Vol. 170, No. 2, 1990
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
July 31, 1990
Pages
TRANSLOCATION
484-490
OF PHOSPHOLIPASE AZ FROM CYTOSOL TO MEMBRANES IN RAT BRAIN INDUCED BY CALCIUM IONS
Yoshlhlro
YOSHIHARA
and
Yasuyoshi
WATANABE*
Department of Neuroscience, Osaka Bioscience Institute, 6-2-4, Furuedai, Suita-shi, Osaka 565, Japan Received
June
4,
1990
SUMMARY: Phospholipase A2 (PLA2) activities were found in the cytosolic fractions of rat brain. Using the gel filtration chromatography, two major peaks of PLAz activities were demonstrated: PLA2-H (200 - 500 kDa) and PLA2-L (100 kDa). PLA2-L was active at both neutral and alkaline pH and absolutely required Ca 2+ for the activity, while the activity of PLA2-H was detected only at alkaline pH and independent of Ca2+. The activation of PLA2-L by Ca2+ was biphasic; the first observed at 1 - 100 ELM Caz+ and the second at 10 mM Ca2+. In the reconstitution system of partially purified PLA2-L and synaptosomal membranes from rat brain, PLA2-L associated with the membranes in a Ca2+-dependent manner. The association was completed within 5 - 10 min at 25 “C both at 10 PM and 1 mM Ca2+, though amount of PLA2-L translocated was dependent on Ca2+ concentrations. These results suggest that Ca2+ promotes the translocation of the cytosolic PLA2-L to membranes where phospholipids, substrate of PLAz, are present. "1990 Academic Pre**, Inc.
In a variety of cells, arachidonic response
to receptor-mediated
phospholipase
A2 (PLA2)
bioactive
substances
central
nervous
conditions, tonic
of arachidonic
the regulatory
Ca2+-dependent lipoxygenase
enzymes manner, (13),
mechanisms
as important
* To whom correspondence
enzymes
(3), body
of senses occurs
reported
in the
the
activation in brain
from cytosol
C (10, 1 l), signal
function
as
(4), neuroendocrine
of
PLAz
the (9).
are still unclear. to membranes
diacylglycerol
(11) and cytidyltransferase
intracellular
In the
hypoxia and convulsion,
acid cascade
kinase
(2).
to
Not only in these physiological
via
to translocate
(14), calpain
to
temperature
(7, 8).
probably
of arachidonic
such as protein
12-lipoxygenase
demonstrated
in
is mainly
converted
and leukotrienes
states such as ischemia,
acid
have been
are
phospholipids
for this process is subsequently
thromboxanes
cycle
but also in the pathological
Several
function
sleep-wake
from membrane
responsible
acid released
eicosanoids
(5, 6) and processing
liberation
However,
The arachidonic
these
to regulate
secretion
The enzyme
as prostaglandins,
system,
neuromodulators hormone
(1).
such
acid can be released
signals.
transduction.
in a
kinase
(12), 5-
(15).
They all
In the present
should be addressed.
The abbreviations used are: aminoethylether)-N,N,N’,N’-tetraacetic
PLAs,
0006-291X/90 $1.50 Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
phospholipase acid.
484
As,
EGTA,
ethylene
glycol
bis(6-
Vol.
170, Nlo. 2, 1990
BIOCHEMICAL
study, we have characterized provide
evidence
MATERIALS
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
two types of PLAz in rat brain cytosol
for the Caz+-induced
translocation
of PLAz-L
(PLAz-H
from cytosol
and -L), and
to membranes.
AND METHODS
Materials: L-u-l -palmitoyl-2-[1-‘4C]-arachidonoyl phosphatidylcholine was obtained from New England Nuclear. HiLoad lo/20 Superdex 2OOpg, Phenyl Superose HR 5/5 Centriprep 10 was from Amicon. All and Mono Q HR 5/5 were purchased from Pharmacia. other chemicals were of reagent grade. Brains from four male Wistar rats were Preparatlon of Enzyme Source: homogenized in 10 vol. of 10 mM Tris-HCI (pH 7.4) containing 1 mM EGTA (buffer A), and centrifuged at 100,000 x g for 60 min. The supernatant (crude cytosol) was used as enzyme source. Partlal Purification of PLA2-H and -L: The obtained supernatant (50 ml) was concentrated to 3 ml with Centriprep 10 device and applied to a HiLoad 16/20 Superdex 200pg column equilibrated with buffer A containing 100 mM NaCI. An aliquot of each fraction was assayed for PLAz activity, and two active fractions were pooled (PLAz-H and L). CaCl2 (final 1 mM) was added to PLA2-L obtained from the Superdex column, and the enzyme was applied to a Phenyl Superose HR 5/5 column equilibrated with 10 mM Tris-HCI (pH 7.4), 1 mM CaClz and 150 mM NaCI. Proteins which adsorbed to the column in a Caz+dependent manner were eluted by removal of Ca2+ with 1 mM EGTA. PLAz-L was further purified by anion exchange chromatography of Mono Q HR 5/5 column. PLA2 activity was measured essentially according to Assay of PLA2 Activity: Teramoto et al. (16). The standard incubation system (100 ~1) contained 100 mM Tris-HCI (pH 6.3), 4 mM CaCl2 and 500 pmol of L-a-l-palmitoyl-2-[1-14C]-arachidonoyl phosphatidylcholine. The reaction was carried out at 37°C for 30 min and stopped by adding 400 11 of Dole’s reagents. The released fatty acid was extracted and its radioactivity was counted by liquid scintilation counter. In some experiments, the reaction product was identified as arachidonic acid by thin layer chromatography. Caz-c-induced Association of PLA2-L to Brain Membranes: Brain synaptosoma membranes were prepared according to Whittaker (17). The partially purified PLA2-L was incubated with brain synaptosomal membrane (100 pg protein) at 25°C in the presence of 0 - 1 mM CaCl2 and 3 mM MgClz in a total volume of 200 ~1. After the indicated time, the reaction mixture was centrifuged at 100,000 x g for 10 min and PLAz activities in supernatant and membrane fractions were measured, respectively.
RESULTS Characterlzatlon
of
Fig. 1 shows Superdex PLAz-H
200pg
Two
a typical
(left panel)
inhibited
b’y increasing
drastically
major
by addition
specific
activities
activated
had a high activity even by Ca2+.
activation
Though
was observed
cytosol
peaks of PLAz
from a HiLoad
activity
were
16/20
observed:
as a broad peak of 200 - 500 kDa, On the other hand,
100 kDa and absolutely of PLA2-H
required
and -L were
PLAz-L Ca2+ for
2.40 and 4.54
at pH 8.3, respectively.
and -L activities
concentration
Cytosol
of 4 mM Ca2+.
mass of about
Effects of Ca2+ on PLA2-H
considerable
Two
acid / 30 min / mg protein
PLA2-H
Brain
profile of rat brain crude
inhibited
After this column,
nmol arachidonic
In Rat
(fractions 43 - 48) appeared
6:3 - 65) had a molecular
the activity.
PLAz
column.
PLAz-H
and its activity was partially
of
elution
gel filtration
and PLA2-L.
(fractons
Types
of Ca2+. PLAz-L
were
in the absence reached
As shown
of Caz+
On the contrary, the maximal
even at 1 v f v f Caz+. 485
compared.
PLAz-L
in Fig. 2A,
and it was slightly (right
panel)
was
activity at 10 mM Ca2+,
Fig. 28 shows pH profiles
of the
Vol.
170, No. 2, 1990
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
A
100
‘O-.0-0 .z-
80
i
‘+0
F P E
80
PLA2-H
‘2
0 a
‘,
60
60 PLAz-L
40
40
20
20
ij s
1.8 0
0 28765432 B
g8765432 Y
q
4 mM
CaC12
: -
5 3.6 0
-/og1ca2+1
-loglCa2+l
:
N 100 L
$
1.4
8 aE B am
80
.
60 .
0.2
02
0 40
60
80
elution
100 volume
120
140
4
6
5
PH7
8
9
10
(ml)
Fig. 1. Sm of PLA H and -I on Hil rind lW70 Suoe rdex 70000 Chrw Concentrated cytosol (s2~l) from four rat brains was loaded onto gel filtration column equilibrated with buffer A containing 100 mM NaCI. Flow rate was 1 ml/min, and 4 ml fractions were collected. An aliquot (80 ~1) of each in the presence (open column) or absence (hatched under Materials and Methods. Dashed line indicates weight markers (kDa) are indicated by arrowheads. Fig. 2.Effects of CaL Concentratons A. PLAa-H (left panel) and -L (right various concentrations of Can+ at pH accurate concentration of free Can+. or with 4 mM CaCla, respectively.
two enzyme
activities.
PLAz-H
dependent
(Al and DH fB) on PI AZ-H and -I Activim panel) from the Superdex column were assayed with 8.3. Caa+-EGTA buffers were used to determine the B. PLAa-H and -L were assayed at various pH without
activity was four times
than at neutral pH (pH 7.5), while (pH 7.5 - 9.5) including
neutral
PLAz-L pH.
showed
higher
maximal
From these results,
at alkaline
pH (pH 8.7 - 9.5)
activity in rather broad we focused
on PLAz-L
pH range which
is
on Caz+ and active at neutral pH.
Caz+-dependent
Binding
of
PLAz-L
Fig. 3 shows the hydrophobic
to Phenyl
chromatography
column was applied to a Phenyl Superose enzyme
which
this step,
adsorbed
however,
activity is probably PLAz-L
fraction was assayed for PLAa activity column) of 4 mM CaCla as described the absorbance at 280 nm. Molecular
activtiy
activity
was eluted of PLAz-L
due to the dissociation
recovered
HR 5/5
of PLAz-L.
PLA2-L
HR 5/5 column in the presence
to the column
the total
Superose
by removal decreased
of activator
to 80 - 90 % by adding 486
from the Superdex of 1 mM CaClz.
The
of Caz+ from the buffer. to 10 - 20 %.
or cofactor an aliquot
Column
This loss of
from the enzyme, of pass-through
In
since
fraction
of
Vol.
170,
No.
2, 1990
BIOCHEMICAL
AND
BIOPHYSICAL
1 mM
1 mM CaCl2
t
RESEARCH
COMMUNICATIONS
EGTA I
PLAP-L 1 1
I ,
I
,
I , 1
I I I
0 10
0
20
elution
30
volume
40
f ml)
Fig. 3.,C$+&pendent Brndrno of PI A I to Phenvl SlLperose HP 5/5 colum PLAs-L from the Superdex gel filtratio?i- column was applied to a Phenyl Superose HR 5/5 column in the presence of 1 mM CaCls at a flow rate of 0.5 ml/min. The adsorbed proteins were eluted by removing Cap+ with 1 mM EGTA. Two-ml fractions were collected and 80 11 of each fraction was assayed for PLAe activity (open column). Dashed line indicates the absorbance at 280 nm.
A
fdpml
o ‘\
oh
J
0
10
100
Ca2+
10 phi
ca2+
1000
(JJMJ
I,
1 mM Ca2+ - 1000
:
i
\O O’\ N.
-.
supernatant -B -_______
irqe
-2 .
membrane
Q” 0,
- 500
\
‘\
-,-0
‘o--------
supernatant
I
,
I
L
10 20 t/me (m/n)
30
0
10
20
30
t/me (m/n)
Membranes. d Association of PI A:, _I wrth Rat B rain Svnaotosomal Mono 0 column was incubated at 25% for 10 min with rat brain PLAa-L from synaptosomal membranes (100 fig protein) with various concentrations of Can+. After PLAs activity in supernatnant ( 0 ) and membrane fractions ( l ) were centrifugation, determined, respectively. Synaptosomal membranes contained basal PLAs activity (65 pmol arachidonic acid released ! 30 min / mg protein). B. PLAs-L was incubated for indicated time with brain membranes with 10 FM (left panel) or 1 mM (right panel) Can+.
487
Vol.
170, No. 2, 1990
Phenyl
Superose
BIOCHEMICAL
column
AND BIOPHYSICAL
to the enzyme.
PLA2-L from the Phenyl Superose
further purified with Mono Q HR 5/5 column step was used in the following Caz+-induced PLAz-L
from
membranes shown
of
Mono
with
in Fig. 4A, PLA2-L associated
absence
of Caz+, the enzyme
However,
the increasing
Time amounts
course
increase
of PLA2-L
of PLA2-L
translocation
% of the enzyme translocated panel).
However,
The translocation
allmost
after Mono Q
with
recovered
rat
brain
synaptosomal
manner.
in the supernatant
a decrease
As
In the fractions.
in the supernatant
PLAz
PLAp activity.
to the membranes differed
Membranes
in a Caz+-dependent
in the membrane
greatly
was
of Ca2+ at 25OC for 10 min.
of Ca 2+ caused
association
to
reconstituted
concentrations
activity was
and the enzyme
Cytosol
to the membranes
concentrations
activity and a simultaneous
from
was
various
column
study.
PLAt-L
Q column
and incubated
(data not shown),
reconstitution
Translocation
RESEARCH COMMUNICATIONS
according
is shown
in Fig. 48.
to Ca2+ concentrations:
The
30 - 40
at 10 PM Ca z+ (left panel) and 70 - 80 % at 1 mM Caz+ (right
the velocity
of translocation
was completed
within
was similar
at both Caz+ concentrations.
5 - 10 min.
DISCUSSION In this study, we presented in rat brain cytosol. from various
low molecular
tissues
and their primary
L in the rat brain
are apparently
and
polypeptide
Caa+
sensitivity.
of PLAa-L
that is, the first activation
resting
state
The
the
and increases
of PLA2-L presence
for the enzyme
PLAz-L
adsorbed
possibility system
(PLAz-H),
Recently,
(19) and spleen
were determined. PLAs,
are reported
and
secretory (20) have
PLAa-H and -
judging
from
molecular
to be a monomeric
of cytosolic
of partially
concentrations
Can + is maintained
free
during
of PLAz-L
to membranes
under
required for
higher
at 10 mM Can+.
(21).
Accordingly,
during
physiological
PLAs-L
neuronal
In
lowers
it is
excitation
conditions.
concentrations
which
of Can+;
at 0.1 - 0.3 PM in the
excitation
free Can+ concentration
of an activator
However,
of Caa+.
Caa+
We
concentration
translocation. to the Phenyl
from a hydrophilic
of PLAs-L
by increasing
at 1 FM Can+ and the second
up to 1 - 2 PM
eluted by removal of Cae+ with EGTA, of the enzyme
biphasically
of cytosolic
that an elevation
the association required
(18). platelet
PLA2s
enzyme
(PLAs-L).
from the secretory
secretory
changed
may lead to the first phase activation speculate
enzyme
amino acid sequences
distinct
was observed
the concentration
suggested
weight
weight
of 14 kDa and active at more than 1 mM Can+.
The activity neurons,
of at least two forms of PLAa
high molecular
such as the pancreas
been characterized
weight
for the existence
One is a Ca 2+-independent
the other is a Ca2+-requiring PLA2s
evidence
suggesting
column
enzyme
from and
cytosol
form.
of Can+ and was
conformational This
to membranes.
synaptosomal 488
in the presence
the Ca a+-induced
form to a hydrophobic
translocation purified
Superose
membranes,
result
In the
change
led us to a reconstitution
indeed,
PLAa-L
Vol.
170, No. 2, 1990
BIOCHEMICAL
depolar/zat/on
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
*+
‘,,.
Ca
arachidonic
acid
eicosanoids
Fig. 5.A
Possible Model for Activation of PI &L 1) Neuronal excitation leads to a rise in intracellular Can+. 2) The increased Caa+ chanoes the conformation of PLAa-L to a hydrophobic form. 3) The hydrophobic PLAa-L associa?es with membranes. 4) The membrane-bound PLAa-L hydrolyzes phospholipid to release arachidonic acid.
translocated
to
the
Channon
and Leslie
the macropharge have
membranes
Ca 2+- and
in
(22) reported
Ca 2+-dependent
cell line RAW 264.7.
not characterized
time-dependent
the enzyme
association
Although in detail,
and
suggested
to be important
protein
kinase
calpain
(11) and cytidyltransferase
signal
C (10, 1 l),
transduction
diacylglycerol
similar
mechanism.
PLA2-L
A possible
signal
5- and
translocation
enzymes
have been
transduction,
such
12-lipoxygenases
of PLA2-L
as
(13, 14),
may also play some important
model for the activation
in
system and
of PLA;!
Some translocating (12),
recently,
of PLA;, with membranes
mechanisms
in intracellular kinase
(15).
Most
they used crude homogenate
seem to exist both in the brain and macropharge. discovered
manners.
roles in
is shown
in
Fig. 5. Further
to examine
studies
on
PLA2-L
are
the role in physiological
now
in progress
and pathological
to determine
the
primary
structure
and
conditions.
ACKNOWLEDGMENTS: This work 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, ON0 Medical Research Foundation, Sankyo Life Science Foundation and Sankyo Co., Ltd.
REFERENCES 1. Waite, M. (1987) The Phospholipases, pp.11 1 - 113, Plenum Publishing Corp. New York 2. Sammuelson, B., Goldyne, M.. Branstrom, E., Hamberg, M., Hammarstrom, S., and Malmsten, C. (1978) Ann. Rev. Biochem. 47, 997 - 1029 3. Hayaishi, 0. (1989) J. Biol. Chem. 263, 14593 - 14596 4. Wolfe, L.S., and Coceani, F. (1979) Ann. Rev. Physiol. 41, 669 - 684 5. Kinoshita, F., Nakai,Y., Katakami, H., Imura, H., Shimizu, T., and Hayaishi, 0. (1982) Endocrinol. 110, 2207 - 2209 6. Ojeda, S. R., Jameson, H. E., and McCann, S. M. (1977) Endocrinol. 100, 1585 - 1594 7. Horiguchi, S., Ueno, IX, Hyodo, M., and Hayaishi, 0. (1986) Eur. J. Pharmacol. 122, 173 179
489
Vol.
170, No. 2, 1990
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
8. Watanabe, Y., Mori, K., Imamura, K., Takagi, S. -F., and Hayaishi, 0. (1986) Brain Res. 378, 216 - 222 9. Bazan, N. G. (1976) Function and Metabolism of Phospholipids in Central and Peripheral Nervous System, pp.317 - 335, Plenum Press, New York 10. Kraft, A. S., and Anderson, W. B. (1983) Nature 301, 621 - 623 11. Melloni, E., Pontremoli, S., Michetti, M., Sacco, O., Sparatore, B., Salamino, F., and Horecker, B. L. (1985) Proc. Natl. Acad. Sci. USA 82, 6435 - 6439 12. Maroney, A. C., and Macara, I. G. (1989) J. Biol. Chem. 264, 2537 - 2544 13. Rouzer, C. A., and Sammuelson, B. (1987) Proc. Natl. Acad. Sci. USA 84, 7393 - 7397 14. Baba, A., Sakuma, S., Okamoto,H., Inoue, T., and Iwata, H. (1989) J. Biol. Chem. 264, 15790 - 15795 15. Cornell, R., and Vance, D. E. (1987) Biochim. Biophys. Acta 919, 26 - 36 16. Teramoto, T., Tojo, H., Yamano, T., and Okamoto, M. (1983) J. Biochem. (Tokyo) 93, 1353 - 1360 17. Whittaker, V. P. (1959) Biochem. J. 72, 694 - 706 18. Puijk, W. C., Verheij, H. M., and de Haas, G. H. (1977) Biochim. Biophys. Acta 492, 254 259 19. Hayakawa, M., Kudo, I., Tomita, M., Nojima, S., and Inoue, K. (1988) J. Biochem. (Tokyo) 104, 767 - 772 20. Ishizaki, J., Ohara, O., Nakamura, E., Tamaki, M., Ono, T., Kanda, A., Yoshida, N., Teraoka, H., Tojo, H., and Okamoto, M. (1989) Biochem. Biophys. Res. Commun. 162, 1030 - 1036 21. Balow, R. M., Tomkinson, B., Ragnarsson, U., and Zetterqvist, 0. (1986) J. Biol. Chem. 261, 2409 - 2417 22. Channon, J. Y., and Leslie, C. C. (1990) J. Biol. Chem. 265, 5409 - 5413
490
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
July 31, 1990
Pages
TRANSLOCATION
484-490
OF PHOSPHOLIPASE AZ FROM CYTOSOL TO MEMBRANES IN RAT BRAIN INDUCED BY CALCIUM IONS
Yoshlhlro
YOSHIHARA
and
Yasuyoshi
WATANABE*
Department of Neuroscience, Osaka Bioscience Institute, 6-2-4, Furuedai, Suita-shi, Osaka 565, Japan Received
June
4,
1990
SUMMARY: Phospholipase A2 (PLA2) activities were found in the cytosolic fractions of rat brain. Using the gel filtration chromatography, two major peaks of PLAz activities were demonstrated: PLA2-H (200 - 500 kDa) and PLA2-L (100 kDa). PLA2-L was active at both neutral and alkaline pH and absolutely required Ca 2+ for the activity, while the activity of PLA2-H was detected only at alkaline pH and independent of Ca2+. The activation of PLA2-L by Ca2+ was biphasic; the first observed at 1 - 100 ELM Caz+ and the second at 10 mM Ca2+. In the reconstitution system of partially purified PLA2-L and synaptosomal membranes from rat brain, PLA2-L associated with the membranes in a Ca2+-dependent manner. The association was completed within 5 - 10 min at 25 “C both at 10 PM and 1 mM Ca2+, though amount of PLA2-L translocated was dependent on Ca2+ concentrations. These results suggest that Ca2+ promotes the translocation of the cytosolic PLA2-L to membranes where phospholipids, substrate of PLAz, are present. "1990 Academic Pre**, Inc.
In a variety of cells, arachidonic response
to receptor-mediated
phospholipase
A2 (PLA2)
bioactive
substances
central
nervous
conditions, tonic
of arachidonic
the regulatory
Ca2+-dependent lipoxygenase
enzymes manner, (13),
mechanisms
as important
* To whom correspondence
enzymes
(3), body
of senses occurs
reported
in the
the
activation in brain
from cytosol
C (10, 1 l), signal
function
as
(4), neuroendocrine
of
PLAz
the (9).
are still unclear. to membranes
diacylglycerol
(11) and cytidyltransferase
intracellular
In the
hypoxia and convulsion,
acid cascade
kinase
(2).
to
Not only in these physiological
via
to translocate
(14), calpain
to
temperature
(7, 8).
probably
of arachidonic
such as protein
12-lipoxygenase
demonstrated
in
is mainly
converted
and leukotrienes
states such as ischemia,
acid
have been
are
phospholipids
for this process is subsequently
thromboxanes
cycle
but also in the pathological
Several
function
sleep-wake
from membrane
responsible
acid released
eicosanoids
(5, 6) and processing
liberation
However,
The arachidonic
these
to regulate
secretion
The enzyme
as prostaglandins,
system,
neuromodulators hormone
(1).
such
acid can be released
signals.
transduction.
in a
kinase
(12), 5-
(15).
They all
In the present
should be addressed.
The abbreviations used are: aminoethylether)-N,N,N’,N’-tetraacetic
PLAs,
0006-291X/90 $1.50 Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
phospholipase acid.
484
As,
EGTA,
ethylene
glycol
bis(6-
Vol.
170, Nlo. 2, 1990
BIOCHEMICAL
study, we have characterized provide
evidence
MATERIALS
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
two types of PLAz in rat brain cytosol
for the Caz+-induced
translocation
of PLAz-L
(PLAz-H
from cytosol
and -L), and
to membranes.
AND METHODS
Materials: L-u-l -palmitoyl-2-[1-‘4C]-arachidonoyl phosphatidylcholine was obtained from New England Nuclear. HiLoad lo/20 Superdex 2OOpg, Phenyl Superose HR 5/5 Centriprep 10 was from Amicon. All and Mono Q HR 5/5 were purchased from Pharmacia. other chemicals were of reagent grade. Brains from four male Wistar rats were Preparatlon of Enzyme Source: homogenized in 10 vol. of 10 mM Tris-HCI (pH 7.4) containing 1 mM EGTA (buffer A), and centrifuged at 100,000 x g for 60 min. The supernatant (crude cytosol) was used as enzyme source. Partlal Purification of PLA2-H and -L: The obtained supernatant (50 ml) was concentrated to 3 ml with Centriprep 10 device and applied to a HiLoad 16/20 Superdex 200pg column equilibrated with buffer A containing 100 mM NaCI. An aliquot of each fraction was assayed for PLAz activity, and two active fractions were pooled (PLAz-H and L). CaCl2 (final 1 mM) was added to PLA2-L obtained from the Superdex column, and the enzyme was applied to a Phenyl Superose HR 5/5 column equilibrated with 10 mM Tris-HCI (pH 7.4), 1 mM CaClz and 150 mM NaCI. Proteins which adsorbed to the column in a Caz+dependent manner were eluted by removal of Ca2+ with 1 mM EGTA. PLAz-L was further purified by anion exchange chromatography of Mono Q HR 5/5 column. PLA2 activity was measured essentially according to Assay of PLA2 Activity: Teramoto et al. (16). The standard incubation system (100 ~1) contained 100 mM Tris-HCI (pH 6.3), 4 mM CaCl2 and 500 pmol of L-a-l-palmitoyl-2-[1-14C]-arachidonoyl phosphatidylcholine. The reaction was carried out at 37°C for 30 min and stopped by adding 400 11 of Dole’s reagents. The released fatty acid was extracted and its radioactivity was counted by liquid scintilation counter. In some experiments, the reaction product was identified as arachidonic acid by thin layer chromatography. Caz-c-induced Association of PLA2-L to Brain Membranes: Brain synaptosoma membranes were prepared according to Whittaker (17). The partially purified PLA2-L was incubated with brain synaptosomal membrane (100 pg protein) at 25°C in the presence of 0 - 1 mM CaCl2 and 3 mM MgClz in a total volume of 200 ~1. After the indicated time, the reaction mixture was centrifuged at 100,000 x g for 10 min and PLAz activities in supernatant and membrane fractions were measured, respectively.
RESULTS Characterlzatlon
of
Fig. 1 shows Superdex PLAz-H
200pg
Two
a typical
(left panel)
inhibited
b’y increasing
drastically
major
by addition
specific
activities
activated
had a high activity even by Ca2+.
activation
Though
was observed
cytosol
peaks of PLAz
from a HiLoad
activity
were
16/20
observed:
as a broad peak of 200 - 500 kDa, On the other hand,
100 kDa and absolutely of PLA2-H
required
and -L were
PLAz-L Ca2+ for
2.40 and 4.54
at pH 8.3, respectively.
and -L activities
concentration
Cytosol
of 4 mM Ca2+.
mass of about
Effects of Ca2+ on PLA2-H
considerable
Two
acid / 30 min / mg protein
PLA2-H
Brain
profile of rat brain crude
inhibited
After this column,
nmol arachidonic
In Rat
(fractions 43 - 48) appeared
6:3 - 65) had a molecular
the activity.
PLAz
column.
PLAz-H
and its activity was partially
of
elution
gel filtration
and PLA2-L.
(fractons
Types
of Ca2+. PLAz-L
were
in the absence reached
As shown
of Caz+
On the contrary, the maximal
even at 1 v f v f Caz+. 485
compared.
PLAz-L
in Fig. 2A,
and it was slightly (right
panel)
was
activity at 10 mM Ca2+,
Fig. 28 shows pH profiles
of the
Vol.
170, No. 2, 1990
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
A
100
‘O-.0-0 .z-
80
i
‘+0
F P E
80
PLA2-H
‘2
0 a
‘,
60
60 PLAz-L
40
40
20
20
ij s
1.8 0
0 28765432 B
g8765432 Y
q
4 mM
CaC12
: -
5 3.6 0
-/og1ca2+1
-loglCa2+l
:
N 100 L
$
1.4
8 aE B am
80
.
60 .
0.2
02
0 40
60
80
elution
100 volume
120
140
4
6
5
PH7
8
9
10
(ml)
Fig. 1. Sm of PLA H and -I on Hil rind lW70 Suoe rdex 70000 Chrw Concentrated cytosol (s2~l) from four rat brains was loaded onto gel filtration column equilibrated with buffer A containing 100 mM NaCI. Flow rate was 1 ml/min, and 4 ml fractions were collected. An aliquot (80 ~1) of each in the presence (open column) or absence (hatched under Materials and Methods. Dashed line indicates weight markers (kDa) are indicated by arrowheads. Fig. 2.Effects of CaL Concentratons A. PLAa-H (left panel) and -L (right various concentrations of Can+ at pH accurate concentration of free Can+. or with 4 mM CaCla, respectively.
two enzyme
activities.
PLAz-H
dependent
(Al and DH fB) on PI AZ-H and -I Activim panel) from the Superdex column were assayed with 8.3. Caa+-EGTA buffers were used to determine the B. PLAa-H and -L were assayed at various pH without
activity was four times
than at neutral pH (pH 7.5), while (pH 7.5 - 9.5) including
neutral
PLAz-L pH.
showed
higher
maximal
From these results,
at alkaline
pH (pH 8.7 - 9.5)
activity in rather broad we focused
on PLAz-L
pH range which
is
on Caz+ and active at neutral pH.
Caz+-dependent
Binding
of
PLAz-L
Fig. 3 shows the hydrophobic
to Phenyl
chromatography
column was applied to a Phenyl Superose enzyme
which
this step,
adsorbed
however,
activity is probably PLAz-L
fraction was assayed for PLAa activity column) of 4 mM CaCla as described the absorbance at 280 nm. Molecular
activtiy
activity
was eluted of PLAz-L
due to the dissociation
recovered
HR 5/5
of PLAz-L.
PLA2-L
HR 5/5 column in the presence
to the column
the total
Superose
by removal decreased
of activator
to 80 - 90 % by adding 486
from the Superdex of 1 mM CaClz.
The
of Caz+ from the buffer. to 10 - 20 %.
or cofactor an aliquot
Column
This loss of
from the enzyme, of pass-through
In
since
fraction
of
Vol.
170,
No.
2, 1990
BIOCHEMICAL
AND
BIOPHYSICAL
1 mM
1 mM CaCl2
t
RESEARCH
COMMUNICATIONS
EGTA I
PLAP-L 1 1
I ,
I
,
I , 1
I I I
0 10
0
20
elution
30
volume
40
f ml)
Fig. 3.,C$+&pendent Brndrno of PI A I to Phenvl SlLperose HP 5/5 colum PLAs-L from the Superdex gel filtratio?i- column was applied to a Phenyl Superose HR 5/5 column in the presence of 1 mM CaCls at a flow rate of 0.5 ml/min. The adsorbed proteins were eluted by removing Cap+ with 1 mM EGTA. Two-ml fractions were collected and 80 11 of each fraction was assayed for PLAe activity (open column). Dashed line indicates the absorbance at 280 nm.
A
fdpml
o ‘\
oh
J
0
10
100
Ca2+
10 phi
ca2+
1000
(JJMJ
I,
1 mM Ca2+ - 1000
:
i
\O O’\ N.
-.
supernatant -B -_______
irqe
-2 .
membrane
Q” 0,
- 500
\
‘\
-,-0
‘o--------
supernatant
I
,
I
L
10 20 t/me (m/n)
30
0
10
20
30
t/me (m/n)
Membranes. d Association of PI A:, _I wrth Rat B rain Svnaotosomal Mono 0 column was incubated at 25% for 10 min with rat brain PLAa-L from synaptosomal membranes (100 fig protein) with various concentrations of Can+. After PLAs activity in supernatnant ( 0 ) and membrane fractions ( l ) were centrifugation, determined, respectively. Synaptosomal membranes contained basal PLAs activity (65 pmol arachidonic acid released ! 30 min / mg protein). B. PLAs-L was incubated for indicated time with brain membranes with 10 FM (left panel) or 1 mM (right panel) Can+.
487
Vol.
170, No. 2, 1990
Phenyl
Superose
BIOCHEMICAL
column
AND BIOPHYSICAL
to the enzyme.
PLA2-L from the Phenyl Superose
further purified with Mono Q HR 5/5 column step was used in the following Caz+-induced PLAz-L
from
membranes shown
of
Mono
with
in Fig. 4A, PLA2-L associated
absence
of Caz+, the enzyme
However,
the increasing
Time amounts
course
increase
of PLA2-L
of PLA2-L
translocation
% of the enzyme translocated panel).
However,
The translocation
allmost
after Mono Q
with
recovered
rat
brain
synaptosomal
manner.
in the supernatant
a decrease
As
In the fractions.
in the supernatant
PLAz
PLAp activity.
to the membranes differed
Membranes
in a Caz+-dependent
in the membrane
greatly
was
of Ca2+ at 25OC for 10 min.
of Ca 2+ caused
association
to
reconstituted
concentrations
activity was
and the enzyme
Cytosol
to the membranes
concentrations
activity and a simultaneous
from
was
various
column
study.
PLAt-L
Q column
and incubated
(data not shown),
reconstitution
Translocation
RESEARCH COMMUNICATIONS
according
is shown
in Fig. 48.
to Ca2+ concentrations:
The
30 - 40
at 10 PM Ca z+ (left panel) and 70 - 80 % at 1 mM Caz+ (right
the velocity
of translocation
was completed
within
was similar
at both Caz+ concentrations.
5 - 10 min.
DISCUSSION In this study, we presented in rat brain cytosol. from various
low molecular
tissues
and their primary
L in the rat brain
are apparently
and
polypeptide
Caa+
sensitivity.
of PLAa-L
that is, the first activation
resting
state
The
the
and increases
of PLA2-L presence
for the enzyme
PLAz-L
adsorbed
possibility system
(PLAz-H),
Recently,
(19) and spleen
were determined. PLAs,
are reported
and
secretory (20) have
PLAa-H and -
judging
from
molecular
to be a monomeric
of cytosolic
of partially
concentrations
Can + is maintained
free
during
of PLAz-L
to membranes
under
required for
higher
at 10 mM Can+.
(21).
Accordingly,
during
physiological
PLAs-L
neuronal
In
lowers
it is
excitation
conditions.
concentrations
which
of Can+;
at 0.1 - 0.3 PM in the
excitation
free Can+ concentration
of an activator
However,
of Caa+.
Caa+
We
concentration
translocation. to the Phenyl
from a hydrophilic
of PLAs-L
by increasing
at 1 FM Can+ and the second
up to 1 - 2 PM
eluted by removal of Cae+ with EGTA, of the enzyme
biphasically
of cytosolic
that an elevation
the association required
(18). platelet
PLA2s
enzyme
(PLAs-L).
from the secretory
secretory
changed
may lead to the first phase activation speculate
enzyme
amino acid sequences
distinct
was observed
the concentration
suggested
weight
weight
of 14 kDa and active at more than 1 mM Can+.
The activity neurons,
of at least two forms of PLAa
high molecular
such as the pancreas
been characterized
weight
for the existence
One is a Ca 2+-independent
the other is a Ca2+-requiring PLA2s
evidence
suggesting
column
enzyme
from and
cytosol
form.
of Can+ and was
conformational This
to membranes.
synaptosomal 488
in the presence
the Ca a+-induced
form to a hydrophobic
translocation purified
Superose
membranes,
result
In the
change
led us to a reconstitution
indeed,
PLAa-L
Vol.
170, No. 2, 1990
BIOCHEMICAL
depolar/zat/on
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
*+
‘,,.
Ca
arachidonic
acid
eicosanoids
Fig. 5.A
Possible Model for Activation of PI &L 1) Neuronal excitation leads to a rise in intracellular Can+. 2) The increased Caa+ chanoes the conformation of PLAa-L to a hydrophobic form. 3) The hydrophobic PLAa-L associa?es with membranes. 4) The membrane-bound PLAa-L hydrolyzes phospholipid to release arachidonic acid.
translocated
to
the
Channon
and Leslie
the macropharge have
membranes
Ca 2+- and
in
(22) reported
Ca 2+-dependent
cell line RAW 264.7.
not characterized
time-dependent
the enzyme
association
Although in detail,
and
suggested
to be important
protein
kinase
calpain
(11) and cytidyltransferase
signal
C (10, 1 l),
transduction
diacylglycerol
similar
mechanism.
PLA2-L
A possible
signal
5- and
translocation
enzymes
have been
transduction,
such
12-lipoxygenases
of PLA2-L
as
(13, 14),
may also play some important
model for the activation
in
system and
of PLA;!
Some translocating (12),
recently,
of PLA;, with membranes
mechanisms
in intracellular kinase
(15).
Most
they used crude homogenate
seem to exist both in the brain and macropharge. discovered
manners.
roles in
is shown
in
Fig. 5. Further
to examine
studies
on
PLA2-L
are
the role in physiological
now
in progress
and pathological
to determine
the
primary
structure
and
conditions.
ACKNOWLEDGMENTS: This work 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, ON0 Medical Research Foundation, Sankyo Life Science Foundation and Sankyo Co., Ltd.
REFERENCES 1. Waite, M. (1987) The Phospholipases, pp.11 1 - 113, Plenum Publishing Corp. New York 2. Sammuelson, B., Goldyne, M.. Branstrom, E., Hamberg, M., Hammarstrom, S., and Malmsten, C. (1978) Ann. Rev. Biochem. 47, 997 - 1029 3. Hayaishi, 0. (1989) J. Biol. Chem. 263, 14593 - 14596 4. Wolfe, L.S., and Coceani, F. (1979) Ann. Rev. Physiol. 41, 669 - 684 5. Kinoshita, F., Nakai,Y., Katakami, H., Imura, H., Shimizu, T., and Hayaishi, 0. (1982) Endocrinol. 110, 2207 - 2209 6. Ojeda, S. R., Jameson, H. E., and McCann, S. M. (1977) Endocrinol. 100, 1585 - 1594 7. Horiguchi, S., Ueno, IX, Hyodo, M., and Hayaishi, 0. (1986) Eur. J. Pharmacol. 122, 173 179
489
Vol.
170, No. 2, 1990
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
8. Watanabe, Y., Mori, K., Imamura, K., Takagi, S. -F., and Hayaishi, 0. (1986) Brain Res. 378, 216 - 222 9. Bazan, N. G. (1976) Function and Metabolism of Phospholipids in Central and Peripheral Nervous System, pp.317 - 335, Plenum Press, New York 10. Kraft, A. S., and Anderson, W. B. (1983) Nature 301, 621 - 623 11. Melloni, E., Pontremoli, S., Michetti, M., Sacco, O., Sparatore, B., Salamino, F., and Horecker, B. L. (1985) Proc. Natl. Acad. Sci. USA 82, 6435 - 6439 12. Maroney, A. C., and Macara, I. G. (1989) J. Biol. Chem. 264, 2537 - 2544 13. Rouzer, C. A., and Sammuelson, B. (1987) Proc. Natl. Acad. Sci. USA 84, 7393 - 7397 14. Baba, A., Sakuma, S., Okamoto,H., Inoue, T., and Iwata, H. (1989) J. Biol. Chem. 264, 15790 - 15795 15. Cornell, R., and Vance, D. E. (1987) Biochim. Biophys. Acta 919, 26 - 36 16. Teramoto, T., Tojo, H., Yamano, T., and Okamoto, M. (1983) J. Biochem. (Tokyo) 93, 1353 - 1360 17. Whittaker, V. P. (1959) Biochem. J. 72, 694 - 706 18. Puijk, W. C., Verheij, H. M., and de Haas, G. H. (1977) Biochim. Biophys. Acta 492, 254 259 19. Hayakawa, M., Kudo, I., Tomita, M., Nojima, S., and Inoue, K. (1988) J. Biochem. (Tokyo) 104, 767 - 772 20. Ishizaki, J., Ohara, O., Nakamura, E., Tamaki, M., Ono, T., Kanda, A., Yoshida, N., Teraoka, H., Tojo, H., and Okamoto, M. (1989) Biochem. Biophys. Res. Commun. 162, 1030 - 1036 21. Balow, R. M., Tomkinson, B., Ragnarsson, U., and Zetterqvist, 0. (1986) J. Biol. Chem. 261, 2409 - 2417 22. Channon, J. Y., and Leslie, C. C. (1990) J. Biol. Chem. 265, 5409 - 5413
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