Vol. 91, No. 1, 1979 November
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
14, 1979
Pages
ISOLATION
OF HOMOGENEOUS
PHOSPHOLIPASE
63-71
A2
FROM HUMAN PLATELETS Rafael
J.
Apitz-Castro', Mahendra
Manuel A. Masl, Kumar Jain2
Maria
' Centro de Biofisica y Bioqucmica, Instituto Investigaciones Cientificas (IVIC), Apartado (Venezuela), and Department of Chemistry, ware, Newark, DE 19711 (USA) Received
September
The
has
been
ane
Az(TXA2)
ated tal
situ
a variant gation of tially
a key
prostaglandins
from
membrane
of of
is
as
and
deficiency
step
(3). A2
and this
for
in
the the
A2
To its
better
activity
regulation, from
platelets of
arachidonic
in
Also been
which
we have blood
liber-
a congeniobserved
platelet the
human
thrombox-
acid
has
understand
enzyme
blood
(1,2).
syndrome,
has been homogenate the extract. protein of platelet
synthesis
phospholipids
phospholipase
impaired
characterized
A2 activity
from
Hermansky-Pudlak
phospholipase
and
Venezolano de 1827, Caracas 101 University of Dela-
A2 from human blood platelets by treatment of the platelet affinity chromatography of is a soluble, heat-stable enzyme inhibits ADP-induced
phospholipase
implicated
--in
Cruz'
24, 1979
Summary: Phospholipase purified to homogeneity with H2S04, followed by The platelet phospholipase MW 44000. The purified aggregation. Introduction.
R.
putative purified
in
aeererole and
par-
platelets.
Materials and Methods. The following published procedures were used for obtaining the materials used in this study. Egg phosphatidyl-choline was p:epared from hen egg yolks (4): The labelled substrate. 2-1 C linolinoyl-phosphatidylcholine, was prepared by acylation of lysophosphatidylcholine (obtained by hydrolysis of egg phosphatidylcholine by phospholipase A2 from Naja naja venom) with rat liver homogenate (5). The specific activity of the product was ~1 mCi/mmol and all (>98%) the label was released in the fatty acid fraction byphospholipase A2 from The lecithin analog, rac-l-octa-decNaja naja or bee venoms. 9-enyl-2-hexadecylglycero-3-phosphocholine (Calbiochem) was oxidized with potassium periodate and potassium permanganate (6,7) to yield rat-1-(9-carboxy)nonyl-2-hexadecylglycero-30006-291X/79/210063-09$01.00/0 63
Copyright All rights
@ I979 by Academic Press. Inc. of reproduction in any form reserved.
Vol. 91, No. 1, 1979
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
phosphocholine, which was coupled to AH-Sepharose 4B (Sigma) The phosphorus content to yield the affinity adsorbant (8). of the affinity gel was ~2.5 nmol/ml of the swollen and packed gel. Platelet rich plasma from freshly drawn human blood was obtained as described elsewhere (9). Phospholipase AZ activity was measured by either of the following two procedures: (a) The radiolabelled substrate dissolved in wet diethylether was incubated with the enzyme (lo), and the products were separated by thin layer chromatography on silica gel plates (Merck) in chloroform + methanol + water (65:35:6), where the fatty acid, diacylphosphatidylcholine and lysophosphatidylcholine migrate at different rates (Rf ^1 0.95, 0.33 and 0.10 respectively. The radioactivity in appropriate spots was visualized with a Beta-Camera LB 290 A (Berthold, Germany) which gave a photographic record. For quantitation of the radioactivity in the spots, the appropriate regions on the t.1.c. plate, after extracting the scraped silica gel were counted. The substrate and products could also be separated by column chromatography on neutral alumina (Woelm) where fatty acid is eluted by chloroform, phosphatidylcholine by chloroform + methanol (7:3), and lysophosphatidylcholine by methanol. (b) Phospholipase A2 activity was routinely measured titrimetrically as described elsewhere (11, 12). Unless stated otherwise the titrations were carried out with 2.5 mM sodium hydroxide at 37'C, pH 9.5 in aqueous solution containing 100 mM KCl, 20 mM CaC12 and 500 uM egg phosphatidylcholine as unilamellar vesicles obtained by sociation in a bath type sonicator (Heat Systems). Control experiments demonstrated that under these conditions there is no detectable proteolytic activity as measured with Azoalbumin (Sigma) (13) even in the whole platelet homogenate. With appropriate precautions we could routinely measure rates of proton release < nmollmin. Results: elets ets
The was
from
lo-40%
overall
essentially 200
ml
linear
stilled homogenate
After
23000
g)
with
and
water. by
residue
KCl)
and
all
up
to
of The
0.16
N H2SOs
was
applied
to
the
in
and
homogenized
pH 7.5,
the
in
affinity
column
64
I
platelon
a
in
dis-
total
The volume
centrifuged
(20
(36
dialyzed
hrs)
min,
and
finally
against
the
dialysis
bag
supernatant
solution
plat-
B homogenizer. 40 ml
was
precipitate and
separated
in
from
Typically,
extensively
1 mM Tris,
A2
were
type
homogenate
white
dissolved
14)
Kontes
centrifugation, was
steps.
glass
the
phospholipase
plasma (9,
supernatant
changes
distilled
The
an
two
rich gradient
made
the
in
platelet
30 minutes
several
removed
of
with was
of
achieved
glycerol
water
(15).
purification
was
(20
mM CaC12
(40
ml
packed
was
lyophilized. and
100
swollen
mM
Vol. 91, No. 1, 1979
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
06
Fig.
l.-
volume)
Elution profile obtained from the affinity column obtained as described in Methods. In this particular experiment, 1.2 ml fractions were collected. Rate is arbitrarily expressed as divisions per minute.
pre-equilibrated
washed
with
solution
at
nm.
The
280
with
the
KCl,
pH 8.0).
and
for
moving
the
in I
till
adsorbed front
the
same
the
solution.
effluent
showed
phospholipase of
the
A typical phospholipase
II
profile
A activity
is
column
no
A activity
solution
elution
The
was
absorption
was
eluted
(40
mM EDTA,
100
for
the
protein
presented
in
mM
(A280) Fig.
1.
B
A
d’
5 3 ‘4 0” -3 I
I
I
I
I
I
123456 MIGRATION (cm) Fig.
2.-
SDS-polyacrylamide pattern of purified platelet phospholipase (PPL) in a 4-40X acrylamide gradient. 15 ugr. protein were loaded on the gel (right). Comparative migration of PPL with regard to known MW protein standards (4-40% acrylamide): cytochrome C (12.5 K), soybean trypsin inhibitor (21.5 K), ovalbumin (43.5
K),
and
bovine
serum
6.5
albumin
(68
K).
BIOCHEMICAL
Vol. 91, No. 1, 1979
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
0.90
0.33
Fig.
The
3.-
Hydrolysis of C-2 radiolabelled phosphatidylcholine by purified platelet phospholipase. Reaction was performed in diethylether and the products separated by thin layer chromatography, as described in Methods. Rf for fatty acid is 0.87, for phosIn this system, phatidylcholine is 0.35 and lysophosphatidylcholine do not migrate from the origin (16).
fractions
dialyzed
gradient
showed of
ular
weight
was
iour
of
enzyme
the
MW,
constituting
was
sometimes
The
rate
of
enhanced
tain
2-position,
in
the and
gel
when
the
1% of
and
The
of
in
band
purified
the
ab-
molec-
the
behav-
of
higher
protein,
excluded.
additives
n-hexanol.
The in
pro-
estimated with
total
On
purified
presence
phosphatidylcholine
presence
no
the
coincides
the
pooled,
lyophilised.
A minor
SDS was
dissolved
then
2).
which
were
SDS,
filtration.
of
and
in
(Fig.
than
hydrolysis in
both
dalton,
in
seen
detergents
the
44000
and
gel
dodecylsulfate
less
phatidylcholine in
band,
activity
water,
polyacrylamide
a single
sodium
enzymatic
against
sence
is
the
extensively
a 4-40% tein
containing
by like
labelled
is
66
found
diethylether,
A2 cer-
2-linolenoylphos-
diethylether
label
phospholipase
releases in
the
the
fatty
lysophosphatidyl-
acid
Vol. 91, No. 1, 1979
choline
fraction
have
(Fig.
isolated
is
phospholipase
shown
in
of
rich
Table
I
is
te
the
the
rate
of
It
enzymatic
activity
has
obtained
preparation. the
Table
530
to
be
rate of
et of
al.
I.
Phospholipase
substrate
of
activator
(%
None
Similar
a partially found
enzyme,
of
Human
to
be
after
% of
Initial
Activity* nmol*min-l*rng-l
100
100
0.4
10
58
2.3
0.03
40
530
column
is in
these
measured with the presence
of
unilamellar egg 45 mM n-hexanol
67
condiwith
is the
at maximal
results
independent cofactor
Steps. Phospholipase
Initial)
of
purified
substrate,
Platelets
The
nature
enzyme
Ca 2+ , and .
phosphatidylcholine at pH 9.5.
phos-
linearly
and
dialysis
Activity vesicles
with was
the
the
2+
the egg
(n-hexanol).
Activity
of
varies
pH for
(16)
the
Under
y 5 mM Ca
of
10 mM CaC12. to
for
replatelet
sonicated
published).
hydrolysis
addition
the
at
with and
requirement
the
activity
sensitive
the
overall
from
-1
smin
optimum
achieved
The
be
of
absolute
Franson
Treatment
*
homo-
phospholipase
= 40%
specific
quite
(to
The
is
-1
Protein
Affinity eluate
in
the
with
mM n-hexanol
hydrolysis
an
The
nmolemg
activator
or
N H2S04
we both
identified
purification
Purification
1.6
enzyme
however,
of
fold
overall
45
found
by
sequence
(Ca2+ 1,
is
> 3000
fold.
concentration.
9.4-9.5.
of
be
purification
was
500,000
is
and
protein
been
overall
containing
lipid
the
Az,
could
The
protein
activity
tions
the
over
phatidylcholine
the
A2 activities
activity.
lyophillized
enzymic
that
phospholipase
platelets
the
plasma
suggests
RESEARCH COMMUNICATIONS
(3).
homogenized
covery
AND BIOPHYSICAL
This
exclusively
platelets
from
3).
A1 and
genized As
BIOCHEMICAL
Various
have
Vol. 91, No. 1, 1979
Discussion.
In
procedure
for
human
platelets.
(44000
dalton)
from
BIOCHEMICAL
the
preceding
a complete The
molecular
differs
membranes it
is
modified
by
n-hexanol,
or
For
the
on
than
a soluble
the
zymes
for
compared
to
hundred
fold
specificity,
the
the
purified
and
other
implicated
in
action
or
pig
diethyldetergents.
fold
to
to
a difference the
en-
In is
contrast, several
ADP-induced due
lower
pancreatic
enzyme
is
the
1000
(11,12).
platelet
a difference
in
in
enzyme.
A:! may
4.
The
data
The
second
bating
the
platelets
one
first
amounts
platelet
that
inhibits wave
help
the
state
Studies to
resolve
wave of
the
(17,18).
with these
the
second
wave
of
aggregation
is
u 0.0015
units
3 minutes)
aggregation.
enzyme.
68
contrast,
effect
shown (=
is
by
the
or
aggrega-
inhibited of
of
in
3 ug
ADP-induced
periods which
In
is enzyme
of
A2 has The
aggregation platelet
(1
of
phospholipase
the
with Longer
endogenous
aggregation on
shows
minute.
the
of
platelet
phospholipase
tion.
larger
venom
to
other bilayers
in
about
presented
sheep
from
in
is
phospholipase
the
units)
block
is
due
from
bilayers
platelet
this
obtained
phospholipid
enzyme
from
enzyme
or
dissolved
inhibiting
and
platelet
0.0015
than
in
A2
enzyme
on
first
possibilities.
platelet Fig.
the
that
substrate
Physiologically, been
acts
conditions
effective
the
which
snake
the
enzyme
as micelles
platelet
enzymes,
We suspect
in
it
identical
aggregation. substrate
the
the
substrate
venom,
other
more
for
(13)
Like
dispersed
the
bee
under the
purified
egg-phosphatidylcholine of
measured
the
the
25000)
(8).
substrate
the
of
(
that
not
Interestingly
(20)
These
such
As
due
platelets. ic
at
phospholipase
likely
i,
6
The effect of purified platelet ADP-induced platelet aggregation A) Concentration dependence, Bee venom phospholipase (BVPL) comparison.
naturally
of
5
(min.)
aggregation
fatty
effect
4
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
the
outer
the
platelet
sources.
In
Vol. 91, No. 1, 1979
our
experiments,
outer of the
besides
surface, the
other
enzyme
and
membrane,
could
Indeed,
preliminary
substrate
and
lyzed
BIOCHEMICAL
by
the
the
factors also
from
AND BIOPHYSICAL
action arising the
contribute
platelet
activator enzyme
the
from
enzyme
to
this
changes
the
of
that rate
the
at
least
the
substrate behaviour
depending of
on
specificity
particular
suggest used,
only
substrate
organization
experiments the
of
RESEARCH COMMUNICATIONS
hydrolysis several
in (21).
upon
the
catahundred
fold.
Acknowledgments. Work supported in part by CONICIT (Grant i/ 0665 to R.A.C.) and by a travel grant from U.S. National Science Foundation (to M1K.J.). We-wish to thank the personnel of the Blood Bank of the Hospital Universitario (Caracas) for the generous supply of fresh platelet-rich plasma. One of US (M.K.J.) would like tQ thank Atala Apitz for a generous hospitality and useful discussions. We acknowledge excellent technical assistance of F. Triana, T. Cardenas, V. Michelena and Mrs. A. Istok, Highly purified Naja Naja phospholipase was a gift of Dr. Flor Barnola.
References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10 . 11. 12. 13.
Blackwell, G.J., Duncombe, W-G., Flower, R.J., Parsons, (1977) Br. J. Pharmac. 59, 353-366. M.F., and Vane, J-R. Silver, M.J., Bills, T.K., and Smith, J.B. (1978) In: Platelets, Ed. G. de Gaetano, and S. Garattini, pp. 213225, Raven Press, New York. Breton-Gorius J,, Trugnan, G., Castro-Mamaspina,, Rendu, F., H ., Andrieu, J.M., Bereziat, G., Lebret, M., and Caen, J.P. (1978) Amer. J. Hematology 4, 387-399. Singleton, W.S., Gray, M-S., Brown, M.L., and White, J.L. (1965) J. Amer. Oil Chemist's Sot. 42, 53-56. Waite, M., and Van Deenen, L.L.(1967) Biochim. Biophys. Acta 137, 498-517. Shimojo, T., Abe, M., and Ohta, M. (1974) J. Lipid Res. 15, 525-527. Rock, C.O., and Snyder, F. (1975) J. Biol. Chem. 250, 6564-6566. Kramer, R.M., Wcthrich, C., Bollier, C., Allegrini, P.R., and Zahler, P. (1978) Biochim. Biophys, Acta 507, 381-394 Apitz-Castro, R., Ramirez, E., Maingon, Rh., Murciano, A., Biochim. Biophys. Acta 455, 371-382. and Ribbi, A. (1976) Biochim. Coles, E., McIlwain, D.L., and Rapport, M.M. (1974) Biophysic. Acta 337, 68-78. Biochem. Biophys. Upreti, G.C., and Jain, M.K. (1978) Arch. 188, 364-375. Jain, M.K., and Apitz-Castro, R. (1978) J. Biol. Chem. 253, 7005-7010. Proc. Natl. Kaplan, L., Weiss, J., and Elsbach, P. (1978) Acad. Sci. 75, 2955-2958.
70
Vol. 91, No. 1, 1979
14. 15. 16. 17. 18. 19. 20. 21. 22.
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
Murata, Y., Stake, M. and Suzuki, T. (1963) J. Biochem. (Tokyo) 53, 431-437. Barber, A.J., and Jamieson, G.A. (1970) J. Biol. Chem. 245, 6357-6395. Kates, M. (1972) In: Laboratory Techniques in Biochemistry and Molecular Biology, Vol. 3. Ed. T.S. Work and E. Work, North Holland Publishing Co., Amsterdam. PP. 551-553, Beckerdite, S., Mooney, C., Weiss, J., Francon, R., and Elsbach, P. (1974) J. Exp. Med. 140, 396-409. Circulation 57 (Supp. II), Franson, R., and Jesse, R.(1978) 11-124. Marcus, A.J. (1978) J. Lipid Res. 19, 793-826. Vincent, J.E., and Zijlstra, F.J. (1976) Prostaglandins 12, 971-979. Iatridis, S.G., Datridis, P.G., Tsiala-Paraschou, E., Markidou, S.G., and Ragatz, B.H. (1976) Haemostasis 5, 165-175. Verger, R., and De Haas, G.H. (1976) Ann. Rev. Biophys. Bioengg. 5, 77-117.
71