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.

386

Inositol 1,4,5-trisphosphate affinity chromatography.

Inositol 1,4,5-trisphosphate (IP3) affinity columns were made by coupling IP3 analogs to a supporting matrix. Sepharose 4B. IP3 5-phosphatase activity...
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