Vol.
176,
May
15, 1991
No.
BIOCHEMICAL
3, 1991
AND
RESEARCH
BIOPHYSICAL
COMMUNICATIONS
1501-1508
Pages
PURIFICATION AND PROPERTIES OF CYTOCHROMEP-45O(SCC) MITOCHONDRIA
Masahiro
1 Department
School Received April
Kuwada',
Rie Kitajimal,
Haruo Suzuki*and
FROM PIG TESTIS
Shigeo Horiel
of Biochemistry and *Department of Biophysical Chemistry, l-15-1 Kitasato University, Sagamihara, of Medicine, Kanagawa 228, Japan 4, 1991
Summary: Cytochrome P-450 was purified from pig testis mitochondria to The purified preparation a specific content of 13.1 n mol/mq of protein. was found to contain a single species of P-450, on sodium dodecyl with an apparent molecular sulfate polyacrylamide gel electrophoresis, weight of about 53000 L 2000. The cholesterol side chain-cleavage system could be reconstituted by mixing the purified cytochrome P-450, adrenodoxin reductase, adrenodoxin, cholesterol and NADPH. The rate of n mol/min/n mol of conversion of cholesterol to preqnenolone was 6.2 P-450 under the conditions employed. The absorption spectrum of the oxidized cytochrome P-450 had maxima at 416, 530 and 568 nm. The reduced CO-complex of the cytochrome P-450 exhibited an absorption maximum P-450 was subjected to microsequence analysis and at 448 nm. The purified its NH -terminal amino acid sequence was found to show considerable 0 1991 Academic Press, Inc. homolo$y with that of bovine adrenal P-45O(SCC).
Many laboratories
have reported
chrome P-45O(SCC) of procedures
from adrenal
It
glands. which
catalyzes
hormones.
enzyme in rat testicular capacity of rat
in the adrenal the conversion
is the initial
steroid
rate-limiting The presence
testis
testis
isolation
P-45O(SCC)
cortex
mitochondria
by Mochizuki
side-chain.
But,
et al.
except (9),
1501
for
role
endocrine
to preqnenolone,
side-chain
by Shimizu
showing the presence (8).
an important
in the synthesis
of the cholesterol
the cholesterol
plays
of cholesterol reaction
of cyto-
by means of a variety
and steroid-producing
et al.
were shown by Bass et al.
ed by Mason and Estabrook its
and purification
mitochondria
was established
mitochondria to cleave
cortex
Cytochrome
(1,2,3,4,5).
in steroidogenesis
the isolation
of various cleavage
in 1961 (6) and
in 1973 (7) to have the
A CO-difference
spectrum
of P-450 was also a preliminary
the purification
report
reportof
and characteriza-
0006-291X/91 $1.50 Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol.
176,
tion
No.
of pig
probably easily
BIOCHEMICAL
3, 1991
testicular
because
P-45O(SCC)
pig
to inactive
precise
P-420
properties
remained
of
bovine
testicular
not
RESEARCH
been
described.
tends
COMMUNICATIONS
This
to be converted
adrenocortical
mitochondrial
we report to practical
sulfate-polyacrylamide purified
the
purified
with
that
sequence
have
P-45O(SCC)
than
communication, mitochondria
of the
BIOPHYSICAL
P-45O(SCC). cytochrome
is more Thus,
P-45O(SCC)
the have
obscure.
In this testis
testicular
AND
gel
of bovine analysis
homogeneity,
The NH2 -terminal P-45O(SCC)
adrenocortical of
its
purification
electrophoresis
preparation. testicular
the
cDNA
has
also
P-45O(SCC)
of P-45O(SCC) as judged
on sodium
and also
from
the
amino
acid
sequence
been
reported
determined
from
pig
dodecyl
properties of
and compared recently
by
(10,ll).
EXPERIMENTAL
PROCEDURE
mification of P-450 from adult piq testis mitochondria. All purification procedures were carried out below 4°C. Potassium phosphate buffer of the indicated concentrations, pH 7.0 or 6.5, containing 100 PM EDTA, 100 PM dithiothreitol, 10 uM pregnenolone, 2O%(V/V) glycerol and 0.2%(w/V) unless noted otherwise. The testes were Emulgen 913, was used throughout, freshly obtained from adult pigs at a slaughterhouse and stored frozen at -15°C. They were thawed before use, decapsulated and then homogenized with 4 volumes of 250 mM sucrose for 2 min in a Waring-type blender. The homogenate was centrifuged at 1,000 X g for 10 min and the pellet was discarded. The supernatant was then centrifuged at 10,000 x g for 30 min to obtain the mitochondrial pellet. The mitochodrial pellet was subjected to extraction with 0.6%(W/V) sodium cholate as described by Imai and Sato for liver microsomes (12) and the cholate extract was fractionated with polyethyleneglycol (13). A 5O%(W/V) polyethylene glycol solution was added dropwise during stirring and then the stirring was continued for an additional 10 min. The fraction precipitating between 8 and 12%(W/V) polyethylene glycol as collected by centrifugation at 20,000 X g for 30 min. precipitate was dissolved in 100 mM potassium phosphate buffer, pH 7.0, containing 0.3%(W/V) sodium cholate and then applied to an AH-Sepharose 4B column (5.0 X 20 cm), which had been equilibrated with the same buffer. After washing the column with the 100 mM buffer, the buffer was replaced by 100 mM potassium phosphate buffer supplemented with 0.2%(W/V) Emulgen 913. Some P-450 was observed in the void volume, but most of this hemoprotein was eluted with the 100 mM buffer containing 0.2%(W/V) Emulgen. The eluted solution was condensed with a membrane filter and then dialyzed overnight against 10 mM buffer, pH 7.0, containing 0.2%(W/V) Emulgen 913. The dialyzed fraction was passed through a DEAE-Toyopearl column (I.5 y 70 cm) equilibrated with the 10 mM buffer, and then the non-adsorbed P-450 fraction was again condensed and dialyzed against 33 mM buffer, pH 6.5, fraction was applied to containing O.OS%(W/V) Emulgen 913. The dialyzed a CM-Toyopearl column(2 X 20 cm) and the column was washed with 4 column P-450 was then eluted with a 600 ml linear volumes of the 33 mM buffer. The resulting P-450 fraction gradient to 500 mM KC1 in the 33 mM buffer. was applied once more to the DEAE- and CM-Toyopearl columns, respectively, and Emulgen 913 was finally removed by means of treatment with Bio BEADS SM-2 and dialysis. Enzyme assays; The cholesterol side-chain cleavage activity of the P-45O(SCC) preparation was measured according to the method of Sugano 1502
Vol.
176,
No.
BIOCHEMICAL
3, 1991
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
et al. The reaction was allowed to proceed for 5 min at 37OC. (14). Steroids were extracted with dichloromethane and then analyzed by HPLC. NHz-terminal amino acid microsequence analysis; The NH2-terminal amino acid sequence was determined with an Applied Biosystems Model 470A Gas-Phase Protein/Peptide Sequencer (15,16). Other assays: The protein content was determined according to a modificatoin of Lowry's method (17). Absorption spectra were obtained with a Hitachi 557 spectrophotometer. SDS gel electrophoresis was performed by the method of Laemmli (18). Chemicals: AH-Sepharose 4B, 2',5'-ADP-Sepharose and standard proteins for molecular weight estimation were purchased from Pharmacia. DEAE- and CM-Toyopearl were products of TOSOH Company (Tokyo). Emulgen 91 (polyoxyethylene nonylphenol ether) was kindly supplied by Kao Chemicals co. (Tokyo). All other chemicals used were of the best grade commercially available. RESULTS A summary is
given
in
part
al.
of the in
on the
methods
(1) * The procedure
Toyopearl buffer,
column
used
involves
pH 7.0,
and the
successive
columns
b
were
Stage
of Purification
AH-Sepharose
48
DEAE-Toyopearl (non adsorbed) CM-Toyopearl (1st)
the
present
et al.
performed
cytochrome
4B,
mitochondria study
with
the
the Most
pig
(wd
Total P-450 (n mol)
1660
352
0.212
163
173
1.06
49.1
3.54
19.6
19.5
69.1
3.98
41.0
10.3
CM-Toyopearl PSV
1.56
20.5
13.1
were obtained
from 3 kg of
1503
10 mM of bound
10 mM buffer.
Specific content (n moVmg protein)
et
on DEAE-
1
protein
based
DEAE-Toyopearl
remained
of P-45O(SCC) from adult testicular mitochondria
is
and Shikita
with
respectively.
c reductase
equilibrated
Total
(19)
twice
DEAE-Toyopearl (non adsorbed)
The mitochondria testis tissue.
testis
AH-Sepharose
pH 6.5,
Table
Purification
pig
The chromatographies
buffer,
mM
5 and testicular DEAE-Toyopearl column
in
by John
chromatographies.
33
from
developed
previously
and CM-Toyopearl
cytochrome
on the
of P-45O(SCC)
1. The procedure
Table
and CM-Toyopearl
of
purification
Recovery (%)
100
11.6
5.82
the
Vol.
176,
No.
BIOCHEMICAL
3, 1991
AND
BIOPHYSICAL
RESEARCH
0.05
COMMUNICATIONS
,,’
/
500
400
300 g
!i D
200 y
100 1
0
20
10
30
FRACTION
Fig.
The
1.
P-45O(SCC)
against
mM KCL in the preparation the
was
final
amide
the
was
applied
to
33 mM buffer
and
eluted
33 mM buffer 13.1
preparation
gel
NUMBER
Chromatography of testicular P-450 on CM-Toyopearl. The nonabsorbed P-450 fraction obtained on DEAE-Toyopearl chromatography was dialyzed overnight against 33 mM buffer, pH 6.5, and then applied to a CM-Toyopearl column. P-450 was eluted with 600 ml of the 33 mM buffer in which the KCL concentration was increased from 0 to 500 mM KCL in a linear gradient. Fractions of 10 ml were collected. Fraction numbers 37-45 were collected and aliquots of them were subjected to analysis for A417, P-450 content and electrophoretic pattern. The pooled fractions were stored at - 20°C in the presence of 4O%(W/V) glycerol.
non-absorbed
dialysis
50
40
electrophoresis.
(Fig.
1).
n mol/
mg
of
P-45O(SCC)
of
The
protein.
CM-Toyopearl with
specific It
gave
The results
the
can
a single
a linear
column gradient
content
of
the
be
in
Fig.
seen
after to final 2 that
band on SDS-polyacryl-
shown in Fig.
2 were obtained
..-L.-ad
-.
Fig.
2.
SDS-polyacrylanide gel electrophoresis of testicular P-45O(SCC). Left lane: molecular weight standards (a-lactalbunin(Mr=14,400), trypsin inhibitor(M =20,100), carbonic anhydrase(Mr=30,000), ovalbumin(M,=43,000f, bovine serum albunin(M =67,000) and and phosphorylase b(M,=94,000), from the botEom to the top. Fraction numbers 38 and 43, respectiveCenter and right lanes: from the CM-Toyopearl column (shown in Fig. 1) ly, eluted and concentrated with an Amicon Type B-15 membrane filter.
1504
500
Vol.
176,
01
No.
BIOCHEMICAL
3, 1991
500
400
Fig.
with
the
gel.
but
molecular
results its
weight
The results
contamination
by P-450(118)
The oxidized
were
mobility
also
shown
in
Fig.
those was
2 also
which
having,
obtained
with
of P-45O(SCC)
spectra
of
of
P-45O(SCC)
form
the
purified
with
of
(nm)
an 8 to
standard
estimated rule
has
out
a molecular
a low-spin
type
P-450(SCC)
(Fig.
The CO-difference
showed
3A).
a maximum
The conversion
system
cholesterol
5 min at
was
6.2
absorption
37°C
15%
proteins,
to be appreciable weight
n mol of
pregnenolone
analyzed
of
shown
in Fig.
3.
530 and 568 nm, substrate-free
of
the
dithionite-reduced
3B). was
of NADPH,
and molecular
are
416,
spectrum
to pregnenolone
consisting
and then
at
spectrum
448 nm (Fig.
of cholesterol
a reconstituted
for
at
preparation
gave peaks
indicating
ditions
7 0
600
500
49000.
The absorption
reductase,
COMMUNlCATlONS
WAVELENGTH
similar
53000 + 2000.
form
’
400
(nm)
Sy comparing
apparent
of about
RESEARCH
Absorption spectra of purified P-45O(SCC) from pig testicular contained 410 nM P-45O(SCC) mitochondria. A: The cuvette in 33 mM buffer. Oxidized form ( ) i Dithionitereduced form (--------); Dithionite-reduced and CO-complexed form (-----). B: CO-difference spectrum.
3.
a 15% gel,
gradient
BIOPHYSICAL
1 100 -0.041’
600
WAVELENGTH
AND
also
adrenodoxin,
oxygen.
The mixture
by HPLC.
The specific
formedf'minln
employed. 1505
mol of P-450
investigated
in
adrenodoxin was
incubated
activity under
the
con-
Vol.
176,
No.
3, 1991
BIOCHEMICAL
Pig trst~cular
Ilc
4.
Automated a gas-phase
Thr
Lys - Thr
Pro
Ser
Pro
are
bovine
acid
the
pig
COMMUNICATIONS
Phe -M
Asp.
Asn
Gly
Xaa
- Ile -m
Ty,
P-450(x-C) Lys
Thr
. Pro
Arg
Pro
Ser (_
Glu
Ile
Pro
Ser.
Pro.
Gly
- Asp
Asn
Gly - Trp - Leu
-IA\nl
analysis
the
mitochondrial
with Fig.
the
gap
4, along
(the
the
was
which
was
then
known
carried
out
of the
introduced Thus
was
the
to obtain
cycles sequence the
NH2-terminal into
pig
the
best
processing
of point
P-45O(SCC)
homologous
with
that
pig
testis
was of
P-45O(SCC).
DISCUSSION The molecular mitochondria
characteristics were
adrenocortical P-45O(SCC) 54000
similar
was
on sodium
found dodecyl
to have
an apparent
spectra
of
adrenocortical
similar.
However,
P-450
P-45O(SCC)
was
stable,
The bovine
an apparent
was
molecular
also
weight
and testicular
that
53000
was
of pig 1506
of bovine testicular
weight
the
+ 2000.
observed
and pig
of
electrophoresis
P-45O(SCC)
The CO-complex but
gel in
bovine adrenocortical
molecular
shown of
a difference
from
from
sulfate-polyacrylamide
P-45O(SCC)
preparations.
purified
of P-45O(SCC)
(4,5,20,21).
to exhibit
testicular
the
to those
mitochondria
and pig
also
of P-45O(SCC)
present
(6)
study
The absorption preparations
in
the
were
stabilities
of
adrenocortical P-45O(SCC)
was
of
fit,
portion
testicular
considerably
using
of pig
NH2-terminal
In order
position
peptide.
adrenocortical
with (5,lO).
first
P-45O(SCC))
a sequence
P-45O(SCC)
of NH -terminal sequencing 2 automated Edman degradation
regular
P-45O(SCC)
leader
to have
of
The results
in
testicular
bovine
-Pro
Thr
(SCC)
shown
a one-amino
the
Arg
Ser
adrenocortical
shown
adrenal
Gly
Pro
Ilc
sequencer. P-450
(15,16)
RESEARCH
Comparison of the NH2-terminal sequence of pig testicular P-45O(SCC) determined in the present study with that of bovine adrenal cortex P-45O(SCC) previously reported by Morohashi et al. (10) - The amino acid residues conserved in the two P-450s are enciosed in boxes. sequence
testicular
BIOPHYSICAL
P-45O(SCC)
Ser
HovIne
Fig.
AND
rela-
of
Vol.
176,
tively
not
tending
shown).
CO-difference
extract
showed
The peak
was
6.2
n mol/n
is
considerably
value
2 to
16,
P-4SO(SCC)
assay
have
also
in
the
P-4SO(SCC)
homology
was
hours
why and the
no peak
cholate
at
chromatography
448 nm.
on
acid
to be slightly
in
only
one of the
from
in
pig in
testicular
of organ-specific
of
highest
present
value
prepa-
reported
for
of values,
adrenocortical
ranging
P-45O(SCC),
especially,
on the
in the
to the
P-45O(SCC)
employed.
specific
The instability
detergent activity
of pig
apparently
sequence
low
testicular
specific
the
case
gave
have
not
amino
P-450(17a)
P-4SO(SCC)
of
between
been
presented.
of new born residues
and species-specific
differences. 1507
It
from
has testis
is
difference the
NH2
has been
pig
adrenocortical
resulted
the
adrenocortical
Therefore,
bovine
probably
Although
P-45O(SCC)
of bovine
(21).
adreno-
different
results.
same
has
considerable
that
acid
bovine
with
adrenocortical
of P-450(17a)
study
that
although
the
from
20 NH2 -terminal
present
from
P-450(SCC)
determinations
of pig
data
testicular
22 residues),
different
precise
of pig
Repeated
adrenocortical the
of the
a variety
to differ
P-4SO(SCC)
reported
been reported
of
conditions
difference
sequence
observed.
amino
(ZO),
the
employed,
study
(3 out
of testicular
P-45O(SCC)
acid
present
also
species
activity
bovine
observed
was
protein
However, for
sequence
content.
terminal
and pig
acid
the
contributed
amino
cortical
observed
amino
to be significant.
The NH2-terminal
samples
the
under
than
conditions
and specific
shown
P-450
reported
the
likely
may
of
lower
been
Therefore, not
activity
explains
practically
after
The specific
P-4SO(SCC).
have
on the
is
a few
mitochondria
of a single
mol
adrenocortical
value
observed
existence
preparation.
(22).
testicular
COMMUNICATIONS
within
partly
420 nm, but
at
only
present
used
been
peak
pig
RESEARCH
to P-420
instability
one NH2 -terminal
the
depending
BIOPHYSICAL
4B.
indicated
bovine from
of
only
ration This
spectra
that
the
AND
be converted
this
at 448 nm was
The fact found
to
Perhaps
a high
AH-Sepharose
in
BIOCHEMICAL
3, 1991
unstable,
(data the
No.
also that
difference P-45O(SCC)
a combination Unfortunately,
Vol.
176,
No.
The NH2 -terminal been reported. testicular in
the
BIOCHEMICAL
3, 1991
sequence
of
bovine
The NH2-terminal P-45O(SCC).
nucleotide
the
NH2-terminal
the
action
of
This
sequence isoleucine
AND
BIOPHYSICAL
testicular
isoleucine deletion
absent
may indicate
removed
COMMUNICATIONS
P-45O(SCC) is
of P-45O(SCC). was
RESEARCH
But during
it
in
has
not
pig
a species-difference is the
also
possible
preparation,
that through
aminopeptidase. REFERENCES
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22.
Shikita, M., and Hall, P.F. (1973) J. Biol. Chem. 248, 5598-5604. Ramseyer, J., and Harding, B.W. (1973) Biochem. Biophys. Acta 315, 306-316. Wang, H.P,, and Kimura, T. (1976) J. Biol. Chem. 251, 6068-6074. Suhara, K., Gomi, T., Sato, H., Itagaki, S., and Katagiri, M. (1978) Arch. Biochem. Biophys. 190, 290-299. Sugano, S., Okamoto, M., Ikeda, H., Takizawa, N., and Horie, S. (1989) Biochim. Biophys. Acta 994, 235-245. Shimizu, K., Hayano, M., Gut, M., and Dorfman, R-1. (1961) J. Biol. Chem. 236, 695-699. Bass, J.J., Bell, J.B.G., and Lacy, D. (1973) J. Endocr. 56, 321-322. Mason, J.I., and Estabrook, R.W. (1973) Ann. N.Y. Acad. Sci. 212, 406-419. Mochizuki, H., Suhara, K., and Katagiri, M. (1990) Seikagaku (Tokyo) 62, 869 (Abstract in Japanese). Morohashi, K., Fujii-Kuriyama, Y., Okada,Y., Sagawa, K., Hirose, T Inayama, S., and Omura, T. (1984) Proc. Natl. Acad. Sci. 81, 46i7-4651. Oonk, R., Krasnow, J., Beattie, W., and Richards, J. (1989) J. Biol. Chem. 264, 21934-21942. Imai, Y., and Sato, R. (1974) Biochem. Biophys. Res. Commun. 60, 8-14. Haugen, D. A., and Coon, M.J. (1976) J. Biol. Chem. 251, 7929-7939. Sugano, S., Morishima, N., Ikeda, H., and Horie, S. (1989) Analyt. 182, 327-333. Hewick, R.M., Hunkapiller, M.W., Hood, L.E., and Dreyer, W.J. (1981) J. Biol. Chem. 256, 7990-7997. Suzuki, H., and Kawamura-Konishi, Y. (1988) Biochem. Int. 17, 577-583. Lowry, 0. H., Rosebrough, N.J., Farr, A.L., and Randall, R.J. (1951) J. Biol. Chem. 193, 265-275. Laemmli, U.K. (1970) Nature 227, 680-685. John, A-B., and Karl, M.D. (1982) J. Biol. Chem. 257, 1269612704. Iwahasi, K., Tugi, M., Hiwatashi, A., Simizu, T., Miyatake, A., and Ichikawa, Y. (1988) Seikagaku (Tokyo) 60, 803 (Abstract in Japanese). Nakajin, S., Shinoda, M., Haniu, M., Shvely J. E., and Hall, P. F. (1984) J. Biol. Chem. 259, 3971-3976. Takikawa, O., Gomi, T., Suhara, K., Itagaki, E., Takemori, S., and Katagiri, M. (1978) Arch. Biochem. Biophys. 190, 300-306.
1508
176,
May
15, 1991
No.
BIOCHEMICAL
3, 1991
AND
RESEARCH
BIOPHYSICAL
COMMUNICATIONS
1501-1508
Pages
PURIFICATION AND PROPERTIES OF CYTOCHROMEP-45O(SCC) MITOCHONDRIA
Masahiro
1 Department
School Received April
Kuwada',
Rie Kitajimal,
Haruo Suzuki*and
FROM PIG TESTIS
Shigeo Horiel
of Biochemistry and *Department of Biophysical Chemistry, l-15-1 Kitasato University, Sagamihara, of Medicine, Kanagawa 228, Japan 4, 1991
Summary: Cytochrome P-450 was purified from pig testis mitochondria to The purified preparation a specific content of 13.1 n mol/mq of protein. was found to contain a single species of P-450, on sodium dodecyl with an apparent molecular sulfate polyacrylamide gel electrophoresis, weight of about 53000 L 2000. The cholesterol side chain-cleavage system could be reconstituted by mixing the purified cytochrome P-450, adrenodoxin reductase, adrenodoxin, cholesterol and NADPH. The rate of n mol/min/n mol of conversion of cholesterol to preqnenolone was 6.2 P-450 under the conditions employed. The absorption spectrum of the oxidized cytochrome P-450 had maxima at 416, 530 and 568 nm. The reduced CO-complex of the cytochrome P-450 exhibited an absorption maximum P-450 was subjected to microsequence analysis and at 448 nm. The purified its NH -terminal amino acid sequence was found to show considerable 0 1991 Academic Press, Inc. homolo$y with that of bovine adrenal P-45O(SCC).
Many laboratories
have reported
chrome P-45O(SCC) of procedures
from adrenal
It
glands. which
catalyzes
hormones.
enzyme in rat testicular capacity of rat
in the adrenal the conversion
is the initial
steroid
rate-limiting The presence
testis
testis
isolation
P-45O(SCC)
cortex
mitochondria
by Mochizuki
side-chain.
But,
et al.
except (9),
1501
for
role
endocrine
to preqnenolone,
side-chain
by Shimizu
showing the presence (8).
an important
in the synthesis
of the cholesterol
the cholesterol
plays
of cholesterol reaction
of cyto-
by means of a variety
and steroid-producing
et al.
were shown by Bass et al.
ed by Mason and Estabrook its
and purification
mitochondria
was established
mitochondria to cleave
cortex
Cytochrome
(1,2,3,4,5).
in steroidogenesis
the isolation
of various cleavage
in 1961 (6) and
in 1973 (7) to have the
A CO-difference
spectrum
of P-450 was also a preliminary
the purification
report
reportof
and characteriza-
0006-291X/91 $1.50 Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol.
176,
tion
No.
of pig
probably easily
BIOCHEMICAL
3, 1991
testicular
because
P-45O(SCC)
pig
to inactive
precise
P-420
properties
remained
of
bovine
testicular
not
RESEARCH
been
described.
tends
COMMUNICATIONS
This
to be converted
adrenocortical
mitochondrial
we report to practical
sulfate-polyacrylamide purified
the
purified
with
that
sequence
have
P-45O(SCC)
than
communication, mitochondria
of the
BIOPHYSICAL
P-45O(SCC). cytochrome
is more Thus,
P-45O(SCC)
the have
obscure.
In this testis
testicular
AND
gel
of bovine analysis
homogeneity,
The NH2 -terminal P-45O(SCC)
adrenocortical of
its
purification
electrophoresis
preparation. testicular
the
cDNA
has
also
P-45O(SCC)
of P-45O(SCC) as judged
on sodium
and also
from
the
amino
acid
sequence
been
reported
determined
from
pig
dodecyl
properties of
and compared recently
by
(10,ll).
EXPERIMENTAL
PROCEDURE
mification of P-450 from adult piq testis mitochondria. All purification procedures were carried out below 4°C. Potassium phosphate buffer of the indicated concentrations, pH 7.0 or 6.5, containing 100 PM EDTA, 100 PM dithiothreitol, 10 uM pregnenolone, 2O%(V/V) glycerol and 0.2%(w/V) unless noted otherwise. The testes were Emulgen 913, was used throughout, freshly obtained from adult pigs at a slaughterhouse and stored frozen at -15°C. They were thawed before use, decapsulated and then homogenized with 4 volumes of 250 mM sucrose for 2 min in a Waring-type blender. The homogenate was centrifuged at 1,000 X g for 10 min and the pellet was discarded. The supernatant was then centrifuged at 10,000 x g for 30 min to obtain the mitochondrial pellet. The mitochodrial pellet was subjected to extraction with 0.6%(W/V) sodium cholate as described by Imai and Sato for liver microsomes (12) and the cholate extract was fractionated with polyethyleneglycol (13). A 5O%(W/V) polyethylene glycol solution was added dropwise during stirring and then the stirring was continued for an additional 10 min. The fraction precipitating between 8 and 12%(W/V) polyethylene glycol as collected by centrifugation at 20,000 X g for 30 min. precipitate was dissolved in 100 mM potassium phosphate buffer, pH 7.0, containing 0.3%(W/V) sodium cholate and then applied to an AH-Sepharose 4B column (5.0 X 20 cm), which had been equilibrated with the same buffer. After washing the column with the 100 mM buffer, the buffer was replaced by 100 mM potassium phosphate buffer supplemented with 0.2%(W/V) Emulgen 913. Some P-450 was observed in the void volume, but most of this hemoprotein was eluted with the 100 mM buffer containing 0.2%(W/V) Emulgen. The eluted solution was condensed with a membrane filter and then dialyzed overnight against 10 mM buffer, pH 7.0, containing 0.2%(W/V) Emulgen 913. The dialyzed fraction was passed through a DEAE-Toyopearl column (I.5 y 70 cm) equilibrated with the 10 mM buffer, and then the non-adsorbed P-450 fraction was again condensed and dialyzed against 33 mM buffer, pH 6.5, fraction was applied to containing O.OS%(W/V) Emulgen 913. The dialyzed a CM-Toyopearl column(2 X 20 cm) and the column was washed with 4 column P-450 was then eluted with a 600 ml linear volumes of the 33 mM buffer. The resulting P-450 fraction gradient to 500 mM KC1 in the 33 mM buffer. was applied once more to the DEAE- and CM-Toyopearl columns, respectively, and Emulgen 913 was finally removed by means of treatment with Bio BEADS SM-2 and dialysis. Enzyme assays; The cholesterol side-chain cleavage activity of the P-45O(SCC) preparation was measured according to the method of Sugano 1502
Vol.
176,
No.
BIOCHEMICAL
3, 1991
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
et al. The reaction was allowed to proceed for 5 min at 37OC. (14). Steroids were extracted with dichloromethane and then analyzed by HPLC. NHz-terminal amino acid microsequence analysis; The NH2-terminal amino acid sequence was determined with an Applied Biosystems Model 470A Gas-Phase Protein/Peptide Sequencer (15,16). Other assays: The protein content was determined according to a modificatoin of Lowry's method (17). Absorption spectra were obtained with a Hitachi 557 spectrophotometer. SDS gel electrophoresis was performed by the method of Laemmli (18). Chemicals: AH-Sepharose 4B, 2',5'-ADP-Sepharose and standard proteins for molecular weight estimation were purchased from Pharmacia. DEAE- and CM-Toyopearl were products of TOSOH Company (Tokyo). Emulgen 91 (polyoxyethylene nonylphenol ether) was kindly supplied by Kao Chemicals co. (Tokyo). All other chemicals used were of the best grade commercially available. RESULTS A summary is
given
in
part
al.
of the in
on the
methods
(1) * The procedure
Toyopearl buffer,
column
used
involves
pH 7.0,
and the
successive
columns
b
were
Stage
of Purification
AH-Sepharose
48
DEAE-Toyopearl (non adsorbed) CM-Toyopearl (1st)
the
present
et al.
performed
cytochrome
4B,
mitochondria study
with
the
the Most
pig
(wd
Total P-450 (n mol)
1660
352
0.212
163
173
1.06
49.1
3.54
19.6
19.5
69.1
3.98
41.0
10.3
CM-Toyopearl PSV
1.56
20.5
13.1
were obtained
from 3 kg of
1503
10 mM of bound
10 mM buffer.
Specific content (n moVmg protein)
et
on DEAE-
1
protein
based
DEAE-Toyopearl
remained
of P-45O(SCC) from adult testicular mitochondria
is
and Shikita
with
respectively.
c reductase
equilibrated
Total
(19)
twice
DEAE-Toyopearl (non adsorbed)
The mitochondria testis tissue.
testis
AH-Sepharose
pH 6.5,
Table
Purification
pig
The chromatographies
buffer,
mM
5 and testicular DEAE-Toyopearl column
in
by John
chromatographies.
33
from
developed
previously
and CM-Toyopearl
cytochrome
on the
of P-45O(SCC)
1. The procedure
Table
and CM-Toyopearl
of
purification
Recovery (%)
100
11.6
5.82
the
Vol.
176,
No.
BIOCHEMICAL
3, 1991
AND
BIOPHYSICAL
RESEARCH
0.05
COMMUNICATIONS
,,’
/
500
400
300 g
!i D
200 y
100 1
0
20
10
30
FRACTION
Fig.
The
1.
P-45O(SCC)
against
mM KCL in the preparation the
was
final
amide
the
was
applied
to
33 mM buffer
and
eluted
33 mM buffer 13.1
preparation
gel
NUMBER
Chromatography of testicular P-450 on CM-Toyopearl. The nonabsorbed P-450 fraction obtained on DEAE-Toyopearl chromatography was dialyzed overnight against 33 mM buffer, pH 6.5, and then applied to a CM-Toyopearl column. P-450 was eluted with 600 ml of the 33 mM buffer in which the KCL concentration was increased from 0 to 500 mM KCL in a linear gradient. Fractions of 10 ml were collected. Fraction numbers 37-45 were collected and aliquots of them were subjected to analysis for A417, P-450 content and electrophoretic pattern. The pooled fractions were stored at - 20°C in the presence of 4O%(W/V) glycerol.
non-absorbed
dialysis
50
40
electrophoresis.
(Fig.
1).
n mol/
mg
of
P-45O(SCC)
of
The
protein.
CM-Toyopearl with
specific It
gave
The results
the
can
a single
a linear
column gradient
content
of
the
be
in
Fig.
seen
after to final 2 that
band on SDS-polyacryl-
shown in Fig.
2 were obtained
..-L.-ad
-.
Fig.
2.
SDS-polyacrylanide gel electrophoresis of testicular P-45O(SCC). Left lane: molecular weight standards (a-lactalbunin(Mr=14,400), trypsin inhibitor(M =20,100), carbonic anhydrase(Mr=30,000), ovalbumin(M,=43,000f, bovine serum albunin(M =67,000) and and phosphorylase b(M,=94,000), from the botEom to the top. Fraction numbers 38 and 43, respectiveCenter and right lanes: from the CM-Toyopearl column (shown in Fig. 1) ly, eluted and concentrated with an Amicon Type B-15 membrane filter.
1504
500
Vol.
176,
01
No.
BIOCHEMICAL
3, 1991
500
400
Fig.
with
the
gel.
but
molecular
results its
weight
The results
contamination
by P-450(118)
The oxidized
were
mobility
also
shown
in
Fig.
those was
2 also
which
having,
obtained
with
of P-45O(SCC)
spectra
of
of
P-45O(SCC)
form
the
purified
with
of
(nm)
an 8 to
standard
estimated rule
has
out
a molecular
a low-spin
type
P-450(SCC)
(Fig.
The CO-difference
showed
3A).
a maximum
The conversion
system
cholesterol
5 min at
was
6.2
absorption
37°C
15%
proteins,
to be appreciable weight
n mol of
pregnenolone
analyzed
of
shown
in Fig.
3.
530 and 568 nm, substrate-free
of
the
dithionite-reduced
3B). was
of NADPH,
and molecular
are
416,
spectrum
to pregnenolone
consisting
and then
at
spectrum
448 nm (Fig.
of cholesterol
a reconstituted
for
at
preparation
gave peaks
indicating
ditions
7 0
600
500
49000.
The absorption
reductase,
COMMUNlCATlONS
WAVELENGTH
similar
53000 + 2000.
form
’
400
(nm)
Sy comparing
apparent
of about
RESEARCH
Absorption spectra of purified P-45O(SCC) from pig testicular contained 410 nM P-45O(SCC) mitochondria. A: The cuvette in 33 mM buffer. Oxidized form ( ) i Dithionitereduced form (--------); Dithionite-reduced and CO-complexed form (-----). B: CO-difference spectrum.
3.
a 15% gel,
gradient
BIOPHYSICAL
1 100 -0.041’
600
WAVELENGTH
AND
also
adrenodoxin,
oxygen.
The mixture
by HPLC.
The specific
formedf'minln
employed. 1505
mol of P-450
investigated
in
adrenodoxin was
incubated
activity under
the
con-
Vol.
176,
No.
3, 1991
BIOCHEMICAL
Pig trst~cular
Ilc
4.
Automated a gas-phase
Thr
Lys - Thr
Pro
Ser
Pro
are
bovine
acid
the
pig
COMMUNICATIONS
Phe -M
Asp.
Asn
Gly
Xaa
- Ile -m
Ty,
P-450(x-C) Lys
Thr
. Pro
Arg
Pro
Ser (_
Glu
Ile
Pro
Ser.
Pro.
Gly
- Asp
Asn
Gly - Trp - Leu
-IA\nl
analysis
the
mitochondrial
with Fig.
the
gap
4, along
(the
the
was
which
was
then
known
carried
out
of the
introduced Thus
was
the
to obtain
cycles sequence the
NH2-terminal into
pig
the
best
processing
of point
P-45O(SCC)
homologous
with
that
pig
testis
was of
P-45O(SCC).
DISCUSSION The molecular mitochondria
characteristics were
adrenocortical P-45O(SCC) 54000
similar
was
on sodium
found dodecyl
to have
an apparent
spectra
of
adrenocortical
similar.
However,
P-450
P-45O(SCC)
was
stable,
The bovine
an apparent
was
molecular
also
weight
and testicular
that
53000
was
of pig 1506
of bovine testicular
weight
the
+ 2000.
observed
and pig
of
electrophoresis
P-45O(SCC)
The CO-complex but
gel in
bovine adrenocortical
molecular
shown of
a difference
from
from
sulfate-polyacrylamide
P-45O(SCC)
preparations.
purified
of P-45O(SCC)
(4,5,20,21).
to exhibit
testicular
the
to those
mitochondria
and pig
also
of P-45O(SCC)
present
(6)
study
The absorption preparations
in
the
were
stabilities
of
adrenocortical P-45O(SCC)
was
of
fit,
portion
testicular
considerably
using
of pig
NH2-terminal
In order
position
peptide.
adrenocortical
with (5,lO).
first
P-45O(SCC))
a sequence
P-45O(SCC)
of NH -terminal sequencing 2 automated Edman degradation
regular
P-45O(SCC)
leader
to have
of
The results
in
testicular
bovine
-Pro
Thr
(SCC)
shown
a one-amino
the
Arg
Ser
adrenocortical
shown
adrenal
Gly
Pro
Ilc
sequencer. P-450
(15,16)
RESEARCH
Comparison of the NH2-terminal sequence of pig testicular P-45O(SCC) determined in the present study with that of bovine adrenal cortex P-45O(SCC) previously reported by Morohashi et al. (10) - The amino acid residues conserved in the two P-450s are enciosed in boxes. sequence
testicular
BIOPHYSICAL
P-45O(SCC)
Ser
HovIne
Fig.
AND
rela-
of
Vol.
176,
tively
not
tending
shown).
CO-difference
extract
showed
The peak
was
6.2
n mol/n
is
considerably
value
2 to
16,
P-4SO(SCC)
assay
have
also
in
the
P-4SO(SCC)
homology
was
hours
why and the
no peak
cholate
at
chromatography
448 nm.
on
acid
to be slightly
in
only
one of the
from
in
pig in
testicular
of organ-specific
of
highest
present
value
prepa-
reported
for
of values,
adrenocortical
ranging
P-45O(SCC),
especially,
on the
in the
to the
P-45O(SCC)
employed.
specific
The instability
detergent activity
of pig
apparently
sequence
low
testicular
specific
the
case
gave
have
not
amino
P-450(17a)
P-4SO(SCC)
of
between
been
presented.
of new born residues
and species-specific
differences. 1507
It
from
has testis
is
difference the
NH2
has been
pig
adrenocortical
resulted
the
adrenocortical
Therefore,
bovine
probably
Although
P-45O(SCC)
of bovine
(21).
adreno-
different
results.
same
has
considerable
that
acid
bovine
with
adrenocortical
of P-450(17a)
study
that
although
the
from
20 NH2 -terminal
present
from
P-450(SCC)
determinations
of pig
data
testicular
22 residues),
different
precise
of pig
Repeated
adrenocortical the
of the
a variety
to differ
P-4SO(SCC)
reported
been reported
of
conditions
difference
sequence
observed.
amino
(ZO),
the
employed,
study
(3 out
of testicular
P-45O(SCC)
acid
present
also
species
activity
bovine
observed
was
protein
However, for
sequence
content.
terminal
and pig
acid
the
contributed
amino
cortical
observed
amino
to be significant.
The NH2-terminal
samples
the
under
than
conditions
and specific
shown
P-450
reported
the
likely
may
of
lower
been
Therefore, not
activity
explains
practically
after
The specific
P-4SO(SCC).
have
on the
is
a few
mitochondria
of a single
mol
adrenocortical
value
observed
existence
preparation.
(22).
testicular
COMMUNICATIONS
within
partly
420 nm, but
at
only
present
used
been
peak
pig
RESEARCH
to P-420
instability
one NH2 -terminal
the
depending
BIOPHYSICAL
4B.
indicated
bovine from
of
only
ration This
spectra
that
the
AND
be converted
this
at 448 nm was
The fact found
to
Perhaps
a high
AH-Sepharose
in
BIOCHEMICAL
3, 1991
unstable,
(data the
No.
also that
difference P-45O(SCC)
a combination Unfortunately,
Vol.
176,
No.
The NH2 -terminal been reported. testicular in
the
BIOCHEMICAL
3, 1991
sequence
of
bovine
The NH2-terminal P-45O(SCC).
nucleotide
the
NH2-terminal
the
action
of
This
sequence isoleucine
AND
BIOPHYSICAL
testicular
isoleucine deletion
absent
may indicate
removed
COMMUNICATIONS
P-45O(SCC) is
of P-45O(SCC). was
RESEARCH
But during
it
in
has
not
pig
a species-difference is the
also
possible
preparation,
that through
aminopeptidase. REFERENCES
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22.
Shikita, M., and Hall, P.F. (1973) J. Biol. Chem. 248, 5598-5604. Ramseyer, J., and Harding, B.W. (1973) Biochem. Biophys. Acta 315, 306-316. Wang, H.P,, and Kimura, T. (1976) J. Biol. Chem. 251, 6068-6074. Suhara, K., Gomi, T., Sato, H., Itagaki, S., and Katagiri, M. (1978) Arch. Biochem. Biophys. 190, 290-299. Sugano, S., Okamoto, M., Ikeda, H., Takizawa, N., and Horie, S. (1989) Biochim. Biophys. Acta 994, 235-245. Shimizu, K., Hayano, M., Gut, M., and Dorfman, R-1. (1961) J. Biol. Chem. 236, 695-699. Bass, J.J., Bell, J.B.G., and Lacy, D. (1973) J. Endocr. 56, 321-322. Mason, J.I., and Estabrook, R.W. (1973) Ann. N.Y. Acad. Sci. 212, 406-419. Mochizuki, H., Suhara, K., and Katagiri, M. (1990) Seikagaku (Tokyo) 62, 869 (Abstract in Japanese). Morohashi, K., Fujii-Kuriyama, Y., Okada,Y., Sagawa, K., Hirose, T Inayama, S., and Omura, T. (1984) Proc. Natl. Acad. Sci. 81, 46i7-4651. Oonk, R., Krasnow, J., Beattie, W., and Richards, J. (1989) J. Biol. Chem. 264, 21934-21942. Imai, Y., and Sato, R. (1974) Biochem. Biophys. Res. Commun. 60, 8-14. Haugen, D. A., and Coon, M.J. (1976) J. Biol. Chem. 251, 7929-7939. Sugano, S., Morishima, N., Ikeda, H., and Horie, S. (1989) Analyt. 182, 327-333. Hewick, R.M., Hunkapiller, M.W., Hood, L.E., and Dreyer, W.J. (1981) J. Biol. Chem. 256, 7990-7997. Suzuki, H., and Kawamura-Konishi, Y. (1988) Biochem. Int. 17, 577-583. Lowry, 0. H., Rosebrough, N.J., Farr, A.L., and Randall, R.J. (1951) J. Biol. Chem. 193, 265-275. Laemmli, U.K. (1970) Nature 227, 680-685. John, A-B., and Karl, M.D. (1982) J. Biol. Chem. 257, 1269612704. Iwahasi, K., Tugi, M., Hiwatashi, A., Simizu, T., Miyatake, A., and Ichikawa, Y. (1988) Seikagaku (Tokyo) 60, 803 (Abstract in Japanese). Nakajin, S., Shinoda, M., Haniu, M., Shvely J. E., and Hall, P. F. (1984) J. Biol. Chem. 259, 3971-3976. Takikawa, O., Gomi, T., Suhara, K., Itagaki, E., Takemori, S., and Katagiri, M. (1978) Arch. Biochem. Biophys. 190, 300-306.
1508
