Vol.
174,
No.
January
1, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Pages
15, 1991
SOLUBILIZATION
OF PROSTAGLANDIN PORCINE TEMPORAL
FROM Hiroshi Department
Received
Morii
of
November
30,
and
D2 BINDING CORTEX
Yasuyoshi
Neuroscience, Osaka Suita-shi, Osaka 565,
364-371
PROTEIN
Watanabe* Bioscience Japan
Institute,
1990
Prostaglandin (PG) D2 binding activity was retained at the highest level in the P2 fraction prepared from porcine temporal cortex with the use of buffer containing mannitol and quinacrine. Then, the activity in this fraction was solubilized with maximal recovery by 10 mM CHAPS. The specific PGD2 binding time-dependently increased and was saturated at around 70 nM. Scatchard plots were fitted to a straight line with a Kd value of 20 nM and Bmax of 120 fmol/mg protein. The binding sites showed high specificity for PGD2. In addition, heat and trypsin treatments remarkably decreased the binding activity. These results suggest that the specific binding protein for 0 1991 AcademcPESB, Inc. PGD2 can be solubilized from these membranes.
Prostaglandin mammalian
brain
hypothermia, analgesia Shimizu
modulation (L).
binding and
to be located
olfactory
bulb,
* To whom
and
correspondence
Ail
rights
0
qf
$1.50 1991 by Academic reproduction
in any
Press. form
on its
activity of PGD2
(4-7)
good
induction, and
in the synaptic derivatives,
we
Further,
using
(3).
cortex,
the
binding
preoptic
agreement
with
be addressed.
IIK.
364
CHAPS,
the
mechanism,
demonstrated
cerebral
in
release,
molecular
for PGD2
indicating
prostaglandin; sulfonate.
resrrwd.
such as sleep
binding
of the
prostanoids
hormone
a variety
we
areas
should
study
specific
analysis,
hypothalamus,
Abbreviations: PG, dimethylammoniol-l-propane 0006-291X/91 Copyright
Using
major
actions
PGD2
to be highly
in discrete
the
function,
of
a specific
image
of
central
course
of rat brain.
showed this
one
olfactory
the
detected
is
various of
During
fraction
autoradiography sites
D2
and exerts
et al. (2)
membrane later
(PG)
3-[(3-cholamidopropyl)
area, the
Vol.
174,
No.
1, 1991
BIOCHEMICAL
above-mentioned solubilizing
central PGD2
EXPERIMENTAL
AND
actions
binding
of
protein
BIOPHYSICAL
PGD2.
RESEARCH
COMMUNICATIONS
In this study,
we succeeded
in
in an active form.
PROCEDURES
Materials [5,6,8,9,12,14,15-3HlPGD2 was purchased from DuPont New England Nuclear. Quinacrine, deoxyribonuclease I, ribonuclease, trypsin, proteinase K (fungal), and phospholipase A2 (Naja naja venom) were CHAPS came from Dojindo Laboratories. Unlabeled purchased from Sigma. PGs were generous gifts from Ono Pharmaceutical Company. Preparation of P2 fraction and solubilization with CHAPS - P2 fraction was prepared from frozen porcine temporal cortex as described by Yumoto et al. (&), with the use of homogenizing buffer containing 0.3 M mannitol, 0.5 mM quinacrine, 1 mM PMSF, 2 mM 2-mercaptoethanol, 1 mM EDTA, and 10 mM HEPES at pH 7.5. CHAPS was dropwisely added with stirring to 5 mg protein/ml P2 fraction in the homogenizing buffer to give a final concentration of 10 mM and the suspension was then stirred at 4 “C for 30 min. After centrifugation at 100,OOOg x 60 min, the supernatant was collected and referred to as the solubilized fraction. Binding assay - PGD2 binding was measured as described by Yumoto et al. (&). Unless stated otherwise, the reaction mixture contained a sample having 2 mg/ml protein, 20 nM [3 H] P G D 2, 1 mM EDTA, 2 mM 2mercaptoethanol, 1 mM PMSF, 0.5 mM quinacrine, 0.1 M NaCl, 0.3 M mannitol, and 50 mM Tris-HCl (pH 7.5). In the case of the solubilized fraction, the CHAPS concentration in the reaction mixture was adjusted to 5 mM. The reaction was started by addition of [3H]PGD2 at 37 “C. Nonspecific binding was determined from the measurement of [3H]PGD2 binding in the presence of 100 uM umabeled PGD2. The specific binding was calculated by substraction of the nonspecific binding from the total binding. All values were expressed as the means of triplicate determinations. Protein determination Protein concentration was determined by the method of Bradford (9) with bovine gamma globulin as standard.
RESULTS
AND
P G D2
binding
buffers from
DISCUSSION
-
to the
Specific
different
proteases,
P2
PGD2
pigs,
temporal
among
various
We therefore
prepared activity
because
is one of the richest
mammalian
brain
to improve
in
varied
the binding
and phospholipases
cortex
sought
binding
possibly
glycosidases,
Porcine
fraction
regions the method 365
various among
cortical
activity
as reported
Yumoto,
for isolation
specimens
was sensitive
by Shimizu
sources of PGD2 (N.
homogenizing
binding
unpublished
to
et al. (3. activity results)
of the P2 fraction
Vol.
174,
No.
1.
Table
BIOCHEMICAL
1, 1991
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Effects of the addition of various substances to the homogenizing buffer on PGD2 binding activity PGD2
binding activity
-sugars +sugars (0.3 M) sucrose mannitol a-methyl mannoside glucose galactose glycerol (lO%(w/v))
7.3 14.5 28.3 13.8 10.7 16.9 15.2
phospholipase quinacrine
47.3
inhibitor (0.5 mM)
fmol/mg
protein
P2 membrane was prepared by the method of Yumoto et al. (8). The homogenizing buffer contained 1 mM EDTA, 2 mM p-mercaptoethanol, 1 mM PMSF, 20 uM indomethacin, 10 mM HEPES, and sugars or glycerol as listed in the table. The pH was adjusted to 7.5 at 4 “C. In the experiments using phospholipase inhibitor, 0.3 M mannitol was added to the homogenizing buffer.
from
frozen
porcine
temporal
its
highest
values
showed containing
mannitol
preferred of
over
soluble
among
sucrose
a potent
the binding
activity
inhibitors
prepared
the sugars
in
the
tested
osmotic
(Table
of
almost
no
(data
in the homogenizing
Mannitol
has been the loss
enhanced
various
leupeptin,
protease
PMSF,
shown).
buffer
(llJ.
further
However,
not
buffer
mitochondoria
A2 (10),
bestatin,
fraction in
and to prevent
brain
of mannitol.
effect
prepared
1).
pressure
aprotinin,
to the P2
was
of phospholipase
(antipain,
the P2 fraction
binding
fraction
preparation
inhibitor
had
PGD2
the
in the presence
tested
phosphoramidon)
when
to maintain
substances
Quinacrine,
cortex.
and
Therefore,
we
mannitol
and
containing
quinacrine. Solubilization Specific
of PGD2
specific
binding
various
concentrations
activity
of the binding
activity
to the P:!
in the reaction
The
activity
binding
activity
was solubilized
of CHAPS.
concentration binding
PGD2
Since fraction
mixture
CHAPS
fraction 366
over
CHAPS
the P2
fraction
5 mM
decreased
in the binding
at 5 mM
of the solubilized
from
with
increased
with
assay, we kept
in all cases (data
-
the its
not shown).
dependent
on the
Vol.
174,
No.
BIOCHEMICAL
1, 1991
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
100
0
IO [CHAPS]
20 (mM)
FIG.l. Solubilization of specific PGD2 binding activity from P2 fraction of porcine temporal cortex with CHAPS. P2 fraction (5 mg protein/ml) was incubated for 30 min at 4 “C with CHAPS at the concentrations indicated in the figure. After centrifugation at 100,OOOg x 60 min, the supernatant was concentrated overnight with a CentriCell (nominal molecular weight limit=10 kDa, Polysciences Inc.) overnight. Subsequently, the binding activity of the supernatant ( 0 ) and of the resuspendal pellet ( 0 ) and the protein content of the supernatant ( A ) were determined. In the binding assay of the supernatant, the CHAPS concentration was kept at 5 mM.
CHAPS
concentration
decreased over mM CHAPS
used in the homogenizing
15 mM
(Fig.
1).
buffer
The maximal
activity
with 40 fmol PGD2 bound/mg protein.
I
I
20
40 Time
up to 10 mM and then was obtained
The recovery
at 10
was 38%, a
60
(min)
FIG.2. Time course of specific PGD2 binding to the solubilized fraction. The solubilized fraction (2 mg protein /ml) was incubated with 20 nM [3H]PGD2 at 37 “C. Aliquots were removed at the indicated times and the binding activities were determined. The specific binding ( 0 ) was calculated by substraction of the nonspecific binding ( A ) from the total binding ( 0 ). 367
Vol.
174,
No.
1,
BIOCHEMICAL
1991
0
AND
20
BIOPHYSICAL
RESEARCH
60
40 [PGDZ]
80
100
(WI)
FIG.3. Specific PGD2 binding to the solubilized fraction concentrations of PGD2. The solubilized fraction (2 mg incubated with PGD2 at various concentrations from 2 to Specific binding activities were replotted as a Scatchard
low
relatively
value
Characteristics
but similar
of
[3H]
Fig. 2 shows the time fraction.
The
equilibrium
at around
reached
its
binding
course of specific
specific
equilibrium
to that reported
PG D 2
binding
whereas
within
5 min
Specificity
of [3H]PGD2
binding at 10 nM was measured ), P~Fhc ( A 1, PGE2 (= )I.
the
binding with
the specifc
binding
not
PGs]
368
fraction
and
reached
to rhe P2 fraction The
nonspecific
(M)
binding to the solubilized in the presence
(8).
to the solubilized time
shown).
was Inset:
receptor
solubilized
increased
p[unlabeled
FIG.4.
to
(data
at various protein/ml) 100 nM. plot.
for the PGE2
[3H]PGD2
slowly
50 min,
COMMUNICATIONS
of
fraction.
unlabeled
[3H]PGD2
PGs [PGD2
(e
-
Vol.
174,
No.
BIOCHEMICAL
1, 1991
Table
2.
Effects
AND
BIOPHYSICAL
RESEARCH
of various treatments on [3H]PGD2 solubilized fraction
COMMUNICATIONS
binding to the
Residual PGD2 binding activity
treatment
0 95 105 10 46 32
heat (95 “C, 5 min) deoxyribonuclease I (0.5 mg/ml) ribonuclease (0.5 mg/ml) trypsin ( 50 ug/ml) proteinase K (50 ug/ml) phospholipase A2( 50 uglml) +20 uM indomethacin P-galactosidase (100 U/ml)
%
58
The solubilized fraction (4 mg protein/ml) was boiled for 5 min or preincubated with various enzymes listed above at 37 “C for 15 min. For the treatment with deoxyribonuclease I, 5 mM MgC12 was further added and for that with phopholipase A2, 5 mM CaC12 was added. Results were expressed as percent of control. Control experiments were carried out in the absence of enzymes and the value of PGD2 binding in the absence and the presence of Ca or Mg was 36, 34, or 39 fmol/mg protein, respectively. binding
showed
specific
binding
30%.
After
receptor
relatively to the total
following
did
not
from
2 to 100 nM
increased
dependent
70 nM.
The
squares
method.
protein,
respectively.
rat brain
binding
assay
potency
was
(Fig.
[3H]PGD2
binding
of
were well
significant
of
the
approximately
the 13H]PGD2-
extent
60-min
during
concentrations
fitted
ratio
(data not shown).
binding
and Bmax
value
equilibrium,
concentration
value
membrane
of
[3H] PGD2
to the solubilized and was saturated
to a straight were
21 nM
showed almost
line and
fraction at around
by the least 119 fmol/mg
the same value
as that of
(2).
PG’s added
markedly
observed
a low
PGD2
PGD2
The Kd value
were
respectively.
Specific
plots
unlabeled
the
at various
on [3H]PGD2
to the reaction
inhibitory,
as shown
in the following
ICY50 values for PGD2, nM
to any
of unlabeled
3).
Kd
consequently
reached
dissociate
measured
Scatchard
synaptic
Various
was
The
and
bindin g exhibited
the addition
P G D 2 binding
level
of [3H]PGD2
the binding
complex
period
high
PGF2o,
order:
and PGE2
This
order
of
sites
is almost
the
the 369
mixture
in the [3H JPGD2
in Fig.
4.
PGD2
> PGF2o
were obtained relative
same
affinities
as that
The
inhibitory
> PGEx.
The
at 18, 708, and 8900 of
found
PG’s
for
the
with
synaptic
Vol.
BIOCHEMICAL
174, No. 1, 1991
membrane
prepared
binding
activity
affinity,
high
Effects
of
solubilized
from
rat brain
exhibited
-
In
in PGD2
binding,
enzymes
or
heat
abolished
by boiling
treatment
with
ribonuclease appears treatment
treatment
possibly the
addition
found
of
in this
to
no
the
K
PGD2 high
investigation
provide
with
was
various
completely
However,
deoxyribonuclease PGD2
activity
These
In
support
to
the
stability, basis
binding I or activity the
by 68% in
P-Galatosidase results
and/or
1).
The
Furthermore,
inhibitor.
by 42%.
the
binding
protein.
the binding
of its molecular
the
molecule
decreased
(Table
the
the
markedly
inhibitor
receptor
to
at 95 “C for 5 min.
phospholipids
to the P2 fraction
will
binding
conformation.
a phospholipase
protein
specific
a cyclooxygenase
requires
to afford
was treated
receptor
activity
of
fraction
decreased
the binding
binding
nature
Therefore,
specific A2
the
fraction
effect.
its stable
study
and further
The
proteinase
activity
the binding
P GD 2 receptor protein
solubilized
of indomethacin,
to maintain
enhanced
the
phosphoIipase
binding
to examine
2).
had
also decreased
the
order
%, respectively.
due
with
specific
of the receptor:
[3 H ] P G D 2
the solubilized
or
or 54
be
the presence
that
trypsin
on
(Table
of
treatment to
the solubilized
characteristics
treatments
involved
by 90
Thus,
RESEARCH COMMUNICATIONS
and saturablility.
various fraction
activity
(2).
the following
specificity,
with
AND BIOPHYSICAL
suggest
carbohydrates
of this
homogenizing
possibility, buffer
By use of the conditions
the solubilization for
the
purification
of active of
the
properties.
ACKNOWLEDGMENTS We are indebted to Dr. 0. Hayaishi, the Director of our institute, for valuable discussion. This study was supported in part by the Special Coordination Funds for Promoting Science and Technology from the Science and Technology Agency, Japan, and by grants from the Naito Foundation and Sankyo Co., Ltd. REFERENCES 1. 2.
S., and Hayaishi, 0. (1989) ProstagEandins Ito, s., Narumiya, Leukotrienes, and Essential Fatty Acids, 37, 219-234. Shimizu, T., Yamashita, A., and Hayaishi, 0. (1982) J. Biol. Chem., 257, 13570-13575. 370
Vol.
174,
No.
1, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Tokumoto, H., Watanabe, Y., Yamashita, A., Arai, Y., and Hayaishi, 0. (1986) Brain Res., 362, 114-121. 4. Yamashita, A., Watanabe, Y., and Hayaishi, 0. (1983) Proc. Natl. Acad. Sci. USA, 80, 6114-6118. 5. Watanabe, Y., Yamashita, A., Tokumoto, H., and Hayaishi, 0. (1983) Proc. Natl. Acad. Sci. USA, 80, 4542-4545. 6. Watanabe, Y., Tokumoto, H., Yamashita, A., Narumiya, S., Mizuno, N., and Hayaishi, 0. (1985) Bruin Res., 342, 110-116. 7. Watanabe, Y., Watanabe, Y., Hamada, K., Bommelaer-Bayet, M.-C., Dray, F., Kaneko, T., Yumoto, N., and Hayaishi, 0. (1989) Bruin Res., 478, 143148. N., Watanabe, Y., Watanabe, K., Watanabe, Y., and Hayaishi, 0. 8. Yumoto, (1986) J. Neurochem., 46, 125-132. 9. Bradford, M.M. (1976) Anal. Biochem., 72, 248-254. Biophys. Acta, 187, 486-491. 10. Markus, H.B. and Ball, E.G. (1969) Biochim. 11. Ozawa, K., Seta, K., Araki, H., and Handa, H. (1967) J. Biochem., 61, 512514. 3.
371
174,
No.
January
1, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Pages
15, 1991
SOLUBILIZATION
OF PROSTAGLANDIN PORCINE TEMPORAL
FROM Hiroshi Department
Received
Morii
of
November
30,
and
D2 BINDING CORTEX
Yasuyoshi
Neuroscience, Osaka Suita-shi, Osaka 565,
364-371
PROTEIN
Watanabe* Bioscience Japan
Institute,
1990
Prostaglandin (PG) D2 binding activity was retained at the highest level in the P2 fraction prepared from porcine temporal cortex with the use of buffer containing mannitol and quinacrine. Then, the activity in this fraction was solubilized with maximal recovery by 10 mM CHAPS. The specific PGD2 binding time-dependently increased and was saturated at around 70 nM. Scatchard plots were fitted to a straight line with a Kd value of 20 nM and Bmax of 120 fmol/mg protein. The binding sites showed high specificity for PGD2. In addition, heat and trypsin treatments remarkably decreased the binding activity. These results suggest that the specific binding protein for 0 1991 AcademcPESB, Inc. PGD2 can be solubilized from these membranes.
Prostaglandin mammalian
brain
hypothermia, analgesia Shimizu
modulation (L).
binding and
to be located
olfactory
bulb,
* To whom
and
correspondence
Ail
rights
0
qf
$1.50 1991 by Academic reproduction
in any
Press. form
on its
activity of PGD2
(4-7)
good
induction, and
in the synaptic derivatives,
we
Further,
using
(3).
cortex,
the
binding
preoptic
agreement
with
be addressed.
IIK.
364
CHAPS,
the
mechanism,
demonstrated
cerebral
in
release,
molecular
for PGD2
indicating
prostaglandin; sulfonate.
resrrwd.
such as sleep
binding
of the
prostanoids
hormone
a variety
we
areas
should
study
specific
analysis,
hypothalamus,
Abbreviations: PG, dimethylammoniol-l-propane 0006-291X/91 Copyright
Using
major
actions
PGD2
to be highly
in discrete
the
function,
of
a specific
image
of
central
course
of rat brain.
showed this
one
olfactory
the
detected
is
various of
During
fraction
autoradiography sites
D2
and exerts
et al. (2)
membrane later
(PG)
3-[(3-cholamidopropyl)
area, the
Vol.
174,
No.
1, 1991
BIOCHEMICAL
above-mentioned solubilizing
central PGD2
EXPERIMENTAL
AND
actions
binding
of
protein
BIOPHYSICAL
PGD2.
RESEARCH
COMMUNICATIONS
In this study,
we succeeded
in
in an active form.
PROCEDURES
Materials [5,6,8,9,12,14,15-3HlPGD2 was purchased from DuPont New England Nuclear. Quinacrine, deoxyribonuclease I, ribonuclease, trypsin, proteinase K (fungal), and phospholipase A2 (Naja naja venom) were CHAPS came from Dojindo Laboratories. Unlabeled purchased from Sigma. PGs were generous gifts from Ono Pharmaceutical Company. Preparation of P2 fraction and solubilization with CHAPS - P2 fraction was prepared from frozen porcine temporal cortex as described by Yumoto et al. (&), with the use of homogenizing buffer containing 0.3 M mannitol, 0.5 mM quinacrine, 1 mM PMSF, 2 mM 2-mercaptoethanol, 1 mM EDTA, and 10 mM HEPES at pH 7.5. CHAPS was dropwisely added with stirring to 5 mg protein/ml P2 fraction in the homogenizing buffer to give a final concentration of 10 mM and the suspension was then stirred at 4 “C for 30 min. After centrifugation at 100,OOOg x 60 min, the supernatant was collected and referred to as the solubilized fraction. Binding assay - PGD2 binding was measured as described by Yumoto et al. (&). Unless stated otherwise, the reaction mixture contained a sample having 2 mg/ml protein, 20 nM [3 H] P G D 2, 1 mM EDTA, 2 mM 2mercaptoethanol, 1 mM PMSF, 0.5 mM quinacrine, 0.1 M NaCl, 0.3 M mannitol, and 50 mM Tris-HCl (pH 7.5). In the case of the solubilized fraction, the CHAPS concentration in the reaction mixture was adjusted to 5 mM. The reaction was started by addition of [3H]PGD2 at 37 “C. Nonspecific binding was determined from the measurement of [3H]PGD2 binding in the presence of 100 uM umabeled PGD2. The specific binding was calculated by substraction of the nonspecific binding from the total binding. All values were expressed as the means of triplicate determinations. Protein determination Protein concentration was determined by the method of Bradford (9) with bovine gamma globulin as standard.
RESULTS
AND
P G D2
binding
buffers from
DISCUSSION
-
to the
Specific
different
proteases,
P2
PGD2
pigs,
temporal
among
various
We therefore
prepared activity
because
is one of the richest
mammalian
brain
to improve
in
varied
the binding
and phospholipases
cortex
sought
binding
possibly
glycosidases,
Porcine
fraction
regions the method 365
various among
cortical
activity
as reported
Yumoto,
for isolation
specimens
was sensitive
by Shimizu
sources of PGD2 (N.
homogenizing
binding
unpublished
to
et al. (3. activity results)
of the P2 fraction
Vol.
174,
No.
1.
Table
BIOCHEMICAL
1, 1991
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Effects of the addition of various substances to the homogenizing buffer on PGD2 binding activity PGD2
binding activity
-sugars +sugars (0.3 M) sucrose mannitol a-methyl mannoside glucose galactose glycerol (lO%(w/v))
7.3 14.5 28.3 13.8 10.7 16.9 15.2
phospholipase quinacrine
47.3
inhibitor (0.5 mM)
fmol/mg
protein
P2 membrane was prepared by the method of Yumoto et al. (8). The homogenizing buffer contained 1 mM EDTA, 2 mM p-mercaptoethanol, 1 mM PMSF, 20 uM indomethacin, 10 mM HEPES, and sugars or glycerol as listed in the table. The pH was adjusted to 7.5 at 4 “C. In the experiments using phospholipase inhibitor, 0.3 M mannitol was added to the homogenizing buffer.
from
frozen
porcine
temporal
its
highest
values
showed containing
mannitol
preferred of
over
soluble
among
sucrose
a potent
the binding
activity
inhibitors
prepared
the sugars
in
the
tested
osmotic
(Table
of
almost
no
(data
in the homogenizing
Mannitol
has been the loss
enhanced
various
leupeptin,
protease
PMSF,
shown).
buffer
(llJ.
further
However,
not
buffer
mitochondoria
A2 (10),
bestatin,
fraction in
and to prevent
brain
of mannitol.
effect
prepared
1).
pressure
aprotinin,
to the P2
was
of phospholipase
(antipain,
the P2 fraction
binding
fraction
preparation
inhibitor
had
PGD2
the
in the presence
tested
phosphoramidon)
when
to maintain
substances
Quinacrine,
cortex.
and
Therefore,
we
mannitol
and
containing
quinacrine. Solubilization Specific
of PGD2
specific
binding
various
concentrations
activity
of the binding
activity
to the P:!
in the reaction
The
activity
binding
activity
was solubilized
of CHAPS.
concentration binding
PGD2
Since fraction
mixture
CHAPS
fraction 366
over
CHAPS
the P2
fraction
5 mM
decreased
in the binding
at 5 mM
of the solubilized
from
with
increased
with
assay, we kept
in all cases (data
-
the its
not shown).
dependent
on the
Vol.
174,
No.
BIOCHEMICAL
1, 1991
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
100
0
IO [CHAPS]
20 (mM)
FIG.l. Solubilization of specific PGD2 binding activity from P2 fraction of porcine temporal cortex with CHAPS. P2 fraction (5 mg protein/ml) was incubated for 30 min at 4 “C with CHAPS at the concentrations indicated in the figure. After centrifugation at 100,OOOg x 60 min, the supernatant was concentrated overnight with a CentriCell (nominal molecular weight limit=10 kDa, Polysciences Inc.) overnight. Subsequently, the binding activity of the supernatant ( 0 ) and of the resuspendal pellet ( 0 ) and the protein content of the supernatant ( A ) were determined. In the binding assay of the supernatant, the CHAPS concentration was kept at 5 mM.
CHAPS
concentration
decreased over mM CHAPS
used in the homogenizing
15 mM
(Fig.
1).
buffer
The maximal
activity
with 40 fmol PGD2 bound/mg protein.
I
I
20
40 Time
up to 10 mM and then was obtained
The recovery
at 10
was 38%, a
60
(min)
FIG.2. Time course of specific PGD2 binding to the solubilized fraction. The solubilized fraction (2 mg protein /ml) was incubated with 20 nM [3H]PGD2 at 37 “C. Aliquots were removed at the indicated times and the binding activities were determined. The specific binding ( 0 ) was calculated by substraction of the nonspecific binding ( A ) from the total binding ( 0 ). 367
Vol.
174,
No.
1,
BIOCHEMICAL
1991
0
AND
20
BIOPHYSICAL
RESEARCH
60
40 [PGDZ]
80
100
(WI)
FIG.3. Specific PGD2 binding to the solubilized fraction concentrations of PGD2. The solubilized fraction (2 mg incubated with PGD2 at various concentrations from 2 to Specific binding activities were replotted as a Scatchard
low
relatively
value
Characteristics
but similar
of
[3H]
Fig. 2 shows the time fraction.
The
equilibrium
at around
reached
its
binding
course of specific
specific
equilibrium
to that reported
PG D 2
binding
whereas
within
5 min
Specificity
of [3H]PGD2
binding at 10 nM was measured ), P~Fhc ( A 1, PGE2 (= )I.
the
binding with
the specifc
binding
not
PGs]
368
fraction
and
reached
to rhe P2 fraction The
nonspecific
(M)
binding to the solubilized in the presence
(8).
to the solubilized time
shown).
was Inset:
receptor
solubilized
increased
p[unlabeled
FIG.4.
to
(data
at various protein/ml) 100 nM. plot.
for the PGE2
[3H]PGD2
slowly
50 min,
COMMUNICATIONS
of
fraction.
unlabeled
[3H]PGD2
PGs [PGD2
(e
-
Vol.
174,
No.
BIOCHEMICAL
1, 1991
Table
2.
Effects
AND
BIOPHYSICAL
RESEARCH
of various treatments on [3H]PGD2 solubilized fraction
COMMUNICATIONS
binding to the
Residual PGD2 binding activity
treatment
0 95 105 10 46 32
heat (95 “C, 5 min) deoxyribonuclease I (0.5 mg/ml) ribonuclease (0.5 mg/ml) trypsin ( 50 ug/ml) proteinase K (50 ug/ml) phospholipase A2( 50 uglml) +20 uM indomethacin P-galactosidase (100 U/ml)
%
58
The solubilized fraction (4 mg protein/ml) was boiled for 5 min or preincubated with various enzymes listed above at 37 “C for 15 min. For the treatment with deoxyribonuclease I, 5 mM MgC12 was further added and for that with phopholipase A2, 5 mM CaC12 was added. Results were expressed as percent of control. Control experiments were carried out in the absence of enzymes and the value of PGD2 binding in the absence and the presence of Ca or Mg was 36, 34, or 39 fmol/mg protein, respectively. binding
showed
specific
binding
30%.
After
receptor
relatively to the total
following
did
not
from
2 to 100 nM
increased
dependent
70 nM.
The
squares
method.
protein,
respectively.
rat brain
binding
assay
potency
was
(Fig.
[3H]PGD2
binding
of
were well
significant
of
the
approximately
the 13H]PGD2-
extent
60-min
during
concentrations
fitted
ratio
(data not shown).
binding
and Bmax
value
equilibrium,
concentration
value
membrane
of
[3H] PGD2
to the solubilized and was saturated
to a straight were
21 nM
showed almost
line and
fraction at around
by the least 119 fmol/mg
the same value
as that of
(2).
PG’s added
markedly
observed
a low
PGD2
PGD2
The Kd value
were
respectively.
Specific
plots
unlabeled
the
at various
on [3H]PGD2
to the reaction
inhibitory,
as shown
in the following
ICY50 values for PGD2, nM
to any
of unlabeled
3).
Kd
consequently
reached
dissociate
measured
Scatchard
synaptic
Various
was
The
and
bindin g exhibited
the addition
P G D 2 binding
level
of [3H]PGD2
the binding
complex
period
high
PGF2o,
order:
and PGE2
This
order
of
sites
is almost
the
the 369
mixture
in the [3H JPGD2
in Fig.
4.
PGD2
> PGF2o
were obtained relative
same
affinities
as that
The
inhibitory
> PGEx.
The
at 18, 708, and 8900 of
found
PG’s
for
the
with
synaptic
Vol.
BIOCHEMICAL
174, No. 1, 1991
membrane
prepared
binding
activity
affinity,
high
Effects
of
solubilized
from
rat brain
exhibited
-
In
in PGD2
binding,
enzymes
or
heat
abolished
by boiling
treatment
with
ribonuclease appears treatment
treatment
possibly the
addition
found
of
in this
to
no
the
K
PGD2 high
investigation
provide
with
was
various
completely
However,
deoxyribonuclease PGD2
activity
These
In
support
to
the
stability, basis
binding I or activity the
by 68% in
P-Galatosidase results
and/or
1).
The
Furthermore,
inhibitor.
by 42%.
the
binding
protein.
the binding
of its molecular
the
molecule
decreased
(Table
the
the
markedly
inhibitor
receptor
to
at 95 “C for 5 min.
phospholipids
to the P2 fraction
will
binding
conformation.
a phospholipase
protein
specific
a cyclooxygenase
requires
to afford
was treated
receptor
activity
of
fraction
decreased
the binding
binding
nature
Therefore,
specific A2
the
fraction
effect.
its stable
study
and further
The
proteinase
activity
the binding
P GD 2 receptor protein
solubilized
of indomethacin,
to maintain
enhanced
the
phosphoIipase
binding
to examine
2).
had
also decreased
the
order
%, respectively.
due
with
specific
of the receptor:
[3 H ] P G D 2
the solubilized
or
or 54
be
the presence
that
trypsin
on
(Table
of
treatment to
the solubilized
characteristics
treatments
involved
by 90
Thus,
RESEARCH COMMUNICATIONS
and saturablility.
various fraction
activity
(2).
the following
specificity,
with
AND BIOPHYSICAL
suggest
carbohydrates
of this
homogenizing
possibility, buffer
By use of the conditions
the solubilization for
the
purification
of active of
the
properties.
ACKNOWLEDGMENTS We are indebted to Dr. 0. Hayaishi, the Director of our institute, for valuable discussion. This study was supported in part by the Special Coordination Funds for Promoting Science and Technology from the Science and Technology Agency, Japan, and by grants from the Naito Foundation and Sankyo Co., Ltd. REFERENCES 1. 2.
S., and Hayaishi, 0. (1989) ProstagEandins Ito, s., Narumiya, Leukotrienes, and Essential Fatty Acids, 37, 219-234. Shimizu, T., Yamashita, A., and Hayaishi, 0. (1982) J. Biol. Chem., 257, 13570-13575. 370
Vol.
174,
No.
1, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Tokumoto, H., Watanabe, Y., Yamashita, A., Arai, Y., and Hayaishi, 0. (1986) Brain Res., 362, 114-121. 4. Yamashita, A., Watanabe, Y., and Hayaishi, 0. (1983) Proc. Natl. Acad. Sci. USA, 80, 6114-6118. 5. Watanabe, Y., Yamashita, A., Tokumoto, H., and Hayaishi, 0. (1983) Proc. Natl. Acad. Sci. USA, 80, 4542-4545. 6. Watanabe, Y., Tokumoto, H., Yamashita, A., Narumiya, S., Mizuno, N., and Hayaishi, 0. (1985) Bruin Res., 342, 110-116. 7. Watanabe, Y., Watanabe, Y., Hamada, K., Bommelaer-Bayet, M.-C., Dray, F., Kaneko, T., Yumoto, N., and Hayaishi, 0. (1989) Bruin Res., 478, 143148. N., Watanabe, Y., Watanabe, K., Watanabe, Y., and Hayaishi, 0. 8. Yumoto, (1986) J. Neurochem., 46, 125-132. 9. Bradford, M.M. (1976) Anal. Biochem., 72, 248-254. Biophys. Acta, 187, 486-491. 10. Markus, H.B. and Ball, E.G. (1969) Biochim. 11. Ozawa, K., Seta, K., Araki, H., and Handa, H. (1967) J. Biochem., 61, 512514. 3.
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