Vol. 90, No. 2, 1979 September 27, 1979
AND BIOPHYSICAL
BIOCHEMICAL
CALMODULIN STIMULATES Patrick
HUMAN PLATELET
Y.-K.
PHOSPHOLIPASE A2
Wang+ and Wai Yiu
Departments of Pharmacology+ The University of Tennessee Center Memphis, Tennessee
RESEARCH COMvtUNlCATlONS Pages 473-480
Cheung+
and Biochemistry+ for The Health Sciences 38163
and Department of Biochemistry+ Jude Children’s Research Hospital Memphis, Tennessee 38101
St.
Received
August
6,1979 SUMMARY
Ca 2+ -binding protein, mediating the effect of Calmodulin is a ubiquitous Ca2+ on many enzyme systems and cellular reactions. Phospholipase A2 (phosphatide-2-acyl-hydrolase, EC 3.1.1.4) yhich governs the level of arachidonic acid in human platelets, requires Ca2 for maximum activity. Resylts presented herein suggest that the stimulation of phospholipase A2 by Ca2 is also mediated through calmodulin. This finding adds to the growing list of enzymes whose activities are regulated by calmodulin. INTRODUCTION Phospholipase lyzes
the
fatty
acid
and
(1).
The
steady
low
(2)
step
in
A2 (phosphatide-2-acyl-hydrolase,
deacylation state
generation
figure
Pickett, increased that
al
(4)
markedly
platelet
of this
fatty
The
the
level A, (5,
by which
free
stimulated
which
cata-
to produce all
mammalian
arachidonic
acid
believed
is
to be the
Arachidonic
acid
a free tissues
generally limiting
serves
and prostaglandins,
as a
compounds
Using
ionophore
A23187,
of
intracellular
Ca2
mobilization
of
is 6)
arachidonic by
acid,
+
indicating
intracellular
Ca2+.
investigated the metal requirements of + that Ca2 was required for maximal enzyme
A2 and showed
mechanism
(3).
in
aggregation.
that
phospholipase &
A, is
acid
platelet
platelet
g
of
thromboxanes,
demonstrated the
phospholipase
activity. been
phospholipase
in
present
level
of endoperoxides,
Rittenhouse-Simmons, platelet
is
of
importantly
et -2
of p4osphoglycerides
intracellular
activity
common precursor that
2 position
a lysophosphoglyceride,
and the the
at the
EC 3.1.1.4),
Ca2’
stimulates
phospholipase
A2 has
not
clarified.
+
Current
t Address
address:
correspondence
Department of Pharmacology New York Medical College-Valhalla, New York 10595 to this
author. 0006-291X/79/180473-08$01.00/0 473
Copyright AU rights
@I 1979 by Academic Press, Inc. of reproduction in any form reserved
Vol. 90, No. 2, 1979
Recent was
BIOCHEMICAL
evidence
first
from
discovered
esterase
is Calmodulin
and cellular
reactions, (11,
muscle
and
sarcoplasmic
the
disassembly
Ca2+ in
12),
A, is
important
to
ated
calmodulin.
indeed
brain
reticulum
pholipase
suggesting
including myosin
chain (18),
dependent
that
mediated
the
the
for
(13-16),
lo),
erythrocyte
NAD kinase
(17),
in erythrocytes
(19)
(21,
22),
and
calmodulin
mediates
the
effect
of
of
that
platelet
activity
(5,
membrane 6))
Ca2+ on phospholipase we
present
human platelet
preliminary
phospholipase
it
phos-
was deemed A2 is
medi-
evidence As by Ca 2+
is
calmodulin. AND METHODS
and Reagents
(l-14C)-2-arachidony1 phosphatidylcholine (55 from Applied Sciences; thin-layer chromatography Instruments; (l-14C)-arachidonic acid (56 mCi/mmole) and arachidonic acid from Nuchek Co. Prostaglandins gifts from Dr. U. Axen of Upjohn Co. Preparation
(9,
in
of enzymes
membranes
finding of
report,
MATERIALS Chemicals
ubiquitous
of
maximum
effect
this
stimulation
through
kinase
Ca 2t transport
that
and the
on Gas ’ In
cyclase
which
phosphodi-
Ca2+ on a variety
phosphorylation
evidence
whether
calmodulin,
(23).
reactions,
examine
of
adenylate
light
that
cyclic 3’,5’-nucleotide + protein Ca2 -binding
effects
kinase (20),
increasing
many cellular
by
the
phosphorylase
of the
of
mediates
of microtubules
In view
indicates
a multifunctional
(7, 81,
Ca2+ -ATPase
many laboratories
as an activator
eukaryotes.
skeletal
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
of Human Platelet
mCi/mmole) was purchased plates from Brinkman from New England Nuclear; and thromboxane B2 were
Membranes
Newly expired platelets, collected by centrifugation at 700 x g for 10 min. to remove red blood cells, were sonicated for approximately 20 min. The homogenate was centrifuged at 7,000 x g for 20 min., and the supernatant fluid was centrifuged further at 105,000 x g for 60 min. The sediment was suspended in 50 mN Tris-HCl (pH 8.8) to make a protein concentration about 10 mg/ml. The sample was used fresh or stored at -20’. One unit of human platelets yielded approximately 2 mg of membrane protein. Assay
of Phospholipase
A2 in Platelet
Membranes
Each assay mixture (final volume 1 ml) contained 50 muCi of (l-14C)-2arachidonyl phosphatidylcholine, p$atelet membranes (approximately 1 mg), 50 mM Tris-HCl (pH 8.8), 0.1 mM Ca2 and 1 ull calmodulin. The substrate was dried under N2 and sonicated in 20 ul of ethylether to produce an emulsion. The reaction was started by the addition of platelet membranes. After incubation at 37O for 5 min. with constant shaking, the reaction was terminated by the addition of 0.2 ml of ice-cold 0.1 M HCl and 10 ug of arachidonic acid. Carrier arachidonic acid was added to facilitate the isolation of (l-14C)-arachidonic acid. The reaction mixture was extracted 3 times with 2 ml of n-hexane. The organic phase was separated from the aqueous phase by centrifugation at 5,000 x g for 5 min. After phase separation, the tube was
474
BIOCHEMICAL
Vol. 90, No. 2, 1979
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
chilled in a methanol-dry ice bath. The hexane phase was removed from the frozen aqueous phase with a Pasteur pipette, and was ttansferred to a scintillation vial. The extract was evaporated to dryness under a stream of air. The dry lipid residue was dissolved in 5 ml of a Triton X-100, toluene-based scintillation fluid for counting. Measurement
of Thromboxane
B2 Released
from
Washed Human Platelets
Platelet-rich plasma was passed through a Sepharose-2B column (1.5 x 20 cm) which had been equilibrated with Ca2 -free Tyrode buffer, pH 1.4 (24). The platelets emerged in the void volume of the column, and were well separated from plasma proteins. A fraction of the platelet suspension (0.5 ml) was incubated with (l-14C)-arachidonic acid (2 ug, 0.36 uCi) for IO min. at 37” with constant stirring in a Payton dual channel aggregometer in the presence or absence of 1 uM calmodulin. After aggregation of the platelets, as monitored by a decrease in light transmission, the reaction was terminated by acidification to pH 3.0 with 0.1 M HCl, and the suspension was extracted 3 times with 2 ml of ethylacetate. The organic phase was pooled and dried under N and the reaction product was separated by thin-layer chromatography, using a*iolvent system containing ethylacetate:acetic acid (99:1, v/v) (25). The reaction product was identified with an authentic thromboxane B, on the same plate (Rf value 0.57). This solvent system clearly separates thromboxane B, The radioactive peaks were localfrom PGE2, PGF2 , and 6-keto PGFi (25). ized with a Pack%rd 2730 radiochro%atogram scanner (Packard Instrument Co.). Preparation
and Assay
of Calmodulin
Calmodulin was purified from bovine brain (26, 27). The assayed by its ability to stimulate bovine brain calmodulin-deficient diesterase (26). The use of bovine brain calmodulin is warranted lacks both tissue and species specificity (28). Protein
protein was phosphobecause it
Determination
Protein was determined as a standard.
according
to Lowry
(29)
using
bovine
serum
albumin
RESULTS AND DISCUSSION Human platelets preparations lipase
A2 showed
protein.
When
arachindonyl only
one
a range these
radioactive acid
on
the
presence
In 1). two-fold,
the
presence
acid
(5,
of
platelet
in
accord
in
the
presence
the
co-migrated
the
previous
a plateau amount
findings
of exogenous increased, of EGTA.
per
with mixture
with The
release in
that
Ca 2+
calmodulin,
about acid
stimulates the
to
(l-14C)-2generated
an authentic
level
level
lOO-200%
small
Four
mg of membrane
reaction
arachidonic
The relatively
(30).
of phospho-
incubated
of
equivalent
475
calmodulin as a source
calmodulin
were
5 min.,
of
used
chromatography.
Ca*+,
In the presence 6). released was further
obtained
which
of EGTA reached with
were
membranes for
thin-layer of
amount
which
140 to 675 ng of
product
in
A2
a considerable membranes
phosphatidylcholine
arachidonic acid
contain
of the platelet
increase
sample
of
of
arachidonic
20 min. was
(Fig.
increased
phospholipase of arachidonic above
the
level
by exogenous
Vol. 90, No. 2, 1979
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
I
I
I
I
I
1
20
30
40
50
60
TME
(mid
Fig. 1 - Effect of calmodulin and Ca2+ on phospholipase platelet membranes. Fifty muCi of (l- r4C)-2-arachidonyl was dried in a test tube under N,, and sonicated in produce an emulsion. To this tube was added 50 mM Tris of platelet membranes in a final volume of 1 ml. After constant shaking for the times indicated, the amount released was determined as described under Materials lip$se A2 activity was expressed as arachidonic acid Ca2 , 0.1 mM; and calmodulin, 1 uM. calmodulin
is
presumably
phenomenon
reminiscent
erythrocyte
plasma
these apparent
leveling-off of
contain
reaction
from
and the The
Thus,
reacylation
known
mM.
phospholipase adenylate
represents
to
phospholipase of
to block
A2 by
Ca2+ an
Ca2+,
the
Ka being (32).
the
platelet
membrane
Trifluoperazine At; activity
cyclase
however, to (33)
preparations,
and EGTA had basal and
they
both
level. cyclic
effectively
nucleotide
476
of acid
the
release
was
further
in
phenothiazine
activity
of
calmodulin
to calmodulin
to
1, calmodulin
the
Similar
reacylation
calmodulin
bound
no effect
few tissues
2).
is bound
in Table
The
initial
between
biological
Calmodulin
As shown
the
antidepressive
trifluoperazine 10’.
and
both
calmodulin.
the
Fig.
and of
small;
of arachidonic
equilibrium (see
(9)
Mammalian
level
a
stimulation
after
catalyzes the
an
specifically
of also
reacylation.
trifluoperazine,
of
acid
membranes,
cyclase
extent was
acid
PhosphoEGTA, 1 mM;
the
endogenous
A2; the platelet membranes contained + Ca2 . Atomic absorption spectrophotometric
in 6 different
stimulated
of use
inactive
endogenous 0.8
due
to phosphoglycerides
presence
affinity,
biologically
to
which
the
was
probably
of
of arachidonic
acyltransferase,
the
phospholipase
release
in
adenyiate
The
levels
to phosphoglycerides.
by In
the
high
arachidonic
released.
calmodulin
active mixture
derivative high
in
(31).
exogenous
relatively
activation
examined
(32).
Ca2+-ATPase by
on brain
probably
acid
the
findings
enzymes
of
and Methods.
calmodulin
incubation
an
arachidonic
endogenous
membrane
retained
minutes
to
of previous
particulate
preparations
due
Aa activity.in human phosphatidylcholine 20 ul of ethylether to buffer (pH 8.8), 1 mg incubation at 37O with
with
trifluoperazine
is
itself
relatively
a
activated
high
levels
of
measurement showed that Ca2 + levels ranged from 0.4 on the suppressed
observations phosphodiesterase
basal the have
activity
of
calmodulinbeen (32).
made on These
BIOCHEMICAL
Vol. 90, No. 2, 1979
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
Phospholipids Phospholipase Ca2
Acyltransferase
A,
II Arachidonic Acid
1
Cycle-oxygenase
Endoperoxides
Thromboxane Synthase I Thromboxane
A,
1 Thromboxane
B2
Fig. 2 - A simplified scheme showing the metabolism of arachidonic acid in human platelets. The level of arachidonic acid, which serves as a precursor is governed by its release from and of endoperoxides and thromboxanes, reacylation to phosphoglycerides. Platelet membranes do not further metabolize arachidonic acid. Whole platelets, however, convert arachidonic acid to the endoperoxides and thromboxane A,. Thromboxane A2 is unstable, and is readily hydrated nonenzymatically to form the more stable thromboxane B,.
TABLE EFFECT
OF Ca2+
OF PHOSPHOLIPASE
1
AND CALMODULIN A,
ON THE ACTIVITY
IN HUMAN PLATELET
MEMBRANES
Additions
Activity (f
mol/mg/5
None
18.8
EGTA
21.1
Trifluoperazine
18.0
Ca2+
42.3
Calmodulin
min.)
38.0
Calmodulin,
Ca2+
47.9
Calmodulin,
EGTA
18.9
Calmodulin,
Ca2+,
Trifluoperazine
23.6
Each reaction mixture in a final volume of 1 ml contained 50 muCi of (1_14C)2-arachidonyl phosphatidylcholine, 50 mM Tris-HCl (pH+8.8), approximately 1 mg and where present 0.1 mM Ca2 , 1 uM calmodulin, 1 mM of platelet membranes, EGTA, or 0.15 mM trifluoperazine. TDifluoperazine was dissolved in 50 mM Tris-HCl (pH 8.8). The reaction mixture was incubated for 5 min. at 37O with constant shaking. Phospholipase A2 activity is expressed as fmoles of arachidonic acid released per mg protein for 5 min.
477
Vol. 90, No. 2, 1979
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
Fig. 3 - Increased formation of thromboxane B, by calmodulin in human platelets. Washed platelets (0.5 ml) were incubated with fl-r4C)-arachidonic acid (2 ug, 0.36 uCi) at 37OC in the presence (A) or absence (B) of 1 uM calmodulin, with constant stirring (1,200 rpm) in an aggregometer for 10 min. At the end of the incubation, the reaction product was extracted and separated by thin-layer chromatography as described in Materials and Methods. The radioactivity on the thin-layer plates was scanned by a Packard 2730 radiochromatogram scanner. The’zone corresponding to TXB, was scrapped and extracted with methanol. The extract was dried and the radioactivity was determined in a liquid scintillation counter. Tliromboxane B, zone in Panel A gave 129,360 dpm, that in Panel B 58,560 dpm.
results
support
mediated
the notion
through human
exogenous
with
fication,
and the
donic boxane B,
boxane
One other
Ba from The
05).
lipids
(34). B,
of
solvent
(Fig.
for
products
A majority
The
on phospholipase
the
A, is
used
formed
the was
After the
isolated
in
and 6-keto
increased
of
reaction
was terminated
separated
identified
this
experiment
PGF1o, at
of exogenous approximately
478
from
acid
was
by acidichroma-
as unchanged
arachi-
as thromboxane clearly
which
the
B,
by thin-layer
was recovered
spot,
using
thromboxane
(l- i4C)-arachidonic
and
remaining
presence
was corroborated
formation
radioactivity
radioactivity In
3).
radioactive PGF,o
the
10 min.,
were
system
PGEa,
A, by calmodulin
measuring
acid
platelets
acid.
noted.
by
arachidonic
.
of Ca 2+
stimulation
of phospholipase
platelets
incubated tography
the
calmodulin.
The stimulation washed
that
migrate
origin
is
calmodulin, two-fold.
B,,
separates behind presumably
the
was
throm-
thromboxane phospho-
amount
of
The
effect
thromof
BIOCHEMICAL
Vol. 90, No. 2, 1979
exogenous intact
calmodulin
platelet
on
is
exterior
surface
let
and acts
on the
of
phospholipase added
and
the
thromboxane
of the
synthase (see
The
increase
of
effect
mediated
tion.
2).
of Cazt like
finding
that
The
of
effect
cellular
synthesis
B,
from arachidonic
plateagonist
The arachidonic
thromboxane
product,
A, and
from is
thromboxane supports
A, has been Results
numerous
enzymes
stimulates
noted
the for
presented
mentioned platelet
enzymes
the
rapidly B,
(36).
notion
that
the
provides
a molecular
and
plays
in
suggest
link
between role
in
that
regula-
interwoven
CAMP and the
is
A, adds to
cellular are
of
its
INTRODUCTION,
phospholipase involved
metabolism
a pivotal
some time,
herein under
of CAMP and prostaglandins
controlling thus
the
on
by cycle-oxygenase t Ca2 for activity.
short-lived acid
the
A2 in human platelets.
and metabolism
regulators,
(35).
requires
is
stable
elucidated.
In
in
effect
into
utilized of
A,
a more
its
transported
them
to
calmodulin
(37). calmodulin
glandins,
the
calmodulin-regulated
instances
exerts
platelets
of
Thromboxane
the
calmodulin.
list
intact
B,
a known physiological
to be readily
on phospholipase
has not been
or is
neither
catalyzes
through
The
many
(34),
phospholipase
of Ca2 ',
growing
on the
thromboxane
either
Thrombin,
appears
thromboxane
effect
mode of action
the
acts
Fig.
regulates
The the
also
non-enzymatically
calmodulin
membrane,
RESEARCH COMMUNICATIONS
of
calmodulin
surface.
synthase
endoperoxides converted
plasma
platelets
Thromboxane
formation
interest;
interior
A,,
to
increased
of special
the
acid
the
AND BIOPHYSICAL
two
in
prosta-
classes
cellular
of
regulation.
ACKNOWLEDGMENTS We are Children's
grateful Research
T. K. Narayanan photometry; work
was
(PY-KW), is
for
to
Hospital
Skip for
Skinner
grants
supplies
of calcium
W. H. Lee for
by
and Mr.
generous
the determination
and to Mr. supported
Mr.
preparation
NS 08059
of a Young
Investigator
Award
Kunkel
Heart
of
of human platelets; by atomic
absorption
of platelet
(WYC),
by ALSAC (WYC) and by the American
a recipient
Keith
CA 21765
microsomes. (WYC),
Association
St.
Jude to Dr.
spectroThis
and DE 40591
(PY-KW).
PY-KW
(HL 22075).
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AND BIOPHYSICAL
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