and benzphetamine.

of NADPH-supported P-450,

the

the microsomal

indicated

drug

of ethylmorphine. that

electron (4)

and Estabrook

supplied oxidation

Cytochrome

has not yet

in NADPH-supported

equivalents

the NADPH-supported

(l-4).

used as the

agreement

Hildebrandt

even in

in NADH-supported

hydroxylation

two reducing is

involved

microsomes

and laurate

general

of cytochrome

that

by liver

bs is

even when NADPH is

to the involvement of liver

cytochrome

catalyzed

an essential

in stearyl of liver

accepted

for

by the

by purified

the oxidation

of the

same

0006-291X/79/210207-07$01.00/0 207

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

BIOCHEMICAL

Vol. 91, No. 1, 1979

substrate

by cytochrome

stimulation

by cytochrome

catalyzed

supported

the role

oxidation divalent

and benzo(a)pyrene.

confirmed

the participation

of

reactions. NADH-supported

by mouse liver

microsomes

using

cytochrome

bs,

against

cytochrome

and NADPH-

in the oxidation hand,

of cytochrome

our is

highly and we

of 7-ethoxy-

immunochemical

in microsomal

-bs in the NADPH-dependent

significantly

the

N-demethylation

bs in both

of cytochonne varies

(10)

benzphetamine

oxidation

On the other

the participation

reactions

and Sato

supported

antibodies

of the

The role

monooxygenase

drugs

and monovalent

coumarin

hydroxylation.

which

drug

of several

the participation

support

Imai

of cytochrome

confirmed

not

system,

-bs in NADPH-dependent

We studied

did

Recently

bs of NADPH-dependent

by a reconstituted

cytochrome

purified

P-448.

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

according

to the

study aniline microsomal

substrates

used. MATERIALS

AND METHODS

Male albino mice (DDD strain, 15-25 g body weight) were kept on a commercial rat chow (Nippon Clea Co.). They were starved for 20 h before use, and liver microsomes were prepared as described previously (11). Cytochrome bs was purified from rat liver microsomes by the method of Omura and Takesue (12) as modified by Ito (13). Antibody against cytochrome bs was prepared as described previously (11). The immunoglobulin fraction was purified by ammonium sulfate fractionation and specific antibody was obtained by the affinity chromatography using an antigen-conjugated Sepharose 4B as described previously (11). Monovalent fragment of the purified antibody (Fab') was prepared by a modified procedure of Nisonoff et al. (14), the detail of which will be published elsewhere (15). NADPH-cytochrome c reductase and NADH-cytochrome c reductase activities were determined as described previously (12). ‘I-Ethox&oumarin O-deethylation, benzo(a)pyrene hydroxylation, and aniline hydroxylation were assayed as described by Ullrich and Weber (17), Nebert and Gelboin (18), and Imai When antibody was added to assay mixtures, et al. (19), respectively. microsomes were preincubated with the antibody for 10 min. The concentrations of immunoglobulin in each set of assay mixtures were made up to the same by the addition of control immunoglobulin obtained from non-immunized rabbits. Protein concentrations were determined by the method of Lowry at al. (16) using bovine serum albumin as the standard. RESULTS As shown NADH-cytochrome affecting the Fab'

in Table

I,

c reductase

NADPH-cytochrome was significantly

purified

anti-&

activity

IG and anti-&

of mouse liver

5 reductase

activity,

weaker

that

than

208

Fab'

microsomes although

by the IG.

the

inhibited without inhibition

The specificity

by of

BIOCHEMICAL

Vol. 91, No. 1, 1979

AND BIOPHYSICAL

Table Effects of anti-bs IG and anti-b5 cytochrome c reductase activities

I

Fab' on NADH- and NADPH-supported of mouse liver microsomes.

control IG anti-b5 IG (5 mg/mg MS protk) (X) 100 21.6

NADH-cyt. 2 reductase NADPH-cyt. c reductase

100

RESEARCH COMMUNICATIONS

control Fab' anti-& Fab' (20 mg/mg MS protein) (X) 100 69.6

94.9

100

97.7

The reaction mixture contained microsomes, antibody, 20 nM cytochrome c, 0.5 mM KCN, and 0.1 mM NADH or 0.1 mM NADPH in 2 ml of 0.1 M potassium phosphate buffer (pH 7.5). The amounts of microsomes were 0.05 and 0.1 mg for NADH-supported and NADPH-supported reactions, respectively. Control activities were 0.763 and 0.213 pmoles of cytochrome c reduced per min per mg of microsomal protein for NADH-supported and NADPHsupported reactions, respectively. Neither control IG nor control Fab' affected the activities. MS stands for microsomes. the

antibody

bs's

were

and the antigenic also

described

confirmed

previously

when incubated

inhibited lower

enhanced

reaction

our previous

2).

did

analysis

not

although

only

(ll),

form

as

precipitates

the

extent

0-deethylation action

as reported

by Mannering

et al.

for

but

also

was not

sensitive

NADPH-supported

by the antibody protein

where

benzo(a)pyrene

(Fig.

3) were

the oxidation both

strongly

209

of NADH

by about

portion

the N-demethylation further

of

inhibited

electron

the

donor

to the inhibition

(Fig. by anti-

reaction

of 10 mg antibody

of 7-ethoxycoumarin inhibited.

20 %.

enhanced

nor NADH-supported

even at a concentration

of microsomal

was significantly

on this

as when NADPH was the sole

hydroxylation Neither

(20)

was also

The addition

an inhibitory

microsomes

reaction

of inhibition

reaction.

anti-&s

of 7-ethoxycoumarin

NADPH-supported

exerted

liver

purified

0-deethylation

The corresponding

to the same level

b5 IG (Fig.

and mouse cytochrome

diffusion

observations

of the NADPH-supported

by rat

The aniline

inhibited

antibody

of the NADH-supported

the rate

ethylmorphine reaction

The monovalent

the NADH-supported

1.

that

IG not

of the

double

by the antibody, than

Anti-Es

with

inhibited

as shown in Fig.

rat

the antigen.

In accordance IG strongly

between

by the Ouchterlony

(11).

with

similarity

Therefore,

(Fig.

was per mg 1) and the

1).

Vol. 91, No. 1, 1979

BIOCHEMICAL

AND BIOPHYSICAL

Oo Anti-b5

IG

(mglmg

1 2 MS protein)

RESEARCH COMMUNICATIONS

3

4

5

Fig. 1 Effects of anti-bs IG on 7-ethoxycoumarin 0-deethylation. The reaction mixture contained microsomes, antibody, and 1 mM 7-ethoxycoumarin in 2 ml of 0.1 M Tris-HCl (pH 7.6). Microsomal protein concentrations were 0.1 and 0.2 mg for NADPH-supported and NADH-supported reactions, respectively. The assay was started by the addition of 0.1 r&f NADPH and/or 0.1 rd-4 NADH at 30 'C. Control activities were 4.50, 5.34, and 0.259 nmoles of product formed per min per mg of microsomal protein for NADPH-supported (-), NADPH plus NADH-supported (p--d), and NADH-supported (s--a) reactions, respectively. In order to indicate the synergistic effect of NADH, the control activity in the presence of NADPH plus NADH is shown in the Figure at 120 %. Fig. 2 Effects of anti-bs IG on aniline hydroxylation. The reaction mixture contained 0.4 mg microsomes, antibody, 10 n@l aniline, 0.1 mM EDTA, and NADPH-generating system or 5 mM NADH in 2 ml of 50 mM Tris-HCl (pH 8.0). Incubation was started by the addition of aniline and continued for 20 min. Control activities were 3.16 and 1.16 nmoles of p-aminophenol formed per min per mg of microsomal protein for NADPH-supported (o--o) and NADH-supported (-) reactions, respectively.

A

B

60 40 20 1

0 Anti-b5

2 3 IG (mglmg

4 5 MS protein)

10 Anti-b5Fob’CmglmgMs

15

20 protein)

Fig. 3 Effects of anti-b5 IG (A) and anti-bs Fab' (B) on benzo(a)pyrene hydroxylation. The reaction mixture contained 0.1 mg of microsomes, antibody, 0.08 mM benzo(a)pyrene, 0.1 mM EDTA, and NADPH-generating system

210

BIOCHEMICAL

Vol. 91, No. 1, 1979

antibody drug

against

cytochrome

oxidation

extent

reactions

to all

divalent

antibody

antigen

molecules pathway

from NAD(P)H

antibody

preparation

against

inhibitory

action

by anti-b5

in inhibiting

the hydroxylation both

inhibited b5 Fab'

Fab'

on the drug

of the NADH-cytochrome by a weaker

steric

bs,

anti-b5

reaction

2 reductase

3.

activity

and examined

the native

(Table

due to the decreased

I)

effective

equally

action its

of anti-

lower

and could

molecular

Fig.'s

antibody.

were

inhibitory with

its

was most sensitive

was much less

reactions

corresponds

a monovalent

When we compare

than

The weaker

of the

of the electron

which

antibody

and NADPH-supported

hindrance

Fab',

bs

of the

We prepared

P-450.

as shown in Fig.

3B).

to cytochrome

in an alteration

of the substrate

oxidation

different

the aggregation

of benzo(a)pyrene,

(Fig.

on microsomal

the addition

induced

the monovalent

NADH-supported

by anti-b5

have

to cytochrome

that

suggesting

was specific

resulting

IG,

is

used,

examined,

could

on the oxidation

evident

study

cytochrome

3A and 3B, it

However,

this

in the membrane

transport

action

bs in the reactions.

criteria

to microsomes

RESEARCH COMMUNICATIONS

inhibitory

to substrates

IG used in

immunochemical

to the inhibition

variable

of cytochrome

anti-25

according

bs exerts according

of participation Although

AND BIOPHYSICAL

inhibition be explained

size.

DISCUSSION Antibodies the electron purified

are transport

antibody

The monovalent

to eliminate

of the antigen Our results oxidation

of this fragment

molecules

against

(Fab')

inhibitors

microsomes. cytochrome

In this bs were

in microsomal

of the antibody

the possibility in

site-specific

of intact

cytochrome

demonstrate

of some drugs,

available

pathway

preparations

the participation

in order

the only

in

studying

study,

highly

used in elucidating

drug

oxidation

reactions.

was also

prepared

and used

of the antibody-induced

rearrangement

the membrane. the involvement

such as 7-ethoxycoumarin

or 5 mM NADH in 1 ml of 0.1 started by the addition of 10 min. Control activities (M) and N&Xl-supported

of cytochrome

bs in the

and benzo(a)pyrene,

even

M Tris-HCl (pH 7.5). The incubation was benzo(a)pyrene and carried out at 37 OC for were 0.208 and 0.106 for NADPH-supported (-) reactions, respectively.

211

BIOCHEMICAL

Vol. 91, No. 1, 1979

when the

reducing

However,

cytochrome

aniline.

equivalents

microsomal

drug

reconstituted

(9)

the

component

the

reactions but

they

of the

concluded

systems

Our observations

indicate

the essential

role

anti-&

in

the hydroxylation

of cytochrome

on their

observations

(3)

the

that

using

of cytochrome

the reaction

by NADPH.

intact

was almost

in

k5

using

cytochrome

is

not an

as the source

microsomes

completely

of

strongly

in NADPH-supported

_1?5

of

oxidation inhibited

by

IG. We recently

presented

of NADPH-cytochrome as well

all, aniline

and second

cytochrome

for

the microsomal

b5

reactions

forms

reported

hand,

it

of cytochrome possibly P-450

cytochrome

is very

having

&.. a high

case of

that

by NADPHthe

via

cytochrome

drug

oxidation of

The isolation

of a

to cytochrome

(21).

REFERENCES 1. 2. 3.

Hildebrandt, A. & Estabrook, R. W. (1971) Arch. Biochem. Biophys. 143, 66-79. Jansson, I. & Schenkman, J. B. (1977) Arch. Biochem. Biophys. 178, 89-107. Lu, A. Y. H., West, S. B., Vore, M., Ryan, D. & Levin, W. (1974) J. Biol. Chem. 249, 6701-6709.

212

second

requires

affinities

affinity

both

P-450

which

P-450-catalyzed

to cytochrome

In the

likely

supplied

due to different

P-450

of some, but not

to cytochrome

solely

the

participate,

reaction, is

reaction

confirmed

source.

bs does not

component,

bs is

of cytochrome

other

oxidation

oxidation

donated

the participation

study

electron

hydroxylation

dependency

of microsomal

bs was recently

On the

as an essential

on cytochrome

cytochrome

for

drug

Our present

are possibly

the benzo(a)pyrene

The different

species

in which

c reductase.

cytochrome

multiple

reactions. -bs in

eiectrons

evidence

in NAB&supported

even when NADPH was used as the

hydroxylation,

electron

immunochemical

c reductase

of cytochrome

drugs

first

(11)

as in NADPH-supported

participation

b5.

donated

when NADPH was used

equivalents.

since

are

involvement

based

reducing

of benzo(a)pyrene,

reaction

RESEARCH COMMUNICATIONS

seem to be involved

suggested

oxidation systems,

obligatory

for

bs does not

Lu et al.

AND BIOPHYSICAL

Vol. 91, No. 1, 1979

4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21.

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