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12α-Hydroxylation of 7α-hydroxy-4-cholesten-3-one by a reconstituted system from rat liver microsomes
BIOCHEMICAL
Vol. 54, No. 3, 1973
12a-HYDROXYLATION
OF
RECONSTITUTED
Claes
7a-HYDROXY-4-CHOLESTEN-3-ONE
SYSTEM
Bernhardsson,
FROM
Ingemar
of
Received
Chemistry,
August
BY
LIVER
A
MICROSOMES
Henry
Danielsson
and
Wikvall
Karolinska
20,
RAT
Bjiirkhem,
Kjell Department
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Institutet,
Stockholm,
Sweden
1973
SUMMARY A preparation of partially purified cytochrome P-450 from rat liver microsomes was found to catalyze 12a-hydroxylation of 7a-hydroxy-4-cholesten-3-one in the presence of NADPH and The reaction was stimulated twoto fourphosphatidyl choline. fold by addition of a preparation of cytochrome P-450 reductase. The reaction was inhibited by carbon monoxide to a considerably less extent than other hydroxylations catalyzed by the reconstituted system. In the presence of optimal concentrations of cytochrome P-450 reductase, cytochrome P-450 prepared from livers of starved rats catalyzed the 12a-hydroxylation more efficiently than cytochrome P-450 prepared from livers of normal rats or rats treated with phenobarbital.
12a-Hydroxylation of
bile
is
acids
the
26-hydroxylase,
the
relative
from
cholesterol the
amounts
formed
from
cholesterol
differ
from
most
several is and dent
other It
it
(3)
has of
been
an
increase
an
increased
liver
but
after
treatment
of
acid
(1,2).
The
microsomal has
or
been
rather
the
activity with
of
of
the
biosynthesis
(1).
Together
the
mixed
with
may
regulate acid
12n-hydroxylase
reported
seems
function that
the
in
12a-hydroxylase
to
carbon
12n-hydroxylase
monoxide is
indepen-
treatment P-450 function
(3,'+).
to
oxidases
(4)
12u-hydroxylase
phenobarbital
0 I973 bv Academic Press, Itic. of reproduction in ar?y jbrm reserved.
the
chenodeoxycholic
mixed
1030 Copyright AN rights
and
cytochrome most
in
liver
Phenobarbital
(3).
of
the
insensitive that
bulk
step
12a-hydroxylase
cholic
P-450
activity the
in
suggested
cytochrome
important
microsomal
of
respects.
insensitive
an
in
leads
the
liver
and
oxidases is Starvation
to
in
decreased also
the
BIOCHEMICAL
Vol. 54, No. 3,1973
leads
to
an
liver
(cf.-
increase ref.
hydroxylase that
tive
to
P-450
is
is
probably
the
liver.
up
oxidases
(6).
It
involved
in
the
ver
P-450
most
of
consisting
cytochrome
shown
P-450
mixed
if
cytochrome
the
cytochrome P-450
the
now
been
of
partially
re-
sensi-
other
cytochrome
has
12c(-
in
mechanism
studied
in
a
purified
reductase
from
rat
li-
microsomes.
Materials. described
of
80
by
exposure
previously
of to
treated
with to
30
use
the
giving
material of
was
a
Pyridine
citric
acid
dehydrogenase
a
di-
and
the
Co.,
St.
Louis,
Methods. --
Male
rats
of In
IV)
gas
material
was
chromatography radioactivity
monolauroylglyceryl-3from one
Dr. used
D,L-isocitric
(Type
prepared
tritium
The
specific
gift as
to
was
thin-layer
with
nucleotides,
Chemical
used.
by
prepared
radioactivity acid
(10).
generous
preparation
(8).
specific
acid
purified
mixture
report
a
procedure
and
same
had
lithocholic
Wilzbach
The
the
g were
and
lithocholic
alkali
pCi/mg.
was
(9)
unlabeled
phosphorylcholine and
was
Tritium-labeled
ILCi/mg.
according
prior
PROCEDURE
6P--'H-7a-IIydroxy-4-cholesten-3-one
as
200
was
concerning
EXPERIMENTAL
of
than
the
the
more
that
bulk
reaction
(7,8)
and
It
considerably
concluded
the
of
(5).
salts
in
activity
fourfold
information
system
P-450
12n-hydroxylation,
from
12a-hydroxylation
cytochrome
to
was
the
further
the
was
inorganic
obtain
cytochrome
case,
12cc-hydroxylase with
reconstituted
of
this
increased
the
bulk
in
different
To
the
and
treatment
function
of
5)
is
cently
of
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
were
W.E.M. in
a
I,ands
previous
acid obtained
and from
isoSigma
MO. the
some
Sprague-Dawley experiments
103 1
strain rats
were
weighing treated
about by
in-
Vol. 54, No. 3, 1973
BIOCHEMICAL
traperitoneal once
injection
dailv
for
3 days
sacrifice
er
tochrome (7)
P-450
and
as
of or
rats
cl-m----
P-450
per
mg
i1
Omura
and
Sato
about
200,
when determined
contained
about
450
preparations
35%
cytochrome the
P-450
from
In
experiments
4-cholesten-3-one, to
1.0
P-450
nmole
of
phorylcholine volume
and of
riments
3
ml
given 1.5
cytochrome
P-450
20
37OC
volumes
sequent activity
at
of in
ten-3-one,
formed
of
dissolved
in
IJ-g
an
of
nmoles
and
8
of
20
llg
Protein
(12).
The
fresh
prerats
cytochrome
rats
of
P-
contained
25-
procedure
used
pg
of
of
acetone,
were
6P-jH-7a-hydroxy-
of
cytochrome
units
medium
(8) (14).
added
in In
a the
total expe-
6P-3H-7a-hydroxy-4-cholesP-450
were and
respec-
dilauroylglyceryl-3-phos-
cytochrome
min
prerats
the
system
were
chromatography chromatographic
and
terminated
by
(2:1,
v/v).
and
determination zones
1032
500
Incubations
used.
chloroform-methanol
different
ul 400
Rucher
reductase for
5
NADPH-generating
1,
cytoaccord-
protein
(13).
starved
25
al.
reductase
al.
whereas
1,
mono-
modified
of
assayed
incubation
P-450,
Table
thin-layer in
Fig.
50
et
standard
shown.in
nmoles
mg
al.
and
cytochrome
reductase,
per
P-420
the
--et
phenobarbital-treated
untreated
P-420.
Lu
cy-
phenobarbital-treated
et
from
cytochrome
10%
1.9
Masters
Lowry
and
cytochrome
P-450
units
to
P-450
phenobarbital-
when
and
500
to
cytochrome
and
cytochrome
according
according of
0.9
to
The
and
weight)
prior
to
(8).
starved
starved and
hours
cytochrome
report
The
body
according
0.6,
(11).
mg/kg
72
respectively
200
assayed
as
previous
protein
contained
well prepared
about
untreated,
for
untreated,
contained
from
in
a
from
parations
paration
as
in
(100
starved
were
described
treated
was
were
reductase
preparations
tively
phenobarbital
microsomes
P-450
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
units
of
were
per-
addition
of
Extraction,
subof
on
the
radio-
chromato-
BIOCHEMICAL
Vol. 54, No. 3,1973
plates
were
performed
experiments from
the
the
ether
tn
labeled
and
some
5000
to
under
with
methanol
trimethylsilyl
chromatography with
radioactive
some
using a
product
31;
W-1
was
a
column.
diluted
with
7a,12a-dihydroxy-4-cholesten-3-one specific
acid
was
conditions
incubated
acid product
was
and
radioactivity.
Tritium-la-
with
described
taurodeoxycholic
hydroxylated
In
with the
equipped
constant
the
extracted
radio-gas
the
(4,6).
into
instrument
lithocholic
tem
by
unlabeled
crystallized
was
converted
experiments,
authentic,
previously
product
analyzed
Colman
beled
described
chromatoplates,
(15)
barber
as
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
reconstituted
previously
(8)
and
assayed
the
sys-
for
incubations
conversion
also
as
into
described
the
6p-
previously
(‘6). RESULTS Incubation cytochrome tem
P-450
and
tidy1
of
a
with
from
mixture
starved
of
choline
duct
6@- 'H-7a-hydroxy-4-cholesten-3-one
mono-
resulted the
rats, and
in
thin-layer
a
to
conversion
product
was
analyzed
silyl
ether
5-10s
had
4-cholesten-3-one product
by
radio-gas
chromatography.
by with
the
crystallization authentic
reaction
into
a
the
P-450
reductase. ether
About
qO$
properties
ref.
17).
The
of identity
of
most was
constant
specific
7a,l2a-dihydroxy-4-cholesten-3-one.
1033
about
7a,26-dihydroxy-
7a,l2a-dihydroxy-4-cholesten-y-one to
the
trimethyland
properties
and
of
of
7a,12a-dihydroxy-4-cholesten-3-one chromatographic
7c(,
stimulated
trimethylsilyl
chromatographic
pro-
of was
cytochrome
into
(cf.with
of
converted
of the
addition
sys-
properties
was
had
3-75;
chromatographic
by
radioactivity
NADPH-generating
of
The
fourfold
The
an
di-lauroylglyceryl-3-phospha-
12a-dihydroxy-4-cholesten-3-one. two-
with
of
the
confirmed
radioactivity
together After
three
Vol. 54, No. 3, 1973
BIOCHEMICAL
200
400 Reductase (U)
600
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
0.5 1.0 CytochromeP-450 (nmoles)
600
1.5
15
10
20
30
Time (min) 1 . Effect of cytochrome P-4.50 reductase concentration P-450 concentration (B), and time (C) on 12n(A) t cytochrome hydroxylation of Tn-hydroxy-4-cholesten-T-one by the reconstituted system with cytochrome P-450 from starved rats (cf. Methods). With the exception of the variable factor in each set of experiments the standard incubation conditions were used (cf. Methods). Fig.
crystallizations was ditions
of
employed
the
amount
time
up
the
enzyme
in
90%
about
which
of to
turation
pg
labeled
rate
assays
tase
necessary
of
the
rate
about
30%
the
of
when
min
substrate
was
4%
oxygen,
30
pg) low
amount
optimal
reaction
56%
1.2 was
of
was
Under
nitrogen
1034
of conditions of in
$
4076
was in carbon
of
reduc-
units inhibited
an
sa-
accu-
P-450
400-600
out
with
saturation
reasonably
system
and
and
conversion
was
carried
with
conditions
the
con-
linear
incubation
cytochrome
complete
the
nmoles
no
making
conversion the
Under
about
standard added.
crystals
was
There
very
The
by
to
1).
were (above
the
material.
up
under
substrate
of
12a-hydroxylation
(Fig.
substrate
the
starting
of
substrate
for
12a-Hydroxylation
radioactivity
P-450
difficult.
1) *
containing
that
30
with
with
added
specific
cytochrome
about
5
the
atmosphere monoxide.
(Fig. by
BIOCHEMICAL
Vol. 54, No. 3, 1973
Under
the
cholic
acid
bited of
same
by
conditions,
and
90%
experiments
in
was
of
treated,
ed
of
systems
cient
with
These
results
vation
on
on
cytochrome
untreated,
starved
cytochrome
P-450
from
cytochrome
P-450
reductase
The
rate
of
the
assayed (Table
is in
1).
cytochrome
some The
from
that
12a-hydroxylation
6P-hydroxylation
of
be
the
reside
pheno-
rats,
was
un-
used,
For
tritium-label-
stimulated
by
different,
phe-
reconstiwas
most
effi-
phenobarbital-treated
the
stimulatory
and
of
in
and
from
hp-hydroxylation
P-450
indicate
to
P-450
12a-hydroxyla-
of
known
results
and
rats
6P-hydroxylation
which
the
starved
phenobarbital-treated
acid,
tuted
of
used.
inhi-
summarizes
with
or
was
were
were
comparison
nobarbital,
I
combinations
whether
lithocholic
Table
from
taurodeoxy-
testosterone
rats
starved
reasons
8).
reductase
fastest
regardless
ref.
which
P-450
of
of
(cf.-
barbital-treated tion
7a-hydroxylation
6p-hydroxylation
about
cytochrome
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
rats, effects
of
phenobarbital
the
star-
treatment
cytochrome
P-450
fractions.
partially
purified
DISCUSSION The
present
chrome
P-450
xylation
of
NADPH
and
up
fourfold
to
work preparation
by
450
contains
fraction
xylase
system
ferred
by
a rat
liver
and
addition
of
the
the
in
finding
Suzuki,
from
the
1035
stimulated reductase
the
NADPH
The
of
cytochrome of
P12a-hydro-
are
trans-
involvment
12a-hydroxylation Mitropoulos
that
the
oxidase
reductase. the
P-450
that
electrons
presence
was
cytochrome
terminal
P-450
by
a
12a-hydrothe
reaction
indicate the
reductase suggested
of
results
that
The
cyto-
catalyzes in
choline.
cytochrome
cytochrome
basis
from
phosphatidyl
These
the
that
Ta-hydroxy-4-cholesten-3-one
preparation.
viously
shows
and 12a-hydroxylation
of
has
been
Myant
(3) is
a preon in-
12d-Hydroxylation
reductase
reductase
reductase
reductase
reductase
Cy-tochrome Cytochrome P-450 Cytochrome P-450 Cytochrome P-450
Cytochrome
Cytochrome
Cytochrome Cytochrome
Cytochrome
Cytochrome
Cytochrome Cytochrome
P-450
P-450
P-450 P-450
P-450
P-450
P-450 P-450
from from
of
--
P-450
reductase
71~I-hydroxy-4-cholesten-Y-one --___ from rat liver
from
from
from from
+
+
+
cytochrome
cytochrome
rats
cytochrome rats
cytochrome
cytochrome
cytochrome
+
+
+
microsomes. --___ were
phenobarbital-treated phenobarbital-treated from untreated rats phenobarbital-treated from starved rats phenobarbital-treated from phenobarbital-treated
starved rats starved rats from untreated rats from starved rats from starved rats from starved rats from phenobarbital-treated
from from
untreated rats untreated rats from untreated rats from untreated rats from starved rats from untreated rats from phenobarbital-treated
System
cytochrome
systems
P-450 P-450 reductase P-450 reductase P-450 reductase
--U of
reductase
500
-________
and
reconstituted
---
rats
rats
rats rats
used.
Table and
+ rats
+
+
P-450
P-450
P-450
cytochrome
cytochrome
---
6P-hydroxylation
cytochrome
P-450
P-450
P-450
Irrespective
I
of
of
5.9
4.6
3.9
3*2
15.2
11.3
11.8
6.8
4.9
5.1
4.6
2.8
nmoles
lithocholic 1.5
12a-Hydroxylation of 7a-hydroxy-kcholesten-3-one
source,
%
of
by
different
7.2
1.6
2. 8
1.2
3.1
1 .5
hp-Hydroxylation of lithocholic acid
cytochrome
acid
P-450
BIOCHEMICAL
Vol. 54, No. 3,1973
hibited
partially
results
do not
nature
of
might
dases,
sults
in
indicate
chrome the
Liver
In
microsomes
constituted monoxide
ref.
reconstituted
was
rather
8).
The
the
properties
unknown
cytochrome and
be
a species
bulk
of
cytochrome
indicate
of
re-
cyto-
P-450
in
using the
re-
carbon
with
different
types
the
cytochrome
P-1450
oxidase
the
previous
towards
that
terminal
P-450
by
insensitive
preoxi-
results
12n-hydroxylation
experiments
of
If
previous
is
P-I150
yet
must
with
the
the It
as
present
the
consonance
systems
containing
specific
from
(3,4)
system (cf. -
fraction the
12u-hydroxylation
different
(3-6).
of
P-l&50.
it
present
cytochrome
amounts
that
The
12w-hydroxylation.
cytochrome
strongly
P-450
"intact"
of
the
c.
concerning
purified
small
from
involved
in
partially
contain
cytochrome
conclusions
oxidase
the
different
of
definite
terminal that
paration
addition
permit
the
conceivable
is
by
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
is
responsible
12cc-hydroxylase
for
system.
ACKNOWI,EDGEMENTS The is tions ject
skilful
gratefully supported
technical
assistance
acknowledged. by
the
This Swedish
work Medical
of
Miss
Irene
is
part
of
Research
Ferdman investiga-
Council
(Pro-
13x-218). REFERENCES
1. 2. 3. 4. 5. 6. 7. 8. 9.
Danielsson, H., in The Bile Acids: Chemistry, Physiology and Metabolism (edited by P.P. Nair and D. Kritchevsky) Vol. 2, p. 1, Plenum Press, New York, 1973. BjorkheM, I., Danielsson, II., and Gustafsson, J., FEBS letters, 2, 20 (1973). Suzuki, M., Mitropoulos, K.A., and Myant, N.B., Biochem. Biophys. Res. Commun., 3, 516 (1968). Einarsson, K., Eur. J. Biochem., 3, 101 (1968). Johansson, G., Eur. J. Biochem., 3, 292 (1970). Bjorkhem, I., and Danielsson, II., Biochem. Biophys. Res. Commun., 5J, 766 (1973). Kuntzman, R., West, S., Jacobson, M., and Lu, A.Y.II., Conney, A.H., .J. Biol. Chem., 247, 1727 (1972). Bjljrkhem, I., Danielsson, H., and Wikvall, Ii., Biochem. Biophys. Res. Commun., 53, 609 (1973). Bjiirkhem, I,, El?r. J. Bschem., g, 337 (1969).
1037
Vol. 54, No. 3, 1973
10 . Il. 1 'IL .
17. 14.
15. 16. 17.
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Wilzbach, k.l<., J. Am. Chem. Sot., 79, 1013 ( 1957). Omura, T., and Sato, R., .I. Iliol. Chem., a, '379 (f9w. Williams, (‘.ff.,
1038
Vol. 54, No. 3, 1973
12a-HYDROXYLATION
OF
RECONSTITUTED
Claes
7a-HYDROXY-4-CHOLESTEN-3-ONE
SYSTEM
Bernhardsson,
FROM
Ingemar
of
Received
Chemistry,
August
BY
LIVER
A
MICROSOMES
Henry
Danielsson
and
Wikvall
Karolinska
20,
RAT
Bjiirkhem,
Kjell Department
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Institutet,
Stockholm,
Sweden
1973
SUMMARY A preparation of partially purified cytochrome P-450 from rat liver microsomes was found to catalyze 12a-hydroxylation of 7a-hydroxy-4-cholesten-3-one in the presence of NADPH and The reaction was stimulated twoto fourphosphatidyl choline. fold by addition of a preparation of cytochrome P-450 reductase. The reaction was inhibited by carbon monoxide to a considerably less extent than other hydroxylations catalyzed by the reconstituted system. In the presence of optimal concentrations of cytochrome P-450 reductase, cytochrome P-450 prepared from livers of starved rats catalyzed the 12a-hydroxylation more efficiently than cytochrome P-450 prepared from livers of normal rats or rats treated with phenobarbital.
12a-Hydroxylation of
bile
is
acids
the
26-hydroxylase,
the
relative
from
cholesterol the
amounts
formed
from
cholesterol
differ
from
most
several is and dent
other It
it
(3)
has of
been
an
increase
an
increased
liver
but
after
treatment
of
acid
(1,2).
The
microsomal has
or
been
rather
the
activity with
of
of
the
biosynthesis
(1).
Together
the
mixed
with
may
regulate acid
12n-hydroxylase
reported
seems
function that
the
in
12a-hydroxylase
to
carbon
12n-hydroxylase
monoxide is
indepen-
treatment P-450 function
(3,'+).
to
oxidases
(4)
12u-hydroxylase
phenobarbital
0 I973 bv Academic Press, Itic. of reproduction in ar?y jbrm reserved.
the
chenodeoxycholic
mixed
1030 Copyright AN rights
and
cytochrome most
in
liver
Phenobarbital
(3).
of
the
insensitive that
bulk
step
12a-hydroxylase
cholic
P-450
activity the
in
suggested
cytochrome
important
microsomal
of
respects.
insensitive
an
in
leads
the
liver
and
oxidases is Starvation
to
in
decreased also
the
BIOCHEMICAL
Vol. 54, No. 3,1973
leads
to
an
liver
(cf.-
increase ref.
hydroxylase that
tive
to
P-450
is
is
probably
the
liver.
up
oxidases
(6).
It
involved
in
the
ver
P-450
most
of
consisting
cytochrome
shown
P-450
mixed
if
cytochrome
the
cytochrome P-450
the
now
been
of
partially
re-
sensi-
other
cytochrome
has
12c(-
in
mechanism
studied
in
a
purified
reductase
from
rat
li-
microsomes.
Materials. described
of
80
by
exposure
previously
of to
treated
with to
30
use
the
giving
material of
was
a
Pyridine
citric
acid
dehydrogenase
a
di-
and
the
Co.,
St.
Louis,
Methods. --
Male
rats
of In
IV)
gas
material
was
chromatography radioactivity
monolauroylglyceryl-3from one
Dr. used
D,L-isocitric
(Type
prepared
tritium
The
specific
gift as
to
was
thin-layer
with
nucleotides,
Chemical
used.
by
prepared
radioactivity acid
(10).
generous
preparation
(8).
specific
acid
purified
mixture
report
a
procedure
and
same
had
lithocholic
Wilzbach
The
the
g were
and
lithocholic
alkali
pCi/mg.
was
(9)
unlabeled
phosphorylcholine and
was
Tritium-labeled
ILCi/mg.
according
prior
PROCEDURE
6P--'H-7a-IIydroxy-4-cholesten-3-one
as
200
was
concerning
EXPERIMENTAL
of
than
the
the
more
that
bulk
reaction
(7,8)
and
It
considerably
concluded
the
of
(5).
salts
in
activity
fourfold
information
system
P-450
12n-hydroxylation,
from
12a-hydroxylation
cytochrome
to
was
the
further
the
was
inorganic
obtain
cytochrome
case,
12cc-hydroxylase with
reconstituted
of
this
increased
the
bulk
in
different
To
the
and
treatment
function
of
5)
is
cently
of
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
were
W.E.M. in
a
I,ands
previous
acid obtained
and from
isoSigma
MO. the
some
Sprague-Dawley experiments
103 1
strain rats
were
weighing treated
about by
in-
Vol. 54, No. 3, 1973
BIOCHEMICAL
traperitoneal once
injection
dailv
for
3 days
sacrifice
er
tochrome (7)
P-450
and
as
of or
rats
cl-m----
P-450
per
mg
i1
Omura
and
Sato
about
200,
when determined
contained
about
450
preparations
35%
cytochrome the
P-450
from
In
experiments
4-cholesten-3-one, to
1.0
P-450
nmole
of
phorylcholine volume
and of
riments
3
ml
given 1.5
cytochrome
P-450
20
37OC
volumes
sequent activity
at
of in
ten-3-one,
formed
of
dissolved
in
IJ-g
an
of
nmoles
and
8
of
20
llg
Protein
(12).
The
fresh
prerats
cytochrome
rats
of
P-
contained
25-
procedure
used
pg
of
of
acetone,
were
6P-jH-7a-hydroxy-
of
cytochrome
units
medium
(8) (14).
added
in In
a the
total expe-
6P-3H-7a-hydroxy-4-cholesP-450
were and
respec-
dilauroylglyceryl-3-phos-
cytochrome
min
prerats
the
system
were
chromatography chromatographic
and
terminated
by
(2:1,
v/v).
and
determination zones
1032
500
Incubations
used.
chloroform-methanol
different
ul 400
Rucher
reductase for
5
NADPH-generating
1,
cytoaccord-
protein
(13).
starved
25
al.
reductase
al.
whereas
1,
mono-
modified
of
assayed
incubation
P-450,
Table
thin-layer in
Fig.
50
et
standard
shown.in
nmoles
mg
al.
and
cytochrome
reductase,
per
P-420
the
--et
phenobarbital-treated
untreated
P-420.
Lu
cy-
phenobarbital-treated
et
from
cytochrome
10%
1.9
Masters
Lowry
and
cytochrome
P-450
units
to
P-450
phenobarbital-
when
and
500
to
cytochrome
and
cytochrome
according
according of
0.9
to
The
and
weight)
prior
to
(8).
starved
starved and
hours
cytochrome
report
The
body
according
0.6,
(11).
mg/kg
72
respectively
200
assayed
as
previous
protein
contained
well prepared
about
untreated,
for
untreated,
contained
from
in
a
from
parations
paration
as
in
(100
starved
were
described
treated
was
were
reductase
preparations
tively
phenobarbital
microsomes
P-450
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
units
of
were
per-
addition
of
Extraction,
subof
on
the
radio-
chromato-
BIOCHEMICAL
Vol. 54, No. 3,1973
plates
were
performed
experiments from
the
the
ether
tn
labeled
and
some
5000
to
under
with
methanol
trimethylsilyl
chromatography with
radioactive
some
using a
product
31;
W-1
was
a
column.
diluted
with
7a,12a-dihydroxy-4-cholesten-3-one specific
acid
was
conditions
incubated
acid product
was
and
radioactivity.
Tritium-la-
with
described
taurodeoxycholic
hydroxylated
In
with the
equipped
constant
the
extracted
radio-gas
the
(4,6).
into
instrument
lithocholic
tem
by
unlabeled
crystallized
was
converted
experiments,
authentic,
previously
product
analyzed
Colman
beled
described
chromatoplates,
(15)
barber
as
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
reconstituted
previously
(8)
and
assayed
the
sys-
for
incubations
conversion
also
as
into
described
the
6p-
previously
(‘6). RESULTS Incubation cytochrome tem
P-450
and
tidy1
of
a
with
from
mixture
starved
of
choline
duct
6@- 'H-7a-hydroxy-4-cholesten-3-one
mono-
resulted the
rats, and
in
thin-layer
a
to
conversion
product
was
analyzed
silyl
ether
5-10s
had
4-cholesten-3-one product
by
radio-gas
chromatography.
by with
the
crystallization authentic
reaction
into
a
the
P-450
reductase. ether
About
qO$
properties
ref.
17).
The
of identity
of
most was
constant
specific
7a,l2a-dihydroxy-4-cholesten-3-one.
1033
about
7a,26-dihydroxy-
7a,l2a-dihydroxy-4-cholesten-y-one to
the
trimethyland
properties
and
of
of
7a,12a-dihydroxy-4-cholesten-3-one chromatographic
7c(,
stimulated
trimethylsilyl
chromatographic
pro-
of was
cytochrome
into
(cf.with
of
converted
of the
addition
sys-
properties
was
had
3-75;
chromatographic
by
radioactivity
NADPH-generating
of
The
fourfold
The
an
di-lauroylglyceryl-3-phospha-
12a-dihydroxy-4-cholesten-3-one. two-
with
of
the
confirmed
radioactivity
together After
three
Vol. 54, No. 3, 1973
BIOCHEMICAL
200
400 Reductase (U)
600
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
0.5 1.0 CytochromeP-450 (nmoles)
600
1.5
15
10
20
30
Time (min) 1 . Effect of cytochrome P-4.50 reductase concentration P-450 concentration (B), and time (C) on 12n(A) t cytochrome hydroxylation of Tn-hydroxy-4-cholesten-T-one by the reconstituted system with cytochrome P-450 from starved rats (cf. Methods). With the exception of the variable factor in each set of experiments the standard incubation conditions were used (cf. Methods). Fig.
crystallizations was ditions
of
employed
the
amount
time
up
the
enzyme
in
90%
about
which
of to
turation
pg
labeled
rate
assays
tase
necessary
of
the
rate
about
30%
the
of
when
min
substrate
was
4%
oxygen,
30
pg) low
amount
optimal
reaction
56%
1.2 was
of
was
Under
nitrogen
1034
of conditions of in
$
4076
was in carbon
of
reduc-
units inhibited
an
sa-
accu-
P-450
400-600
out
with
saturation
reasonably
system
and
and
conversion
was
carried
with
conditions
the
con-
linear
incubation
cytochrome
complete
the
nmoles
no
making
conversion the
Under
about
standard added.
crystals
was
There
very
The
by
to
1).
were (above
the
material.
up
under
substrate
of
12a-hydroxylation
(Fig.
substrate
the
starting
of
substrate
for
12a-Hydroxylation
radioactivity
P-450
difficult.
1) *
containing
that
30
with
with
added
specific
cytochrome
about
5
the
atmosphere monoxide.
(Fig. by
BIOCHEMICAL
Vol. 54, No. 3, 1973
Under
the
cholic
acid
bited of
same
by
conditions,
and
90%
experiments
in
was
of
treated,
ed
of
systems
cient
with
These
results
vation
on
on
cytochrome
untreated,
starved
cytochrome
P-450
from
cytochrome
P-450
reductase
The
rate
of
the
assayed (Table
is in
1).
cytochrome
some The
from
that
12a-hydroxylation
6P-hydroxylation
of
be
the
reside
pheno-
rats,
was
un-
used,
For
tritium-label-
stimulated
by
different,
phe-
reconstiwas
most
effi-
phenobarbital-treated
the
stimulatory
and
of
in
and
from
hp-hydroxylation
P-450
indicate
to
P-450
12a-hydroxyla-
of
known
results
and
rats
6P-hydroxylation
which
the
starved
phenobarbital-treated
acid,
tuted
of
used.
inhi-
summarizes
with
or
was
were
were
comparison
nobarbital,
I
combinations
whether
lithocholic
Table
from
taurodeoxy-
testosterone
rats
starved
reasons
8).
reductase
fastest
regardless
ref.
which
P-450
of
of
(cf.-
barbital-treated tion
7a-hydroxylation
6p-hydroxylation
about
cytochrome
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
rats, effects
of
phenobarbital
the
star-
treatment
cytochrome
P-450
fractions.
partially
purified
DISCUSSION The
present
chrome
P-450
xylation
of
NADPH
and
up
fourfold
to
work preparation
by
450
contains
fraction
xylase
system
ferred
by
a rat
liver
and
addition
of
the
the
in
finding
Suzuki,
from
the
1035
stimulated reductase
the
NADPH
The
of
cytochrome of
P12a-hydro-
are
trans-
involvment
12a-hydroxylation Mitropoulos
that
the
oxidase
reductase. the
P-450
that
electrons
presence
was
cytochrome
terminal
P-450
by
a
12a-hydrothe
reaction
indicate the
reductase suggested
of
results
that
The
cyto-
catalyzes in
choline.
cytochrome
cytochrome
basis
from
phosphatidyl
These
the
that
Ta-hydroxy-4-cholesten-3-one
preparation.
viously
shows
and 12a-hydroxylation
of
has
been
Myant
(3) is
a preon in-
12d-Hydroxylation
reductase
reductase
reductase
reductase
reductase
Cy-tochrome Cytochrome P-450 Cytochrome P-450 Cytochrome P-450
Cytochrome
Cytochrome
Cytochrome Cytochrome
Cytochrome
Cytochrome
Cytochrome Cytochrome
P-450
P-450
P-450 P-450
P-450
P-450
P-450 P-450
from from
of
--
P-450
reductase
71~I-hydroxy-4-cholesten-Y-one --___ from rat liver
from
from
from from
+
+
+
cytochrome
cytochrome
rats
cytochrome rats
cytochrome
cytochrome
cytochrome
+
+
+
microsomes. --___ were
phenobarbital-treated phenobarbital-treated from untreated rats phenobarbital-treated from starved rats phenobarbital-treated from phenobarbital-treated
starved rats starved rats from untreated rats from starved rats from starved rats from starved rats from phenobarbital-treated
from from
untreated rats untreated rats from untreated rats from untreated rats from starved rats from untreated rats from phenobarbital-treated
System
cytochrome
systems
P-450 P-450 reductase P-450 reductase P-450 reductase
--U of
reductase
500
-________
and
reconstituted
---
rats
rats
rats rats
used.
Table and
+ rats
+
+
P-450
P-450
P-450
cytochrome
cytochrome
---
6P-hydroxylation
cytochrome
P-450
P-450
P-450
Irrespective
I
of
of
5.9
4.6
3.9
3*2
15.2
11.3
11.8
6.8
4.9
5.1
4.6
2.8
nmoles
lithocholic 1.5
12a-Hydroxylation of 7a-hydroxy-kcholesten-3-one
source,
%
of
by
different
7.2
1.6
2. 8
1.2
3.1
1 .5
hp-Hydroxylation of lithocholic acid
cytochrome
acid
P-450
BIOCHEMICAL
Vol. 54, No. 3,1973
hibited
partially
results
do not
nature
of
might
dases,
sults
in
indicate
chrome the
Liver
In
microsomes
constituted monoxide
ref.
reconstituted
was
rather
8).
The
the
properties
unknown
cytochrome and
be
a species
bulk
of
cytochrome
indicate
of
re-
cyto-
P-450
in
using the
re-
carbon
with
different
types
the
cytochrome
P-1450
oxidase
the
previous
towards
that
terminal
P-450
by
insensitive
preoxi-
results
12n-hydroxylation
experiments
of
If
previous
is
P-I150
yet
must
with
the
the It
as
present
the
consonance
systems
containing
specific
from
(3,4)
system (cf. -
fraction the
12u-hydroxylation
different
(3-6).
of
P-l&50.
it
present
cytochrome
amounts
that
The
12w-hydroxylation.
cytochrome
strongly
P-450
"intact"
of
the
c.
concerning
purified
small
from
involved
in
partially
contain
cytochrome
conclusions
oxidase
the
different
of
definite
terminal that
paration
addition
permit
the
conceivable
is
by
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
is
responsible
12cc-hydroxylase
for
system.
ACKNOWI,EDGEMENTS The is tions ject
skilful
gratefully supported
technical
assistance
acknowledged. by
the
This Swedish
work Medical
of
Miss
Irene
is
part
of
Research
Ferdman investiga-
Council
(Pro-
13x-218). REFERENCES
1. 2. 3. 4. 5. 6. 7. 8. 9.
Danielsson, H., in The Bile Acids: Chemistry, Physiology and Metabolism (edited by P.P. Nair and D. Kritchevsky) Vol. 2, p. 1, Plenum Press, New York, 1973. BjorkheM, I., Danielsson, II., and Gustafsson, J., FEBS letters, 2, 20 (1973). Suzuki, M., Mitropoulos, K.A., and Myant, N.B., Biochem. Biophys. Res. Commun., 3, 516 (1968). Einarsson, K., Eur. J. Biochem., 3, 101 (1968). Johansson, G., Eur. J. Biochem., 3, 292 (1970). Bjorkhem, I., and Danielsson, II., Biochem. Biophys. Res. Commun., 5J, 766 (1973). Kuntzman, R., West, S., Jacobson, M., and Lu, A.Y.II., Conney, A.H., .J. Biol. Chem., 247, 1727 (1972). Bjljrkhem, I., Danielsson, H., and Wikvall, Ii., Biochem. Biophys. Res. Commun., 53, 609 (1973). Bjiirkhem, I,, El?r. J. Bschem., g, 337 (1969).
1037
Vol. 54, No. 3, 1973
10 . Il. 1 'IL .
17. 14.
15. 16. 17.
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Wilzbach, k.l<., J. Am. Chem. Sot., 79, 1013 ( 1957). Omura, T., and Sato, R., .I. Iliol. Chem., a, '379 (f9w. Williams, (‘.ff.,
1038