Effect of carbon tetrachloride-induced soluble protein on microsomal NADPH oxidase activity of rat liver

Effect of carbon tetrachloride-induced soluble protein on microsomal NADPH oxidase activity of rat liver

BiochemicalPhormacologv, Printed in Great Britain. NO.17, PP. 2837-2840,1982. Vol. 31, CKKM-2952/82/172837-04$03.00/O @ 1982 Pergamon Press Ltd. P...

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BiochemicalPhormacologv, Printed in Great Britain.

NO.17, PP. 2837-2840,1982.

Vol. 31,

CKKM-2952/82/172837-04$03.00/O @ 1982 Pergamon Press Ltd.

PRELIMINARY COMMUNICATIONS EFFECT

OF CARBON

ON MICROSOMAL

Tohoru Department

TETRACHLORIDE-INDUCED

NADPH

Hasegawa,*

of Public

OXIDASE

Masana

Health,

(Received Liver

microsomes

communication cytoplasm NADPH

are capable

describes

of rats

(0.5 ml/kg

University

Tomokuni*

Medical

School,

700, Japan

1982;

accepted

18 May

NADPH

that a soluble

intoxicated

of body weight)

250 g body weight) livers

were

with

with

1982)

at slow rates

protein

CC14

stimulates

This

(1).

extracted

The sedimented

min.

HCl buffer resulting

105,OOOg

extraction

Six days

after

from

liver

microsomal

microsomes

resuspended

were

microsomes

saved

and normal

soluble

protein

were

buffer were

it at 105,OOOg

for 60

10 mM Tris-

as Ccl4 microsomes. of Ccl4

supernatant

also

(pH 7.5).

and microsomes

for extraction

105,OOOg

(200-

injection,

in 0.15 M KCl,

in this experiment was

buffer

rats

10 mM Tris-HCl

for 20 min,

by centrifuging

male

the CC14

4 mM Tris-HCl

in 0.15 M KCl,

at 10,OOOg

supernatant

of normal

into Donryu

the supernatant

(pH 7.5) and used

Normal

tein.

was made

centrifuged

from

injected

0.25 M sucrose,

homogenate

It was

sedimented

was

intraperitoneally.

perfused

a 20% liver

(pH 7.5). then

OF RAT LIVER

and Katsumaro

of oxidizing

evidence

that were

Ogata

PROTEIN

oxidation.

cc14

Then

19 April

ACTIVITY

Okayama

Okayama

SOLUBLE

soluble

that was

extracted

The

saved

from normal

profor

rat

liver. Crude nium

extracts

sulfate

(of which Ccl4

precipitation

the pH was

or normal,

saturated. obtain

The

40-70%

supernatant

*Present School,

of CC14,

and normal,

soluble

at 4O with

stirring.

adjusted

by Tris

105,OOOg

supernatant

solution

was

saturation,

solution

address: Nabeshima,

was

the ammonium

Department Saga

until

840-01,

by ammo-

ammonium

gradually

sulfate to the

solution

and the supernatant

was

content

to 70% saturated,

Japan.

added

made

the supernatant

sulfate

of Community

were

Saturated

to 7.5) was

centrifuged

raised

protein

Health

was

40%

saved.

To

of the 40% saturated

The solution

Science,

was

then

Saga Medical

2838

Preliminary co~unicatjons

centrifuged KCl,

at 10,OOOg

10 mM Tris-HCl

at 4O to remove 40 and

ammonium

protein

Figure

ulated

of CC14

ble protein 25-fold.

control).

addition

soluble

in the presence

the boiled

ammonium

with

The mechanism soluble

stimulated

sulfate

involve

a cyanide

sensitive

velocity

of oxidation

of carbon

tein,

monoxide

was

soluble

stimulated

oxidation

precipitate

protein

effect

protein

(21, but that

protein

did not.

was

induced

on solu-

20- to

faster

than

at 200).

On

stimulated

2-

was

stim-

by the addition

of CCL4 microsomes

at 100"

on NADPH

soluble

1 mM),

indicating

than

for 10 min;

oxidation,

by 50%.

The aerobic

protein that

the velocity

However,

by normal

the stimulation carbon

which

was

soluble

on NADPH

oxidase

that

did not (1).

On

induced

by the

is, the effect

by CC14

soluble

soluble

induced

components,

induced

by

did not slow

by normal

oxidation

of

not inhibited

monoxide

oxidation

by a

oxidation

the stimulation

protein,

sensitive

reaction

induced

of oxidation

carbon

of NADPH

monoxide

the stimulating

oxidation

such as cytochrome

component, slowed

NADPH

for.

or CCl4,

induced

involved

oxidation

oxidation

was heated

of microsomal

on the stimulation

that

stim-

as normal

protein

NADPH

was

significant

oxidized

was

oxidation

not the same as that on the stimulation

suggesting

was more

in the presence

On addition

slow oxidation

concentration

protein,

NADPH

at 20').

effect.

monoxide

protein

this

NADPH

is not yet accounted

(final concentration

soluble

this

had no stimulating

by normal,

the CC14

protein

protein

soluble

of stimulation

carbon

as a CC14

microsomes.

cyanide

the contrary,

between

at pH 7.5 in the

(4-6 nmoles/min/mg

faster

a protein

protein

in this experiment

microsomes,NADPH

The stimulated

of normal

overnight

that was obtained

of oxidation

soluble

protein,

was

was dialyzed

oxidized

(same protein

nOriK3l

of Ccl4

protein

precipitate

is consistent

of

soluble

on addition

40-70%

stimulation

The Ccl4

5- to 7-fold.

of ccl4

aerobically

protein

in 0.15 M

protein.

of CC14 microsomes

of normal

to 3-fold;

protein

was

was used

(3.5 mg protein/ml),

In the presence

in the presence

The

soluble

This

soluble

The precipitate

(2-4 nmoles/min/mg

protein

was redissolved

and the solution

saturation

that NADPH

soluble

addition

NADPH

sulfate

3- to 5-fold.

ulated

sulfate.

of Ccl4 microsomes

of normal

the precipitate

(pH 7.5),

or a normal

1 shows

presence

Ccl4

buffer

70% ammonium

soluble

for 10 min,

pro-

by CC14

such as hemo-

by normal

soluble

Preliminary

communications

CONT

RO L (Ccl4

2839

microsomes)

0.6 E s 0 d

m l-

a

= 0.4 z a a a

PROTE IN

I 0 cn m a 0.2

(MINUTES)

T I M E

Fig.

1.

Effect liver

of normal, microsomes

mixture

contained

or CC14, under CC14

aerobic

10 mM Tris-HCl

mal

soluble

of 4.0 ml. Hitachi

The changes

two-wavelength

photometer.

Temperature

was

reaction

mixture

presence

of Ccl4 microsomes;

for 30 set

beam 20'.

difference

+ CO.

reaction

0.1 mM NADPH

and nor-

in a total

CO was bubbled

volume

recorded

recording

in a

spectro-

through

Key:(-)

(.--..)in the presence

ble

by

(2.6 mg protein/ml),

at 340 nm were

(one bubble/set).

(--.--) in the presence

oxidation

The final

(3.5 mg protein/ml)

microsomes; protein

(pH 7.5),

in absorbancy double

on NADPH

microsomes

buffer

protein

protein

conditions.

or normal

0.15 M KCl, or CC14

soluble

the in the

of normal

of Ccl4 microsomes

+ Ccl4

solu-

2840

Preliminary

These

results

impairment of cc14

may be associated

by CC14

protein

was

of microsomal increased

communications

with

some processes

electron

with

transport,

increasing

of recovery because

time after

from the

the stimulation

the CC14

injection.

REFERENCES 1.

2

J.R.

Gillette,

532

(1957).

T. Omura, Fedn

Proc.

B.B.

R. Sato, 24, 1181

Brodie

D.Y.

and B.N.

Cooper,

(1965).

La Du,

0. Rosenthal

J. Pharmac.

exp.

Ther.

and R.W. Estabrook,

119,