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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.
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,
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,