Vol.
168,
April
No.
1, 1990
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Pages
16, 1990
INOSITOL Masato
Fuai Department Pharmacology,
of
**Department
Received
1.4.5TRISPHOSPBATE
Ishimatsu’. Ozaki”
Faculty of Dentistry, Medicine, Kyushu University,
and *Department Fukuoka 812.
Faculty of Engineering. Chemistry. Matsuyama 790. Japan
of Resources
12,
CHROWATOGRAPAY
Hirata. Yutaka Watanabe** . Toyohiro Yanaga. Toshitaka Koga and Shoichiro
Biochemistry. Faculty of
March
AFFINITY
379-386
Ehiae
of Japan
University,
1990
Summary : Inositol 1.4.5-trisphosphate (IPa) affinity columns were made by coupling IPa analogs to a supporting matrix. Sepharose 48. IP, 5-phosphatase activity, IPB 3-kinase activity and IPI binding activity from rat brain were adsorbed on the IP3 columns. and were eluted by increasing KC1 concentration. This purification procedure increased the specific activities of these parameters 5-200-fold. Thus Sepharose 48 immobilized IP3 analogs can specifically interact with IPs-binding proteins. demonstrating that IPJ affinity columns are a good method for purifying such proteins. Furthermore. suggest that IPJ analogs can be linked to other molecules to our results make useful derivatives without loss of their biological activities. 0 1990 Academic
Press,
Inc.
Inositol
1,4.5-trisphosphate
hydrolysis as
of phosphatidylinositol
an
intracellular
route,
formation other
group
whose potential
Ca2* release
the
IPs-recognizing is
necessary activity.
is
activities
IPs
metabolized
is
subsequently (4).
now being
and
two
degraded
Alternatively.
investigated
by chemical
what
(6.7).
Thus, IP,
proteins
in relation
structural
features
modification
free
produces
in the
inositol
of
the
of
the (51,
proteins
receptors
in
by
1.3.4.5-IP,
three
to IPS metabolism.
of these
routes.
results
to
enzymes
related
from
phosphorylation
kinase
role
Ca”
by two known
putative
to determine and
mobilizing
macromolecules:
domain
important
an
by IP3 5-phosphatase.
of IP3 by an ATP-dependent is
the receptor-activated plays
by
(l-3).
which
role
from
messenger
catalyzed
as IP8-interacting
the
for
1.4-IPZ,
phosphatase
listed
sites
dephosphorylation. of
3-hydroxyl
it
store
a product
4,5-bisphosphate.
second
non-mitochondrial One
(IPa).
are
involved
To characterize to their
functions,
IPI
essential
molecule
are to
build
0006-291X/90 379
in
up $1.50
Copyright 0 1990 by Academic Press. Inc. All rights of reproduction in any form reserved.
Vol.
168,
No.
1, 1990
BIOCHEMICAL
structure-activity and with
profiles
the metabolic
Recently,
for
the
such
BIOPHYSICAL
interaction
a series
as 4-azidobenzoyl
of
or 4-aminobenzoyl
IPs
(12).
little
Using
the ability
but the potencies In addition activity other
profiles
applicability rat
brain
analogs.
of the
analogs
through
with
Sepharose was examined
membrane
COMMUNICATIONS
with
are
receptor
sites
these
analogs
above, added 4B to using
such
modifications
4-(5-
reduced
(12).
capable
We have affinity
group
proteins.
us to build
are
fractions
2nd hydroxyl
IPs-recognizable
to allow
substituents.
cytosol
the
and analogs
IP,
bulky
8195).
with
with
the analogs
obtain
which
4-aminocyclohexanecarbonyl
that
to interact
in
as analog
coupled
we found
among the proteins
the
analogs,
(designated
(#209)
as described
molecules
analogs
group
to designing
IPI
(8204).
these
did vary
of IPI
of
benzamidoethyl-Z-hydroxyphenylazo)benzoyl (#206)
RESEARCH
(8-11).
enzymes
we synthesized
substituents
AND
up structure
of attaching now coupled columns
and detergent-extracts
to these
and
the of
fractions.
MATERIALS AND METHODS Materials. 13H] IP, (specific radioactivity: 31 GBq/mmol or 1.67 TBq/mmol) was obtained from Amersham. IPI was prepared by alkaline hydrolysis of the phosphoinositide fraction (Sigma), according to Grado and Ballou (13) , and purified by a SAX column on an HPLC system. Activated CH-Sepharose 4B was purchased from Pharmacia. Chemical synthesis of the IP, analogs will be described elsewhere (14). All other reagents were of the highest grade available. Preparation of the cytosol fraction and detergent-extract from the particulate fraction of the rat brain. Whole brains, excised from Wistar rats of either sex, weighing 200-300 g. were homogenized with a Dounce of Buffer A containing 50 q M KCl. 10 mM Hepes buffer homogenizer in 5 vol. (pH 7.2). 2 mM NaN3. 2 q M EDTA and 10 116113 -mercaptoethanol. The homogenate was centrifuged at 100.000 g for 60 min. and the resultant supernatant was designated as the cytosol fraction. The pellet was resuspended in the same buffer and recentrifuged. This washed precipitate was solubilized with 1% detergent in Buffer A at a density of 50 ag/ml wet weight at 4°C for 6 hrs. as described by Supattapone et al. (15). in which they used Triton X-100 as the detergent. Mainly. we used the detergent polyoxyethylene 10 tridecyl ether (PIOTE) because it has negligible absorbance at 280 nm and thus allows us to monitor protein elution from the IPS column at this wavelength. The extent of extraction by PlOTE was similar to that with the same concentration of Triton X-100. in terms of the specific activities of IPs 5-phosphatase and [“H]IP, binding. The solubilized materials (detergent-extract) were obtained by centrifuging the mixture at 100.000 g for 2 hrs. Protein concentration was determined by the method of Lowry et al (16) using bovine serum albumin as a standard or by the method of Bradford (17). using a Bio-Rad kit. Assay of IPS metabolic enzyme activities. IPs 5-phosphatase activity was assayed in a mixture (0.5 ml) containing 20 mH Hepes buffer (pH 7. O), 5 q M and 5OpM IP. (containing 370 Bq [8H]IPa of lower specific MgClz radioactivity) at 37°C for 5 min. The reaction was terminated by adding the 380
Vol.
168,
No.
1, 1990
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
0 Acllvated CH-Sepharose
48
*
NaN02 HCI
(Na)HOsPO (Na)HO,PO
zd
-
salt-Sepharose
1. Procedure used to prepare henzoyl]-1.4.5-tri-O-phosphono-ayo-inositol-Sepharose See text for details.
producf
$$j$$ :::a I:.:.:.:.:.:.
48
2-O-[4-(5-aminoethyl-2-hydroxyphenylazo) 4B
of 20% trichloroacetic acid. After and extraction with diethylether (4 ml was analyzed by applying the sample to a SAX IP, 3-kinase activity was assayed as previously same
Y OPO,H(Na)
::::::::E
R
(CH*),NHC-
Fig.
C@z
OH
diazofizafion
2-0-[4-(5-aminoe~hyl-2-hydroxyphenylazo)benzoyl~-l,4,5tri-0-phasphono-myo-inositol trisodium
HO
volume
10 ain
(#204
resin).
centrifugation at 3000 rpm for x 3). the reduction of [aH]IP3 column in an HPLC system (18). described (12.18).
Assay for [“H]IP, binding activity of the detergent-extract and its subfraction on an IPI affinity column. Binding assays with soluble fractions were performed as follows: the mixture (0.45 ml) contained 50 mM Tris-HCl buffer (pH 8.3). 1 q M EDTA, 0.98 nM [“HIIP, of a higher specific radioactivity and lo-2OOpg soluble fraction (final detergent concentration was adjusted at 0.2%). Following incubation on ice for 10 min. the mixture was added to 50 ~1 of bovine r-globulin (10 mg/ml) as a carrier protein, followed by the addition of 500121 of polyethyleneglycol 6000 (3O%.w/v). Each tube was left on ice for After centrifuging the mixture at 16.000 g for 10 min, the 30 min. precipitate was dissolved in 1 ml of 0.1 N NaOH, and then counted for 3H-radioactivity. The aH-radioactivity in the precipitate gradually decreased. with an ICsO of 52 nM (mean of three determinations), by adding increasing concentrations of unlabeled IPa (l-1000 nM). Thus. non-specific binding was determined in the presence of 1 PM unlabeled IP3 and was subtracted from the total binding in its absence to determine the specific binding. In a typical assay with 2OOpg detergent-extract. the ‘H-radioactivity of total or non-specific binding was about 900-1100 dpa or 100-300 dpm. respectively. The specific binding was linearly increased by increasing the amount of extract up to 800 pg. Preparation of Sepharose 48 coupled with IPs analogs (i)2-O-[4-(5-aminoethyl-2hydroxyphenylazo)benzoyl]-1,4.5-tri-Ophosphono-•yo-inositol trisodium salt-Sepharose 4B (Fig. 1) : Step 1: Following washing activated CH-Sepharose 4B (1 g) with 1 mH HCl and subsequently with 0.1 M NaHCOa (pH 8.0). the resin was mixed with 2~mol tyramine at room temperature for I hr. collected by
381
Vol.
168,
No.
1, 7990
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
0 t
COzN
3 0
Activated Cl-l-Sepharose
48
OH 2-0-(4-aminocyclohexanecarbonyi)-1,4,5-t&0phosphono-myo-inositol trisodium
Fig. 2. Procedure used to prepare tri-O-phosphono-•yo-inositol-Sepharose details.
salt-Sepharose
2-O-(4-aainocyclohexanecarbonyl)-1.4.548 (#206 resin). See text
48
for
suction filtration, washed with 0.5 M NaCl plus 50 mM Tris-HCl buffer (pH 8.0) and 0.5 M NaCl plus 50 l M fomic acid (pH 4.0). alternatively and finally suspended in 0.1 M NaBCOs (pH 8.0). Step 2: Twenty microliters of concentrated HCI and 5~~01 NaNOI were mixed with 2 ~.rmol 2-O-(cl-aminobenzoyl)1.4.5-tri-O-phosphono-myo-inositol in water cooled to 0 “c by an ice-rater bath and the mixture was left at the same temperature for 30 ain for diazotization. Step 3: The diazotization product at Step 2 was mixed with the product at Step 1. and the mixture was incubated at room temperature for 1 hr. The resin coupled with the IP3 analog was collected by passing the mixture through a sintered glass funnel, followed by washing as described above. Because the IP3 analog used here was previously designated as compound (12). we will call this the #204 resin or a #204-column when refering to #204 it in this paper. (ii)2-0-(4-a~inocyclohexanecarbonyl)-l.4.5-tri-O-phosphonoinositol trisodium salt-Sepharose 48 (Fig. 2) : After first washing the activated CH-Sepharose 48 (1 g) with 1 mY HCl and subsequently with 0.1 Y NaHCOs (pH 8.0). the resin was suspended in 0.1 Y NaHCOa (pa 8.0) and mixed with 3 q g 2-0-(4-a~inocyclohexanecarbonyl)-l.4.5-tri-O-phosphonoinositol. previously designated as compound #206 (12). The mixture was incubated at coupled with the IPa analog #206 was room temperature for 1 hr. The resin collected by passing the mixture through a filter, followed by alternately washing with 0.5 M NaCl plus 50 mM Tris-HCl (pH 8.0) or 0.5 Y NaCl plus 50 nY formic acid (pH 4.0).
RESULTS AND DISCUSSION The cytosol #206-column.
of the
and the adsorbed
potassium
concentrations
protect
the
sample:
activation
substrate
fraction
columns
attack
of this
(12).
chelate
the Mg**
enzyme.
The
were
All
the
phosphatase, the
present
in the IP,
was applied eluted media
presence
which
used contained
(19)
would
prevent
5-phosphatase 382
activity
can utilize
of MgZ+
solution
to either
a #204-
by a step-wise
by IPS 5-phosphatase
requires
cytosolic
brain
proteins
(Fig.3). from
rat
increase
in
2 mM EDTA to present
in the
IPS analogs
and inclusion the activation
(a specific
or
activity
as a
of EDTA to of this of
37
Vol. 168, No. 1, 1990
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
0..50 2 a
-~
25 original
::: ::’ YB::. v .I
25
original
0
JO
cytosol
fraction
Fig.
3. IPa affinity column chromatography of the cytosol fraction from the rat brain. The cytosol fraction (5.6 qg/ml x 5 ml) was applied to a l-ml #204(A) or #206-column (B). equilibrated with buffer A. Ten 3-ml fractions were collected (up to tube #lo). Then 0.2 M KG1 elution was started at fraction 11. followed by 0.5 M KC1 at fraction 16 and 2 M KC1 at fraction 21. The KC1 eluates were collected in l-ml fractions. Fractions 2. 12. 17 and 22 were assayed for IPS 5-phosphatase (m) and IPS 3-kinase (-1. Each value is mean of duplicate determinations. Three other experiments gave essentially similar results.
nmol/mg/min) 0.5
that
M KCl.
On the
number
other
with
was a 3-
hand,
retained to
5-fold
a #206-column
on
the
#204-column
increase
in
adsorbed
383
this the
was specific phosphatase
eluted
with
activity activity
0.2 M and (Fig.
3A). to
the
Vol.
168,
No.
1,
1990
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
-4m
9
1
-2 .E
a 7
P
6
-300 ;
5
2 2 2 -200 o N" u
4
ii
3
; .E
0.8 2a
0 e a d-8
0.5-
p”
-0.6
”
5
0
15
10
25
20
B
original extract
- 0.4
.. .:: ::: :.: ::: II.::
-0
-0
2
d-i 2
-0.6
loo-
0.2
II
Fig.
least little activity
= l-2
-0.4"
P-.:.
fraction
..
.o.21
20.
25
= OF
a" -100
^n E a
I
20
original extract
number
4. IPI affinity column chromatography of detergent-extract of rat brain membrane fraction. The IX-PlOTE extract of the membrane fraction (2.3 mg/ml x 20 ml) was applied to a #204(AI or #206-column (B) equilibrated with Buffer A containing 1% PIOTE. and ten 5-ml fractions were collected. The KC1 elution was performed as described in Fig. 3. Four fractions were assayed for IPa 5-phosphatase (@) and IPs binding (m). Each determination was done in duplicate. Four other experiments gave essentially the same results. The extract with 1% Triton X-100 behaved in the same manner as the PIOTE extract.
extent.
i.e..
activity which
most was
is
mainly
of
eluted
the
activity
with present
0.2 in
passed M KC1
the
384
cytosol
(Fig.
through 3B). fraction
at
50
The
IPS
(5.
20).
mM KC1
and
3-kinase was
also
Vol.
168,
No.
1, 1990
determined
in
#204-column
each
from
or #206-column #204-column to the
#206-column
increased
the
AND
BIOPHYSICAL
The
enzyme
3A).
Both
specific
activities 0.5
(Fig.
COMMUNICATIONS
were
M and
3B).
eluted
activity
adsorb
over
by lo-
other
proteins.
probably
due to the
columns
to fractionate
the
from
2 M KC1 eluted
Purification
enzyme
to non-specifically
IPa-recognizing
RESEARCH
long
enzyme the
#204-
to to-fold.
proteins
in
spacer
a
The
addition
between
the
and IPJ.
brain
used
membrane
both
fraction
1% detergent
(PlOTE
5-phosphatase
The results
on the
essentially
the
total
enzyme
(Fig. or Triton
IP,
5-phosphatase
activity in the
to the
#206-column,
the salt-concentration:
that
mean of 4
The IP3 affinity them
re-equilibration The related
with
#204-column. potencies
of the
of analog
#204
not
while
of
IPI
3-kinase
5-fold
by the high greater
70% of the
the
specific
greater
than
activity
fraction.
and could
be used again of
did The
be eluted salt
than
by the
that
not
[aH]IPB
by raising
concentration
had
of the
extract
were regenerated
2M KCl.
and
analogs
(12)
fractions
with
[““PIIP, 8206
cytosol
(i)
probably
analog
of
of analog
highly
they
chromatography:
#206-column. of the
the
IPI
and membrane
inhibition
in
after
6M urea
examined
column
cytosolic
that
were
the
of the
in the
PM.
was about
were
followed
by
buffer.
of
interaction
was 5.6
detergent-extract
could
the initial
both
but
fraction
thus.
IPs-phosphatase
lOO-200-fold
a solution
results
from
#204-column:
of
determined.
more than
flow-through
eluted
were
membrane
enzyme:
columns,
used contained
determinations).
characteristics
activities
both
was
columns
with
to the
in the
sample
in the
80% of the
from rat
to 2 mM EDTA; and both
cytosolic
on both
the media
in the
on the
the material
activity
binding
the
appearing
was retained
all
activity
for
detergent-extract
addition
0.2 M or 0.5 M KC1 eluate
bind
washing
[3H]IP3
In contrast,
activity.
case,
in
was retained
extract.
(42 fmol/mg.
X-100).
and
in the
a specific
In this
activity
that
binding
4).
same as those
enzyme activity
by
(Fig.
the
appeared
We also
IPI
fraction.
by 2 Y KC1
activities
resin
BIOCHEMICAL
fraction
concentrated 385
phosphatase
(12)).
and
by both
retained
by the in the
(the
Ki value
by erythrocyte
ghosts
(ii)
[aHlIPa these
to be
5-phosphatase
to differences
hydrolysis
was 16 PM
IP,
were
due
the
appeared
The activities binding
two columns
in
the
because
Vol.
168, No. 1, 1990
analogs
%204 and
(12).
Thus.
with
IPs
these
activities:
206
may be synthesized,
useful
the
IB-coupled
activities.
may be linked
AND BIOPHYSICAL
with
proteins:
these
thus
interacted
Sepharose
recognizable
concentrating analogs
BIOCHEMICAL
therefore
to
other
IP,
derivatives
as proposed
proteins
analogs
These
with
were these
results
molecules
RESEARCH COMMUNICATIONS
capable
such as spin-labeled
by Nahorski
and Potter
are
suggest
loss
of
affinities
of interacting
columns
further
without
higher
their
useful that
the
for IP,
biological
or fluorescent-IP3
(21).
Acknowledgments : This work was supported by the Uehara Memorial Foundation and a Grant-in-Aid for Scientific Research from the Ministry of Education. Science and Culture of Japan. We thank Prof. II. Kuriyama for allowing T. I. to join this research oroject. Thanks are also due to M. Ohara for comments on the manuscript and K: Higucbi for secretarial work.
REFERENCES 1. 2. 3. 4. 5. i: a. 9. 10. 11. 12. 13. 14. 15. 16. :;: 19. 20. 21.
Berridge, Y. J.. and 1rvine.R.F. (1984) Nature 312, 315-321. Berridge.M.J. (1987) Ann. Rev. Biochem. 53. 159-193. Williamson. J. R., Cooper.R.H.. Joseph. S. K.. and Th0mas.A. P. (1985) Am. J. Physiol. 248. C203-C216. Yajerus.P.W., Connol1y.T.M.. Bansa1.V.S.. 1nhorn.R.C.. Ross.T.S.. and Lips, D. L. (1988) J. Biol. Chem. 263. 3051-3054. 1rvine.R. F.. Letcher.A. J., Heslop. J. P., and Berridge.Y. J. (1986) Nature 320. 631-634. (1986) Biochew. J. 240, 917-920. Irvine, R. F.. and Mo0r.R.M. Morris. A. P. , Gallacher, D. V., Irvine, R. F., and Peterson. 0-H. (1987) Nature 330. 653-655. Irvine.R.F., Brown.K.0.. and Berridge,Y.J. (1984) Biochem. J. 221, 269-272. Strupish. J.. Cooke.A.M.. Potter, B. V. L., Gigg. R.. and Nahorski. S. R. (1987) Biochea. J. 253. 901-905. Po1okoff.M.A.. Bencen,G.H.. Vacca.J.P., deSo1ms.S.J.. Y0ung.S.D.. and Huff.J.R. (1988) J. Biol. Chem. 263. 11922-11927. Henne.V.. Mayr.G.W., Grabowski.B., K0ppitz.B.. and So1ing.H.D. (1988) Eur. J. Biochem. 174. 95-101. Hirata. M. , Watanabe.Y., Ishimatsu. T.. Ikebe.T., Kimura. Y.. Yamaguchi. K.. 0zaki.S.. and Koga.T. (1989) J. Biol. Chem. 264, 20303-20308. Grado. C.. and Bal1ou.C.E. (1961) J. Biol. Chem. 236.54-60. Hirata. M., Yanaga. F.. Koga, T.. Watanabe, Y.. and Ozaki. S. (1990) J. Biol. Chem. in press. (1988) J. Biol. Supattapone, S., Worley. P. F., Baraban. J.M., and Snyer.S.H. Chem.263, 1530-1534. Lowry. 0. H. . Rosebr0ugh.N. J., Farr. A. L.. and Randall. R. J. (1951) J. Biol. Chew 193, 265-275. Bradf0rd.M. (1976) Anal. Biochem. 72. 248-254. Yamaguchi. K.. Hirata. M. . and Kuriyama.H. (1987) Biochem. J. 244. 787-791. Sasaguri. T. , Hirata.M. and Kuriyama.H. (1985) Biochem. J. 231, 497-503. Kimura. Y., Hirata. M.. Yamaguchi. K., and Koga. T. (1987) Arch. Biochem. Biophys. 257.363-369. Nahorski. S. R.. and Potter, Pharmacol. Sci. 10. B. V.L. (1989) Trends 139-144.
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