Vol.
167,
March
No.
16,
2, 1990
BIOCHEMICAL
BIOPHYSICAL
AND
RESEARCH
COMMUNICATIONS Pages
1990
396-401
PURIFICATION AND CHAR4CI'ERIZATION OF A CYlWOLIC PHOSPHOINOSITIDEPHOSPHOLIPASE C (y Z-TYPE) FROM HUMAN PLATELETS Yoshiko
Bannol, Aiming Yul, Toshihiko Nakashimaz, Yoshimi Tadaomi Takenawaa and Yoshinori NozawalX
Departments
of 'Biochemistry Medicine,
and 'Neurosurgery, Tsukasarnachi-40, Gifu
Department
of 3Pharmacology, Sakae-cho,
Tokyo Metropolitan Itabashi-ku, Tokyo
Received
January
27,
Homma3,
Gifu University 500, Japan Institute 173, Japan
School
of
of Gerontology,
1990
A human platelet cytosolic phosphoinositide-specific phospholipase Summary: C, one of four PLC activity peaks separated by column chromatographies, designated as cPLC-I, was purified to homogeneity. The cPLC-I exhibited an gel electrophoresis and was apparent Mr of 145 kDa by SDS-polyacrylamide immunologically identified to be PLC-y 2. It hydrolyzed PI and PIP2 at optimum Deoxycholate and cholate inhibited the enzyme activity to pH of 5.5-6.0. hydrolyze two substrates. Calcium was required to obtain the maximal activity for PI- and PIPz-hydrolysis at concentration of 1O-3 M and 10-S M, respectively. Hgz+ (1 PM) inhibited strongly the enzyme activity. a1990 Academic Press,
Inc.
Phosphoinositide-specific and
its
isozymes
various
tissues
(PLC-P
, PLG-Y
have
been
size
but
of PI-PLC various (originaly
phospholipase been
and cells
in
from
bovine
(2,3)
tissues by Northern named as PLC-IV to c-src PLC-y
gene
brain they
sequence.
isolated
soluble
and
and are
PI.&a
widely
membrane
four
soluble
from
dissimilar
In addition, its
is
and
cDNAs encoding
that
acid
C (PI-PLC)
from
The
indicating amino
has been
brain
purified (1).
, PLC-S
isolated, also
homologous bovine
have
guinea
not recently
corresponding
only
distributed fractions
PLC isozymes pig
uterus)
in molecular
a new cDNA clone
mRNA was detected
in
PI-PLCs
product,
. Therefore, from
and multiple
human platelet
cytosol
X To whom correspondence
and showed PLC-y
pronounced
previously
sequence
reported
similarity
was designated
platelets
have
Mr forms (5-9). should
been
of PI-PLC
However,
investigated have
precise
been
in shown
multiplicity
several
to be present of
platelet
be addressed.
The abbreviations used are: PI, phosphatidylinositol; PIP2 , phosphatidylinositol 4,5-bisphosphate ; PLC, phospholipase C; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; PMSF, phenylmethylsulfonyl fluoride; Dll', dithiothreitol; EGTA, [ethylenebis(oxyethylenenitrilo)]tetraacetic acid. 0006-291X/90 Copyright All rights
in
hybridization (3,4). Like PLC-y the cDNA clone and here designated as PLC-)/ 2) contained regions
PLC-y1. The laboratories,
of
$1.50
0 1990 by Academic Press, Inc. of reproduction in any form reserved.
396
to as
Vol.
167,
No.
PLCs has activity
not
been
such
laboratories. human
at
In
identified
pH,
the
acidic
and
immunologically
some bioch:emical
properties
pHs,
was
their in
assay
PLC-y
2.
to be PLC-y
multiple
presence
contain A
absence
of
PI-PLC for
of
recognizable
cytosolic
PI-PLC among
activities and
2 was purified
for
different forms
PI-PLC
major
COMMUNICATIONS
conditions were
hydrolyzing
the to
RESEARCH
detergent
we examined
found
were
the
and
study,
neutral against
BIOPHYSICAL
because
by measuring
fraction
antibody
AND
substrate
present
cytosol
cytosolic
specific
established,
as
platelet
PIPz, The
BIOCHEMICAL
2, 1990
PI-PLC
to homogeneity
PI
of and
detergent. by the (cPLC-I) and
its
characterized.
MATERIALS
ANDMfDHoJx
Materials: PI (soybean), PIP2 (bovine brain) and phosphatidylethanolaine (PE, egg yolk) were purchased from Sigma. [3H]PI (spec. act.; 16.6 Ci/mmol) was from Amersham Corporation. [3H]PIP2 (spec. act.; 3.5 Ci/mmol) was from Du Pont-New England Nuclear. Other agents were of highest purity commercially available. Assay for phospholipase C activity: The reaction mixture (50 ~1) contained 20 mM Tris-maleate buffer (pH 5.5), 80 mM KCl, [aHIP (20,000 dpm)/PI (0.4 mM) or [3H]PIPz (17,000 dpm)/PIPz (0.1 mM)/ PE (0.5 mM), and enzyme protein. When assayed in the presence of 0.1 % deoxycholate the reaction was performed in 20 mM Tris-maleate buffer (pH 6.5 - 7.0). CaZ+ concentrations used for PI- and PIPz-ydrolysis were 2 mM and 10 PM, respectively. Free Ca2+ concentrations were adjusted to the desired levels using Ca2+/EGTA buffers The reaction was performed as described previously (11). (10). Purification of Phospholipase C: The cytosolic fraction of outdated human platelet concentrate was prepared as described previously (11) and dialyzed against buffer A [20 mM Tris-HCl (pH 7.4) containing 5 mM EGTA, 1 mM EDTA, 1.0 mM phenylmethylsulfonyl fluoride (PMSF), 1 mM dithiothreitol (MT) and 10 % glycerol]. After centrifugation the dialysate was loaded on Fast Q-Sepharose column and eluted with a linear NaCl gradient (0.1 - 0.4 M). Two activity. fractions (Fr-I, Fr-II) were resolved; Fr-I was eluted at 0.18-0.25 M NaCl and Fr-II at 0.25 - 0.35 M NaCl (Fig. 1). Fr-I recognizable by the anti-PI& y 2 antibody was subjected to further purification by successive column chromatographies. Briefly, Fr-I was loaded on Ultrogel AcA-44 column and eluted with buffer A containing 0.3 M NaCl. The active fraction was dialyzed against buffer B (20 mM Tris-HCl, pH 7.4, 1 mM EDTA, 1 mM EGTA, 0.1 mM PMSF, 11 mM DlT and 10 % glycerol) and then applied onto Mono Q column and eluted with a linear NaCl gradient (0.1-0.3 M). The PLC activity was recovered in two peaks; the first peak eluted at 0.1-0.18 M NaCl and the second peak at 0.25-0.3 M NaCl. The first activity peak, designated as cPLC-I, gave a positive cross-reaction with anti-PLC-y 2 antibody. cPI.C-I was subjected to the heparin-agarose column chromatography followed by elution with a linear NaCl gradient 110.2 - 0.6 M) in buffer B, yielding a single activity peak. When the activity peak was applied onto hydroxyapatite column and eluted with a linear gradient of potassium phosphate (0.1 - 0.4 M), the activity was eluted at 0.25 - 0.3 M potassium phosphate. At the final step of Mono S column chromatography with a linear NaCl gradient (0.1 M - 0.4 M), a single activity peak of cPLC-I was recovered at approx. 0.2 M NaCl, which was coincident with a symmetrical protein peak. Other methods' Analytical polyacrylamide gel electrophoresis was perI* formed by the method of Laemmli (12) in gradient (8-16 %) slab gels containing SDS. Electrophoretic transfer of protein from slab gels to nitrocellulose sheets and subsequent immunoblotting using [ 125Ilprotein A were performed as described (13). Anti-PLC-y 2 antibody was prepared using PLC-y 2 proteins produced by E. coli expression system (3). E. coli crude extracts were loaded on 6 % SDS397
Vol.
167,
No.
BIOCHEMICAL
2, 1990
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
polyacrylamide gel. Following electrophoresis, the band corresponding to PLCy 2 proteins was cut from the gel. The gels including PLC-y 2 proteins were injected into rabbits with Freund complete adjuvant every 3 weeks for 2 months. Antibodies against PLC-fi , PLC-y 1 and PLC-S were also prepared using each type of PLC protein produced by E. coli expression systems (4). Anti-PLCy 2 antibody only reacted with PLC-)/ 2 but not with other PLCs. Similarly, anti-PLC-fi , anti-PLC-y 1 and anti-PLC-6 antibodies reacted specifically with PLC-p , PLC-y 1 and PLC-6 , respectively (manuscript in preparation). RESULTS
into
The
cytosolic
two
fractions
when
measured
activity Fr-I for
(Fr-I, using
for
was recognizable further
between
antibody hydroxyapatite
(Mr.
at
single
(CPLG-I)
subjected
2 it and
the which
to
Fast
pH 5.5
broad
240 kDa)
chromatography,
peak was
a
and
PIPz-hydrolysis
Q-Sepharose
as substrates
of Fr-I,
and
column
first
Fr-I
PI-
upon
PIP2
by anti-PLC-y
purification
catalase
Mono Q
for
Fr-II)
PI and
was higher
chromatography
Its
PLC activities
Fr-II
antibody
but
1). at
pH 7.0,
respectively.
Fr-II
was not.
activity
was eluted
bovine activity was
serum
chromatography PI-hydrolyzing
to
peak
Ultrogel
albumin
at
(Mr. with
chromatographies The overall
Therefore,
AcA-44 the
column position
67 kDa).
peak was recovered cross-reactive
resolved
The
was subjected
subsequent
and Mono S columns.
column
(Fig.
and
were
in the
two
Upon peaks.
anti-PLC-y
on heparin-agarose,
purification
steps
achieved
02 Fig. 1. Fast Q-Sepharose column chromatography of the human platelet cytosol. PI-hydrolysis at pH 5.5 without deoxycholate (0); PI-hydrolysis at pH 7.0 in the presence of deoxycholate (1 r&ml) (A ); PIPz-hydrolysis at pH 6.5 in the presence of deoxycholate (1 m&ml) using [~H)PIPz/PE as substrate (0 ); N&l concentration (-----) ; absorbance at 280 nm (-). Fist. 2, SDS-polyacrylamide gel electrophoresis and immunoblot of cPLGI. The purified cPLC-I was subjected to SDS-polyacrylamide gel and stained with Coomassie Briliant Blue (1) or immunoblotted with anti-PLC-y 2 antibody (2). Molecular mass markers used were: 200 kDa, myosin; 116 kDa, a -galactosidase, 97 kDa, phosphorylase b; 67 kDa, bovine serum albumin; 45 kDa, egg albumin. 398
2
Vol.
167,
No.
approx.
BIOCHEMICAL
2, 1990
6,800-fold
activity
of
and 9.5 The
cytosolic
cleaved
finally
molecular using
mass of
of pH on the
PI and PIP2
exhibited
as
an acidic
Addition
Iof
hydrolysis, This
detergent
(1 mg/ml)
(5.0
with
a
analysis that
2 antibody.
The molecular
mass
using
cPK-I
of cPIC-I
in
- 5.5)
for
Fig.
3-A,
either
PI-
was without
for
inhibited
PI-
is
Wako-
present
in
effect
PIPz-hydrolysis
the
purified
3-B).
with cPLC-I
or PIPZ-hydrolysis.
on pH optimum
by 80 %,
(Fig.
examined
by shifting
PI-hydrolysis
activity
were
whereas
Cholate
for
to it
was
PI-
pH 6.0. rather
inhibitory
and PIP*-hydrolysis.
cPLC-I
concentration
activity (Fig.
reaching
a maximal
at
lo-4
to
of
2 mM of Mn2+,
As shown
that
for
as
13.6
revealed
filtration
that
activity
(1 mg/ml)
affected
-the PIP;!-hydrolyzing
shown).
band
anti-PLC-y gel
was
respectively.
Immunoblot
examined.
indicating
hydrolyzing
pH optimum
enhanced The
2).
by calibrated
substrates.
deoxycholate but
both
of cPLC-I
2 and PLC-8
the
were
total
state.
The effects both
with
145 kDa by SDS-PAGE,
initial
protein
(Fig.
1, PLC-y
purified
the
PI and PIPz,
a single
solely
thus
COMMUNICATIONS
% of
activity
on SDS-PAG
was 140 kDa as determined
a monomeric
1.3
for
, PLC-y
cross-reacted
and
of
displayed
to PLC-p
of cPLC-I
Pack HPLC column
a yield
RESEARCH
The specific
145 kDa estimated
cPIC-I
BIOPHYSICAL
mg of protein
cPLC-I
antibodies
purified cPLC-I
fraction. / min per
The properties of
with
purified
specific
the
purification
the
,umol
AND
lo-3
level
EGTA or
hydrolyze
the at
10-S M and the
EDTA by approx. ,uM)
was without
and Cu2+ caused
3 i lo-
PI
and PIP;!
PIPz-hydrolyzing
activity 60 %
effect weak
or and
o
on
was enhanced activity
was inhibited metal (15
ions
- 20 %).
by addition (data
not
FM)
such
Although
the
(50
A)
;)jq
5.0
6.0 PH
70
6.0
0 1.0 [DETERGENT]
2.0 (w/d)
EG,A--7-6 G-i, -5 -4 -3 LOG [Ca*+l M
-
The buffer Fig. 3. A) pH-dependence of cPLC-I in PIand PIPI-hydrolysis. used was Tris/maleate, pH 4.5-8.0. PI (O,O)or PIP2 (A,A)-hydrolyzing (O,A) or presence (@,A) of activities were measured in the absence B) Effects of deoxycholate (--) or cholate (-----) on deoxycholate (1 mg/ml). CPU-I activity for PI(0) or PIP2(0) hydrolysis. C) Calcium dependence of cPLC-I activity in PI(0) or PIPz(0) hydrolysis.
399
Ca2 +
by Ca2+
was maximal
90 %, respectively other
inhibition
~~~~:~
E 8
was dependent
activity PI-hydrolyzing
The PIP2-hydrolyzing
M.
.?JgZ+ (50 Fez+
to
3-C);
Vol.
167,
No.
enzyme
2, 1990
activity
BIOCHEMICAL
was extremely
the 1 ,u M HgClz, ,uM dithiothreitol. 0.29
inhibitory
mM for
effect
per
min/mg
protein,
to
the
with
were
,~mol
at
by addition
Km values
17.6
COMMUNICATIONS
50 % inhibition
completely
apparent
and Vmax values
RESEARCH
HgZ+
was overcome
plots,
PIP2
BIOPHYSICAL
sensitive
From double-reciprocal and
AND
were
and
of
0.12
10.3
100
mM for
,U mol
PI
cleaved
respectively.
DISCUSSION
In
the
purified
previous
from
67 kDa (7), optima
platelet
PI-
be activated (Mr
and
Because among
well
been
platelet as
have
shown
of
there
hydrolysis
was
higher
activity
chromatographies,
or cholate. purified other
previously was
specificity
are
two
(Fig.
1).
(98
to
to see,
therefore,
(Fr-I
enzymes
whether
in human platelet
did
from 1
Hgz+,
but
not
(14)
PLC-y
2 is
expressing
somehow
high
level
Fr-I
PI-
with
a
showed
an
deoxycholate of
a partially
from
those
of
Moreover, substrate
with for the and it with
for
by stepwise 2,
inhibitory
of pp6Oc-src
and
distinct
(5,7-g).
associated
(PI,
Fr-II
different
in
has human
minus),
Caz*-dependence,
evidence (15,16),
of
at pH 5.5
cross-reactivity
studies provided PLC-)I 1 activation
PI-PJX
by either
cytosols
different
display
substrate,
or
those
of
PI or PIPz
be PLC-y
to
a
conditions
from to
but
in
absence
while
similar (6),
the
exhibiting
activity
purified
platelet
PLC-y
to
cPLC-I
Fr-II)
and was inhibited were
isolated
PI-PLCs
plus
2 antibody,
al.
(6)
cytosolic various
pH
and could
(pH,
examined
identified
et
in
used
and
cPLC-I
neutral
when either
detergent
a greater
with
properties
Recently, several tyrosine kinase in
protein
pHs;
Fr-I
by Manne brain
(5.5)
platelet
fractions
PI-hydrolysis
purified
similar
detergents.
acidic
pH was not.
kDa)
Manne et al.
under
by anti-PLC-y
enzymatical
They had
were
70 kDa and
of deoxycholate
we have
immunologically
for
human.
conditions
activities
Mrs
120 kDa,
detergent of
different
(5),
pH optimum
context,
and
and sensitivity
antibody.
kinase
Its PI-PLC
cPLC-I toward
5.5
hand,
by this
their
at neutral
pH at
other
multiplicity
recognizable
column
presence
assay
In this
neutral
properties
from
the
an acidic
different
by measuring
catalytic
optimum
On the
laboratories,
substrates; that
in
with
bovine
57 kDa (9)
inhibited
understood.
cytosol
PIP2
and
had
which
enzymes
143 kDa from
detergent.
and was rather
used.
PI-PLC
PIPZ-hydrolysis
98 kDa)
detergent) not
cytosol;
by this
deoxycholate was
various
140 - 100 kDa (8)
for
PI-PLC
studies,
behaviors anti-PLC-y
1
involvement of is of interest protein
tyrosine
(17).
ACKNOWLEDGMENT
This Education,
study was supported Science and Culture
in part of Japan. 400
by
a grant
from
the
Ministry
of
Vol.
167,
No.
BIOCHEMICAL
2, 1990
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
REFERENCES
1
Rhee ,'a '9 G *, Suh, P.G., Ryu, S.H. and Lee, K.Y. (1989) Science 244, 546* 550. 2. Ohta, S., Matsui, A., Nozawa, Y. and Kagawa, Y. (1988) FEBS Lett. 242, 31-35. 3. Fmori, Y., Homma, Y., Sorimachi, H., Kawasaki, H., Nakanishi, O., Suzuki, K. and Takenawa, T. (1989) J. Biol. Chem. 264, 21885-21890. 4. Homma, Y., Takenawa, T., Kmori, Y., Sorimachi, H. and Suzuki, K. (1989) Biochem. Biophys. Res. Commun. 164, 406-412. 5. Hakata, H., Kambayashi, J. and Kosaki, G. (1982) J. Biochem. 92, 929935. 6. Manne, V. and Kung, H.F. (1987) Biochem. J. 243, 763-771. 7. Banno, Y., Nakashima, S. and Nozawa, Y. (1986) Biochem. Bioph.ys. Res. Commtm.
136,
Low, M.G., 9. Baldassare, 8.
28,
713-721.
Carroll, J.J.,
R.C. and Cox, Henderson, P.A.
A.C. (1986) and Fisher,
Biochem. J. 237, 139-145. G.J. (1989) Bi0chemistr.y
6010-6016.
10. Raaflaub, J. (1986) Methods Biochem. Anal. 3, 301-325. 11. Banno, Y., Ysda, Y. and Nozawa, Y. (1988) J. Biol. &em. 263, 1145911465. 12. Laemmli, U.K. (1970) Nature 227, 680-685. 13. Towin, H., Staehelin, T. and Gordon, J. (1979) Proc. Natl. Acad. Sci. USA. 76, 4350-4354. 14. Ryu, S.H., Cho, K.S., Lee, K.-Y., Suh, P.-G. and Rhee, S.G. (1987) J. Bicl. Chem. 262, 12511-12518. 15. Meisenhelder,J., Sub, P-G.,Rhee,S.G. and Hunter T. (1989) Cell 57, 11091122. 16. Nishibe, S., Wahl, M.I., Rhee, S.G. and Carpenter, G. (1989) J. Biol. Chem. 264, 10335-10338. 17. Golden, A. and Brugge, J.S. (1989) Proc. Natl. Acad. Sci. KSA. 86, 901-905.
401
167,
March
No.
16,
2, 1990
BIOCHEMICAL
BIOPHYSICAL
AND
RESEARCH
COMMUNICATIONS Pages
1990
396-401
PURIFICATION AND CHAR4CI'ERIZATION OF A CYlWOLIC PHOSPHOINOSITIDEPHOSPHOLIPASE C (y Z-TYPE) FROM HUMAN PLATELETS Yoshiko
Bannol, Aiming Yul, Toshihiko Nakashimaz, Yoshimi Tadaomi Takenawaa and Yoshinori NozawalX
Departments
of 'Biochemistry Medicine,
and 'Neurosurgery, Tsukasarnachi-40, Gifu
Department
of 3Pharmacology, Sakae-cho,
Tokyo Metropolitan Itabashi-ku, Tokyo
Received
January
27,
Homma3,
Gifu University 500, Japan Institute 173, Japan
School
of
of Gerontology,
1990
A human platelet cytosolic phosphoinositide-specific phospholipase Summary: C, one of four PLC activity peaks separated by column chromatographies, designated as cPLC-I, was purified to homogeneity. The cPLC-I exhibited an gel electrophoresis and was apparent Mr of 145 kDa by SDS-polyacrylamide immunologically identified to be PLC-y 2. It hydrolyzed PI and PIP2 at optimum Deoxycholate and cholate inhibited the enzyme activity to pH of 5.5-6.0. hydrolyze two substrates. Calcium was required to obtain the maximal activity for PI- and PIPz-hydrolysis at concentration of 1O-3 M and 10-S M, respectively. Hgz+ (1 PM) inhibited strongly the enzyme activity. a1990 Academic Press,
Inc.
Phosphoinositide-specific and
its
isozymes
various
tissues
(PLC-P
, PLG-Y
have
been
size
but
of PI-PLC various (originaly
phospholipase been
and cells
in
from
bovine
(2,3)
tissues by Northern named as PLC-IV to c-src PLC-y
gene
brain they
sequence.
isolated
soluble
and
and are
PI.&a
widely
membrane
four
soluble
from
dissimilar
In addition, its
is
and
cDNAs encoding
that
acid
C (PI-PLC)
from
The
indicating amino
has been
brain
purified (1).
, PLC-S
isolated, also
homologous bovine
have
guinea
not recently
corresponding
only
distributed fractions
PLC isozymes pig
uterus)
in molecular
a new cDNA clone
mRNA was detected
in
PI-PLCs
product,
. Therefore, from
and multiple
human platelet
cytosol
X To whom correspondence
and showed PLC-y
pronounced
previously
sequence
reported
similarity
was designated
platelets
have
Mr forms (5-9). should
been
of PI-PLC
However,
investigated have
precise
been
in shown
multiplicity
several
to be present of
platelet
be addressed.
The abbreviations used are: PI, phosphatidylinositol; PIP2 , phosphatidylinositol 4,5-bisphosphate ; PLC, phospholipase C; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; PMSF, phenylmethylsulfonyl fluoride; Dll', dithiothreitol; EGTA, [ethylenebis(oxyethylenenitrilo)]tetraacetic acid. 0006-291X/90 Copyright All rights
in
hybridization (3,4). Like PLC-y the cDNA clone and here designated as PLC-)/ 2) contained regions
PLC-y1. The laboratories,
of
$1.50
0 1990 by Academic Press, Inc. of reproduction in any form reserved.
396
to as
Vol.
167,
No.
PLCs has activity
not
been
such
laboratories. human
at
In
identified
pH,
the
acidic
and
immunologically
some bioch:emical
properties
pHs,
was
their in
assay
PLC-y
2.
to be PLC-y
multiple
presence
contain A
absence
of
PI-PLC for
of
recognizable
cytosolic
PI-PLC among
activities and
2 was purified
for
different forms
PI-PLC
major
COMMUNICATIONS
conditions were
hydrolyzing
the to
RESEARCH
detergent
we examined
found
were
the
and
study,
neutral against
BIOPHYSICAL
because
by measuring
fraction
antibody
AND
substrate
present
cytosol
cytosolic
specific
established,
as
platelet
PIPz, The
BIOCHEMICAL
2, 1990
PI-PLC
to homogeneity
PI
of and
detergent. by the (cPLC-I) and
its
characterized.
MATERIALS
ANDMfDHoJx
Materials: PI (soybean), PIP2 (bovine brain) and phosphatidylethanolaine (PE, egg yolk) were purchased from Sigma. [3H]PI (spec. act.; 16.6 Ci/mmol) was from Amersham Corporation. [3H]PIP2 (spec. act.; 3.5 Ci/mmol) was from Du Pont-New England Nuclear. Other agents were of highest purity commercially available. Assay for phospholipase C activity: The reaction mixture (50 ~1) contained 20 mM Tris-maleate buffer (pH 5.5), 80 mM KCl, [aHIP (20,000 dpm)/PI (0.4 mM) or [3H]PIPz (17,000 dpm)/PIPz (0.1 mM)/ PE (0.5 mM), and enzyme protein. When assayed in the presence of 0.1 % deoxycholate the reaction was performed in 20 mM Tris-maleate buffer (pH 6.5 - 7.0). CaZ+ concentrations used for PI- and PIPz-ydrolysis were 2 mM and 10 PM, respectively. Free Ca2+ concentrations were adjusted to the desired levels using Ca2+/EGTA buffers The reaction was performed as described previously (11). (10). Purification of Phospholipase C: The cytosolic fraction of outdated human platelet concentrate was prepared as described previously (11) and dialyzed against buffer A [20 mM Tris-HCl (pH 7.4) containing 5 mM EGTA, 1 mM EDTA, 1.0 mM phenylmethylsulfonyl fluoride (PMSF), 1 mM dithiothreitol (MT) and 10 % glycerol]. After centrifugation the dialysate was loaded on Fast Q-Sepharose column and eluted with a linear NaCl gradient (0.1 - 0.4 M). Two activity. fractions (Fr-I, Fr-II) were resolved; Fr-I was eluted at 0.18-0.25 M NaCl and Fr-II at 0.25 - 0.35 M NaCl (Fig. 1). Fr-I recognizable by the anti-PI& y 2 antibody was subjected to further purification by successive column chromatographies. Briefly, Fr-I was loaded on Ultrogel AcA-44 column and eluted with buffer A containing 0.3 M NaCl. The active fraction was dialyzed against buffer B (20 mM Tris-HCl, pH 7.4, 1 mM EDTA, 1 mM EGTA, 0.1 mM PMSF, 11 mM DlT and 10 % glycerol) and then applied onto Mono Q column and eluted with a linear NaCl gradient (0.1-0.3 M). The PLC activity was recovered in two peaks; the first peak eluted at 0.1-0.18 M NaCl and the second peak at 0.25-0.3 M NaCl. The first activity peak, designated as cPLC-I, gave a positive cross-reaction with anti-PLC-y 2 antibody. cPI.C-I was subjected to the heparin-agarose column chromatography followed by elution with a linear NaCl gradient 110.2 - 0.6 M) in buffer B, yielding a single activity peak. When the activity peak was applied onto hydroxyapatite column and eluted with a linear gradient of potassium phosphate (0.1 - 0.4 M), the activity was eluted at 0.25 - 0.3 M potassium phosphate. At the final step of Mono S column chromatography with a linear NaCl gradient (0.1 M - 0.4 M), a single activity peak of cPLC-I was recovered at approx. 0.2 M NaCl, which was coincident with a symmetrical protein peak. Other methods' Analytical polyacrylamide gel electrophoresis was perI* formed by the method of Laemmli (12) in gradient (8-16 %) slab gels containing SDS. Electrophoretic transfer of protein from slab gels to nitrocellulose sheets and subsequent immunoblotting using [ 125Ilprotein A were performed as described (13). Anti-PLC-y 2 antibody was prepared using PLC-y 2 proteins produced by E. coli expression system (3). E. coli crude extracts were loaded on 6 % SDS397
Vol.
167,
No.
BIOCHEMICAL
2, 1990
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
polyacrylamide gel. Following electrophoresis, the band corresponding to PLCy 2 proteins was cut from the gel. The gels including PLC-y 2 proteins were injected into rabbits with Freund complete adjuvant every 3 weeks for 2 months. Antibodies against PLC-fi , PLC-y 1 and PLC-S were also prepared using each type of PLC protein produced by E. coli expression systems (4). Anti-PLCy 2 antibody only reacted with PLC-)/ 2 but not with other PLCs. Similarly, anti-PLC-fi , anti-PLC-y 1 and anti-PLC-6 antibodies reacted specifically with PLC-p , PLC-y 1 and PLC-6 , respectively (manuscript in preparation). RESULTS
into
The
cytosolic
two
fractions
when
measured
activity Fr-I for
(Fr-I, using
for
was recognizable further
between
antibody hydroxyapatite
(Mr.
at
single
(CPLG-I)
subjected
2 it and
the which
to
Fast
pH 5.5
broad
240 kDa)
chromatography,
peak was
a
and
PIPz-hydrolysis
Q-Sepharose
as substrates
of Fr-I,
and
column
first
Fr-I
PI-
upon
PIP2
by anti-PLC-y
purification
catalase
Mono Q
for
Fr-II)
PI and
was higher
chromatography
Its
PLC activities
Fr-II
antibody
but
1). at
pH 7.0,
respectively.
Fr-II
was not.
activity
was eluted
bovine activity was
serum
chromatography PI-hydrolyzing
to
peak
Ultrogel
albumin
at
(Mr. with
chromatographies The overall
Therefore,
AcA-44 the
column position
67 kDa).
peak was recovered cross-reactive
resolved
The
was subjected
subsequent
and Mono S columns.
column
(Fig.
and
were
in the
two
Upon peaks.
anti-PLC-y
on heparin-agarose,
purification
steps
achieved
02 Fig. 1. Fast Q-Sepharose column chromatography of the human platelet cytosol. PI-hydrolysis at pH 5.5 without deoxycholate (0); PI-hydrolysis at pH 7.0 in the presence of deoxycholate (1 r&ml) (A ); PIPz-hydrolysis at pH 6.5 in the presence of deoxycholate (1 m&ml) using [~H)PIPz/PE as substrate (0 ); N&l concentration (-----) ; absorbance at 280 nm (-). Fist. 2, SDS-polyacrylamide gel electrophoresis and immunoblot of cPLGI. The purified cPLC-I was subjected to SDS-polyacrylamide gel and stained with Coomassie Briliant Blue (1) or immunoblotted with anti-PLC-y 2 antibody (2). Molecular mass markers used were: 200 kDa, myosin; 116 kDa, a -galactosidase, 97 kDa, phosphorylase b; 67 kDa, bovine serum albumin; 45 kDa, egg albumin. 398
2
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167,
No.
approx.
BIOCHEMICAL
2, 1990
6,800-fold
activity
of
and 9.5 The
cytosolic
cleaved
finally
molecular using
mass of
of pH on the
PI and PIP2
exhibited
as
an acidic
Addition
Iof
hydrolysis, This
detergent
(1 mg/ml)
(5.0
with
a
analysis that
2 antibody.
The molecular
mass
using
cPK-I
of cPIC-I
in
- 5.5)
for
Fig.
3-A,
either
PI-
was without
for
inhibited
PI-
is
Wako-
present
in
effect
PIPz-hydrolysis
the
purified
3-B).
with cPLC-I
or PIPZ-hydrolysis.
on pH optimum
by 80 %,
(Fig.
examined
by shifting
PI-hydrolysis
activity
were
whereas
Cholate
for
to it
was
PI-
pH 6.0. rather
inhibitory
and PIP*-hydrolysis.
cPLC-I
concentration
activity (Fig.
reaching
a maximal
at
lo-4
to
of
2 mM of Mn2+,
As shown
that
for
as
13.6
revealed
filtration
that
activity
(1 mg/ml)
affected
-the PIP;!-hydrolyzing
shown).
band
anti-PLC-y gel
was
respectively.
Immunoblot
examined.
indicating
hydrolyzing
pH optimum
enhanced The
2).
by calibrated
substrates.
deoxycholate but
both
of cPLC-I
2 and PLC-8
the
were
total
state.
The effects both
with
145 kDa by SDS-PAGE,
initial
protein
(Fig.
1, PLC-y
purified
the
PI and PIPz,
a single
solely
thus
COMMUNICATIONS
% of
activity
on SDS-PAG
was 140 kDa as determined
a monomeric
1.3
for
, PLC-y
cross-reacted
and
of
displayed
to PLC-p
of cPLC-I
Pack HPLC column
a yield
RESEARCH
The specific
145 kDa estimated
cPIC-I
BIOPHYSICAL
mg of protein
cPLC-I
antibodies
purified cPLC-I
fraction. / min per
The properties of
with
purified
specific
the
purification
the
,umol
AND
lo-3
level
EGTA or
hydrolyze
the at
10-S M and the
EDTA by approx. ,uM)
was without
and Cu2+ caused
3 i lo-
PI
and PIP;!
PIPz-hydrolyzing
activity 60 %
effect weak
or and
o
on
was enhanced activity
was inhibited metal (15
ions
- 20 %).
by addition (data
not
FM)
such
Although
the
(50
A)
;)jq
5.0
6.0 PH
70
6.0
0 1.0 [DETERGENT]
2.0 (w/d)
EG,A--7-6 G-i, -5 -4 -3 LOG [Ca*+l M
-
The buffer Fig. 3. A) pH-dependence of cPLC-I in PIand PIPI-hydrolysis. used was Tris/maleate, pH 4.5-8.0. PI (O,O)or PIP2 (A,A)-hydrolyzing (O,A) or presence (@,A) of activities were measured in the absence B) Effects of deoxycholate (--) or cholate (-----) on deoxycholate (1 mg/ml). CPU-I activity for PI(0) or PIP2(0) hydrolysis. C) Calcium dependence of cPLC-I activity in PI(0) or PIPz(0) hydrolysis.
399
Ca2 +
by Ca2+
was maximal
90 %, respectively other
inhibition
~~~~:~
E 8
was dependent
activity PI-hydrolyzing
The PIP2-hydrolyzing
M.
.?JgZ+ (50 Fez+
to
3-C);
Vol.
167,
No.
enzyme
2, 1990
activity
BIOCHEMICAL
was extremely
the 1 ,u M HgClz, ,uM dithiothreitol. 0.29
inhibitory
mM for
effect
per
min/mg
protein,
to
the
with
were
,~mol
at
by addition
Km values
17.6
COMMUNICATIONS
50 % inhibition
completely
apparent
and Vmax values
RESEARCH
HgZ+
was overcome
plots,
PIP2
BIOPHYSICAL
sensitive
From double-reciprocal and
AND
were
and
of
0.12
10.3
100
mM for
,U mol
PI
cleaved
respectively.
DISCUSSION
In
the
purified
previous
from
67 kDa (7), optima
platelet
PI-
be activated (Mr
and
Because among
well
been
platelet as
have
shown
of
there
hydrolysis
was
higher
activity
chromatographies,
or cholate. purified other
previously was
specificity
are
two
(Fig.
1).
(98
to
to see,
therefore,
(Fr-I
enzymes
whether
in human platelet
did
from 1
Hgz+,
but
not
(14)
PLC-y
2 is
expressing
somehow
high
level
Fr-I
PI-
with
a
showed
an
deoxycholate of
a partially
from
those
of
Moreover, substrate
with for the and it with
for
by stepwise 2,
inhibitory
of pp6Oc-src
and
distinct
(5,7-g).
associated
(PI,
Fr-II
different
in
has human
minus),
Caz*-dependence,
evidence (15,16),
of
at pH 5.5
cross-reactivity
studies provided PLC-)I 1 activation
PI-PJX
by either
cytosols
different
display
substrate,
or
those
of
PI or PIPz
be PLC-y
to
a
conditions
from to
but
in
absence
while
similar (6),
the
exhibiting
activity
purified
platelet
PLC-y
to
cPLC-I
Fr-II)
and was inhibited were
isolated
PI-PLCs
plus
2 antibody,
al.
(6)
cytosolic various
pH
and could
(pH,
examined
identified
et
in
used
and
cPLC-I
neutral
when either
detergent
a greater
with
properties
Recently, several tyrosine kinase in
protein
pHs;
Fr-I
by Manne brain
(5.5)
platelet
fractions
PI-hydrolysis
purified
similar
detergents.
acidic
pH was not.
kDa)
Manne et al.
under
by anti-PLC-y
enzymatical
They had
were
70 kDa and
of deoxycholate
we have
immunologically
for
human.
conditions
activities
Mrs
120 kDa,
detergent of
different
(5),
pH optimum
context,
and
and sensitivity
antibody.
kinase
Its PI-PLC
cPLC-I toward
5.5
hand,
by this
their
at neutral
pH at
other
multiplicity
recognizable
column
presence
assay
In this
neutral
properties
from
the
an acidic
different
by measuring
catalytic
optimum
On the
laboratories,
substrates; that
in
with
bovine
57 kDa (9)
inhibited
understood.
cytosol
PIP2
and
had
which
enzymes
143 kDa from
detergent.
and was rather
used.
PI-PLC
PIPZ-hydrolysis
98 kDa)
detergent) not
cytosol;
by this
deoxycholate was
various
140 - 100 kDa (8)
for
PI-PLC
studies,
behaviors anti-PLC-y
1
involvement of is of interest protein
tyrosine
(17).
ACKNOWLEDGMENT
This Education,
study was supported Science and Culture
in part of Japan. 400
by
a grant
from
the
Ministry
of
Vol.
167,
No.
BIOCHEMICAL
2, 1990
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
REFERENCES
1
Rhee ,'a '9 G *, Suh, P.G., Ryu, S.H. and Lee, K.Y. (1989) Science 244, 546* 550. 2. Ohta, S., Matsui, A., Nozawa, Y. and Kagawa, Y. (1988) FEBS Lett. 242, 31-35. 3. Fmori, Y., Homma, Y., Sorimachi, H., Kawasaki, H., Nakanishi, O., Suzuki, K. and Takenawa, T. (1989) J. Biol. Chem. 264, 21885-21890. 4. Homma, Y., Takenawa, T., Kmori, Y., Sorimachi, H. and Suzuki, K. (1989) Biochem. Biophys. Res. Commun. 164, 406-412. 5. Hakata, H., Kambayashi, J. and Kosaki, G. (1982) J. Biochem. 92, 929935. 6. Manne, V. and Kung, H.F. (1987) Biochem. J. 243, 763-771. 7. Banno, Y., Nakashima, S. and Nozawa, Y. (1986) Biochem. Bioph.ys. Res. Commtm.
136,
Low, M.G., 9. Baldassare, 8.
28,
713-721.
Carroll, J.J.,
R.C. and Cox, Henderson, P.A.
A.C. (1986) and Fisher,
Biochem. J. 237, 139-145. G.J. (1989) Bi0chemistr.y
6010-6016.
10. Raaflaub, J. (1986) Methods Biochem. Anal. 3, 301-325. 11. Banno, Y., Ysda, Y. and Nozawa, Y. (1988) J. Biol. &em. 263, 1145911465. 12. Laemmli, U.K. (1970) Nature 227, 680-685. 13. Towin, H., Staehelin, T. and Gordon, J. (1979) Proc. Natl. Acad. Sci. USA. 76, 4350-4354. 14. Ryu, S.H., Cho, K.S., Lee, K.-Y., Suh, P.-G. and Rhee, S.G. (1987) J. Bicl. Chem. 262, 12511-12518. 15. Meisenhelder,J., Sub, P-G.,Rhee,S.G. and Hunter T. (1989) Cell 57, 11091122. 16. Nishibe, S., Wahl, M.I., Rhee, S.G. and Carpenter, G. (1989) J. Biol. Chem. 264, 10335-10338. 17. Golden, A. and Brugge, J.S. (1989) Proc. Natl. Acad. Sci. KSA. 86, 901-905.
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