Isolation of CA Independent Cytosolic PLA2
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S L Hazen, R J Stuppy and R W Grossglycerophospholipids.absolute f1-2 regiospecificity for diradylcalcium-independent phospholipase withmyocardial cytosolic phospholipase A2. APurification and characterization of canine:
1990, 265:10622-10630.J. Biol. Chem.
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THE
JOURNAL OF BIOLOOKXL CHEMlSTRY
8 1990 by The American Society for Biochemistry and Molecular Biology, Inc.
Vol . 26 5, No. 18, Issue of June 25 , pp. 1062% 10630,199O
Printed in U.S. A.
Purification and Characterization of Canine Myocardial Cytosolic
Phospholipase A2
A CALCIUM-INDEPE NDENT PHOSPHOLIPASE WITH ABSOLUTE
w-2
REGIOSPE CIFICITY FOR DIRADYL
GLYCEROPHOSPHOLIPIDS*
(Received for publicat ion, January 22, 1990)
Stanley L. Hazen, Robert J. Stuppy, and Richard W. Gross+
From the Division
of
Molecular and Cellular Cardiovascular Biochemistry, Washington University School
of
Medicine,
St. Louis, Missouri 63110
Recen tly, we ident if ied a novel calcium-independent,
plasmalogen-select ive phospholipase AZ act iv ity in ea-
nine myocardial cytosol which represents the major
measurable phospholipase AZ act iv ity in myocardial
homogenates (Wolf , R. A., and Gross, R. W. (1985) J.
Biol. Chem. 260, 7295-7303). We now report the
154,000-fold purification of this phospho lipase Az to
homo geneity through utilization of sequen tial an ion
exchange, chrom atofocusing, af f inity, M ono Q, and
hydroxylapat ite chromatographies. The purified en-
zyme had a molecular mas s of 40 kDa, possessed a
specif ic act iv ity of 227 rmol/mg min, had a pH opt i-
mum of 6.4, and catalyzed the regiospecif ic cleavage
of the ~2-2 fat ty acid from diradyl glycerophospholi-
pids. The purified polypept ide was remarkable for i ts
abil i ty to select ively hydrolyze plasmenylcholine in
homogeneous vesicles (subclass rank order: plasmen-
ylcholine > alkyl-ether choline glycerophosp holipid >
phosphat idylcholine) as well as in mixed bi layers com-
prised of equimolar plasmenylcholine/phosphat idyl-
choline. Purif ied myocardial phospholipase AZ also
possessed select iv ity for hydrolysis of phospholipids
containing arachidonic acid at the sn-2 position in
comparison to oleic or palmit ic acid. Taken together,
these results const itute the f irst purif icat ion of a cal-
cium-independent phospholipase with absolute regio-
specif ic ity for cleavage of the sn-2 acyl linkage in
diradyl glycerophospholipids and demonstrate that
myocardial phospholipase AZ has kinet ic characteris-
t ics which are ant ic ipated to result in the select ive
hydrolysis of sarcolemmal phospholipids during myo-
cardial ischemia.
Myocardial ischemia is associated with numerous biochem-
ical alterations which collect ively inf luence, and synergist i-
cal ly contribute to, the accumulat ion of amphiphil ic metabo-
l i tes in ischemic zones (e.g. Refs. l-5). Concom itant with the
onset of myocardial ischemia, phospholipase A, act iv ity is
augmented result ing in the release of unsaturated fat ty acids
and the accumulat ion of lysophospholipids (e.g. Refs. 6-8).
* This research was supported by National Institutes of Health
Grant HL34839 and Monsanto Company. The costs of publication of
this article were defrayed in part by the payment of page charges.
This article must therefore be hereby marked aduertisement in
accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
t Recipient of an Established Investigator Award from the Amer-
ican He&t Association. To whom co&espondence should be ad-
dressed: Division of Molecular and Cellular Cardiovascular Biochem-
istry, Washington University School of Medicine, 660 S. Euclid, Box
8020, St. Louis, MO 63110.
Lysophospholipids are potent membrane perturbing metabo-
l i tes which alter the dynamics of myocardial sarcolemmal
membrane s (9) and precipitate electrophysiologic alterations
in vit ro which are indistinguishable from those present during
myocardial ischemia in uiuo (10, 11). Accordingly, we and
others have suggested that act ivat ion of phospholipase A2 and
the resultant accumulat ion of lysophospholipids is int imately
related to the development of electrophysiologic dysfunct ion
in ischemic myocardium .
Myocardial sarcolemma is predominant ly comprised of
plasmalogen molecular species (12, 13), and the sarcolemmal
membrane is the primary target of accelerated phospholipid
catabolism in myoc ytes subjected to simulated ischemia (14).
In previous studies we demonstrated that the major measur-
able phospholipase AP act iv ity in canine myo cardium is cal-
cium-independent and has direct physical access to the sar-
colemmal membrane (15). Since accelerated sarcolemmal
phospholipid catabolism has been implicated as the biochem-
ical mechan ism underlying electrophysiologic dysfunct ion and
myo cyt ic cell death during m yocardial ischemia, the purifi-
cat ion and characterization of this calcium-independent phos-
pholipase AZ is of obvious importance. We now report the
154,000-fold purif icat ion of canine myocardial cytosolic phos-
pholipase AZ to homogeneity and demonstrate that the puri-
f ied enzyme has kinet ic propert ies which make it the l ikely
enzymic mediator of accelerated sarcolemmal phospholipid
catabolism during myocardial ischemia.
EXPERIMENTAL PROCEDURES
Purification
of
Canine Myocardial Cytosolic Phospholipse AZ-
Mongrel dogs (25-35 kg) fed ad libitum were anesthetized with
intravenous sodium pen tothal (40 mg/kg). Following a left thoracot-
omy, the heart was removed and immediate ly placed i n homogeniza-
tion buffer (0.25 M sucrose, 10 mM imidazole, 10 mM KCL, 5 mM
K[PO,], pH 7.8) at 0 C. Ventricular tissue was rapidly trimmed of
fat , weighed, and placed in fresh ice-cold homogenizat ion buffer (25%
w/v). Myocardium was finely minced (0.2 x 0.4~cm pieces) and
homogenize d utilizing a loose-fitting Potter Elvehjem homogenize r (3
strokes at 2,000 rpm) o n ice. All further purification steps were
performed at 4 C. Nuclei, cellular debris, and mitochondria were
removed by centrifugation at 10,000 X g,., for 20 min, and the
supernatant was subsequently centrifuged at 85,000 X g,.. for 60 min
to separate the cytosolic and microsomal fractions.
The supernatant (cytosol) was initially filtered through glass wool,
dialyzed twice (8 h/dialysis) against 10 liters of buffer 1 (15 mM
imidazole , 5 mM K[PO,], 10% glycerol, pH 7.8), and loaded onto a
previously equilibrate d DEAE-S ephacel column (5 X 7 cm, 3 ml/
min). The column was subsequently washed with buffer 1 containing
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Myocardial Cytosolic Calcium-independent Phospholipase A2 10623
1
m M
DTT, and phospholipase A, activity was eluted by application
of a 100
mM
NaCl stepwise gradient in elution buffet -(IO
mM
imidazole. 10
mM
KCI. 10% alvcecol. 1
mM
DTT.
DH
8.0). Active
fractions were identified , pooled, dialyzed against 26 liters of buffer
2 overnight (10
mM
imidazole, 10
mM
KCl, 25% glycerol, 1
mM
DTT,
pH 8.0) and loaded onto a previously equilibrate d PBE-94 chcoma-
tofocusing column (1.6
x
30 cm, 1.8 ml/min). A shallow pH gradient
was subsequently generated utilizing 10% PB96. 5% PB74 . 25%
glycerol, 1
&M
DTT, pH 7.1. Active &actions from the chcomatofo-
cusing column were identifie d and immediatel y applied to a 1
x
l-cm
N6-[(6-aminohexyl)cacbamoylmethyl]ATP-agacose column pcevi-
ously equilibrate d with buffer 3 (10
mM
imidazole, 25% glycerol, 1
mM
DTT, pH 8.3) (Sigma Lo t No. 124F-78951 or Phacmacia LKB
Biotechnology Inc. Lot No. AG5461101 gave the best yields) at 2 ml/
min. The affinity column was extensively washed in buffer 3, buffer
3 containing 10
mM
adenosine, and buffer 3 containing 10
mM
AMP
prior to further washing with buffer 3 alone (to remove uv absorbing
AMP). P hospholipase A, activity was quantitatively eluted by appli-
cation of buffer 3 containing 1
mM
ATP. The active fractions from
ATP affinity chromatography were directly loaded onto an HR5/5
Mono Q column previously equilibrate d with buffer 4 (20 mM imid-
azole, 25% glycerol, 1
m M
DTT. uH 8.3). and mvocacdial ohosoholi-
pase A2 was subsequently eluted utilizing a nonlinear saIt gradient
(O-450
m M
NaCl). Active fractions were identifie d and immediate ly
applied to a Koken hydcoxylapatite HPLC column (4 mm
x
10 cm)
previously equilibrate d with buffer 5 (10
mM
K[POJ, 25% glycerol, 1
mM
DTT, pH 7.4). Homogeneous myocacdial cytosolic phospholipase
A, was subsequently eluted utilizing a nonlinear K[PO,] gradient (O-
450 mM).
Preparation of Synthetic Phospholipids-Homoge neous l-O-(Z)-
hexadec-1.enyl-GPC was obtained by alkaline methanolysis of bo-
vine heact choline glycecophospholipids, was purified by silicic acid
column chromatography, and was resolved into individua l molecular
species by isocratic reverse-phase HPLC as previously describe d (16).
Synthesis of sn-2-radiolabeled plasmenylcholine was performed by
dicyclohexylcacbodiimide-mediated synthesis of radiolabe led fatty
acid anhydride followed by its condensation to the sn-2 hydcoxyl of
l-0-(Z)-hexadec-l-enyl-GPC utilizing N, N-dimethyl-4-aminopyci-
dine as catalyst (17). Each radiolabe led choline glycecophospholipid
molecular species was initially purified by preparative thin layer
chromatography (15) and subsequently purified by Pactisil SCX-
HPLC chromatography (18). Synthesis and purification of m-2-
radiolabe led phosphatidylch oline and alkyl-ether c holine glyceco-
phospho lipid molecular species were performed similarly util izing the
appropriate radiolabe led fatty acid and lysophosphoglycecide as stact-
ing materials. Specific molecular species of unlabe led phosphatidyl-
choline, plasmenylcholine, or alkyl-ether choline glycecophospholi-
pids were synthesized and purified similarly. To facilitate direct
kinetic comparisons between diacyl, vinyl-ether, and alkyl-ether sub-
classes, radiolabe led molecular species of identical specific activities
were synthesized by utilizing common preparations of freshly synt.he-
sized radiolab eled fatty acyl anhydride and the appropriate lysophos-
pholipid subclass.
The structure and purity of each radiolabe led synthetic product
was confirmed by thin layer chromatography in two solvent systems
(19), straight-phase HPLC (18), and comigcation with authentic
standards on reverse-phase HPLC (13). Gas chcomatogcaphic analy-
sis of each unlabe led plasmenylcholine species synthesized demon-
strated that the sn-1 vinyl-ether group and the sn-2 acyl group were
present in stoichiometcic amounts (+2%). The cegiospecificity of the
synthesized sn-2-labeled ch oline glycecophospholipids was quantified
utilizing Nuja naja phospholipase A2 (12) which demonstrated that
at least 98 and 95% of radioactivity in plasmenylcholine and phos-
phatidylcholine, respectively, was released as 3H-fatty acid during
hydrolysis. The cegiospecificity of purchased sn-1-radiolabeled DPPE
was similarly confirmed since 97% of the radioactivity comigcated
i The abbreviations used ace: DTT, d ithiothceitol; DPPC, l-pal-
mitoyl-2-palmitoyl-an-glyceco-3-phosphocholine; DPPE , l-palmi-
toyl-2-palmitoyl-sn-glyceco-3-phosphoethanolamine; GPC, sn-glyc-
eco-3-phosphocholine; GPE, sn-glyceco&phosphoethanolamine; LPC,
lysophosphatidylcholine; LPE, lysophosphatidyietha nolamine; PA,
phosp hatidi c acid; PAF , l-O-hexadecyl-2-acetyl-sn-glyceco-3-phos-
phocholine; PC, phosphatidylcholine; HPLC, high pressure liquid
chromatography; FPLC, fast protein liquid chromatography; EGTA,
[ethylenebis(oxyethylenenitcilo)]tetcaacetic acid; CHAPS, 3-[(3-cho-
lamidopcopyl)dimethylammonio]-l-pcopanesulfonic acid.
with lysophospholipid after hydrolysis by NC@ naja phospholipase
AZ.
Enzyme Assays-Phospholipase A2 activity in column chcomato-
graphic fractions was routinely assayed by incubating enzyme (5-50
~1) with 2 PM l-0-(Z)-hexadec-l-enyl-2-[9,lO-3H]octadec-9-enoyl-
GPC (introduced by ethanolic injection (10 ~1)) in assay buffer (final
conditions: 100
m M
Tcis, 4
mM
EGTA, 5% glycerol, pH 7.0) at 37 C
for 5 min in a final volume of 200 ~1. Reactions were quenched by
addition of 100 ~1 of butanol, vortexed, and the organic phase was
separated by centcifugation. Released radiolabele d fatty acid was
isolated by application of 25 yl of the butanol phase to channeled
Silica Gel G plates, development in petroleum ether/ethyl ether/
acetic acid (70:30:1), and subsequent quantification by scintillation
spectcometcy. Kinetic assays of phospholipase A2 activity were pec-
formed similarly except that incubations were performed for 1 min
which resulted in linear reaction velocities with respect to both time
and enzyme concentration for each substrate examined.
Hydrolysis of 1,2-dipalmitoyl-(N-methyl-(3H])GPC and l-[l-i4C]
palmitoyl-2-palmitoyl-GPE was assessed similarly except reactions
were quenched with 200 ~1 of butanol and reaction products were
separated by thin layer chromatography utilizing silica OF plates
(Analabs) with a solvent system of CHC13/acetone/MeOH/AcOH/
H,O (6:8:2:2 :1) as previously described (19).
Lysophospholipase activity was assayed by incubating loo-150 ~1
of column eluents with 13
@M
l-[l-4C]palmitoyl lysophosphatidyl-
choline in assay buffer (final volume = 200 ~1) for 5 min at 37 C.
Released [l-Clpalmitic acid was quantified as described above. For
kinetic assays, reactions were performed similarly for 1 min at 37 C.
LPC acyltcansfecase or lysophospholipase-tcansacylase activities
were quantified by production of [C]DPPC from lYZ]LPC in the
presence oc absence of palmitoyl-Cohas described (20). Palmitoyl-
CoA hydrolysis was assessed bv incubating 13
uM
14C1ualmitovl-CoA
__
with column eluant (loo-150 ~1) in assay buffer-in a final reaction
volume of 200 ~1 for 5 min at 37 C (column fractions) or for 1 min
at 37 C (kinetic assays) similar to the method previously described
(21).
Acetyl-CoA, sphingomyelin, dicadyl glycecols, tciolein, PAF, cho-
lestecyloleate, PA, palmitoylcacnitine, and acetylcholine hydrolysis
by myocacdial phospholipase Al were assessed utilizing previously
established techniques (22-31).
Sensitivity of Myocardial Phospholipas e Ai Activity to Chemical
Modification-The ATP affinity column eluate (5 pa) was incubated
with either 1
mM
dithiobisnitcobenzoic acid, 1 rni pacabcomophen-
acylbcomide, or 10
FM
phenylmethylsulfonyl fluoride, dialyzed against
buffer 3 and subsequently assayed as described above.
Thermal Denaturation-The purified protein was incubated at
37 C oc at 60 C in assay buffer for various times prior to addition
of neat saturating concentrations of substrate (3 X K,). After an
addition al 1 min incubation, products were extracted with butanol,
separated by thin layer chromatography and quantified as described
above.
Zodination, Sod ium Dodecyl Sulfate-Polyacrylamide Gel Electropho-
resis, and Autoradiography of Myocardial Phospholipa se A,-Aliquots
of HPLC-hydcoxylapatite active fractions (100 ~1) were reacted with
250 &i 251-Bolton-Huntec reagent (specific activity = 4400 Ci/mol)
overnight at O-4 C (32). Unbound inactivated reagent was removed
during electcophoresis (unbound reagent precedes dye front) in 10%
sodium dodecyl sulfate-polyaccylamide gels prepared by the Laem mli
method (33). Gels were subsequently fixed (three changes) in H,O/
MeOH/AcOH (5:5:1) with packets of mixed bed resin in gauze to
reduce the background intensity of autoradiographs of dried gels.
Protein Determinations-Protein content was determined utilizing
a Bio-Rad protein assay kit (first four steps) oc the Quanti-Gold
method (Diversified Biotech) (fourth through sixth steps) using bo-
vine serum a lbumin as standard.
Sources of Materials-[Hloleic, [Hlpalmitic, [Hlacachidonic
acids, cholestecol-14Cloleate. 13Hltciolein. 251-Bolton-Huntec ce-
agent, [i4C]palmitoyl LPC, [i4C]palmitoyl-CoA, [i4C]-palmitoyl-2-
palmitoyl-GPE, [1,2-14C]DPPC, and 1-[Y-glycecol]PA were puc-
chased from Du Pont-New Englan d Nuclear. [H]Dicadyl glycecols
(sn-2 labeled) were the generous gift of DC. D. A. Ford (Washington
University). All other radiolab eled reagents were purchased from
Amecsham Corp. Bovine heart lecithin, DPPC, and palmitoyl LPC
were purchased from Avanti Polar Lipids. PA and lyso-PA wece
purchased from Secdacy Lipids, while oleic, palmitic, and acachidonic
acids were obtained from Nu Chek Prep. Inc. Naja naja. pancreatic
and bee venom phospholipases A,, nucleotides, glycerol, common
buffer reagents, and the following agacose matrices were obtained
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10624
Myocardial Cytosolic Calcium-independent Phospholipase A2
from Sigma : n-ribose-5-phosphate, AMP (N-linkage), ADP (N6-
linkage),
ATP (N, C-8, and ribose hydroxyl-linkages), GTP (ribose
hydroxyl-linkage), and UTP (ribose hydroxyl-linkage). AG-CoA type
5, AG-ATP types 2-4, Blue Sepharose CL-GB, DEAE-Sephacel, PBE-
94, PB74, PB96, and Mono Q columns were purchased from Phar-
macia LKB Biotechnology Inc. All HPLC columns were purchased
f rom P. J. Cobert . De tergents and molecular weight standards were
purchased from Pierce Chemical Co. Dicyclohexylcarbodiimide, N,N-
dimethyl-4-aminopyridine, deoxycholate, and taurocholate were ob-
tained from Aldrich. All other reagents were obtained from Fisher.
RESULTS
Characterizat ion of Crude Myocardial Cytosolic Phospholi-
pase
AP Activity-As
previously demonstrated (15), the major
measurable phospholipase AZ act iv ity in canine myocardium
was present in the cytosolic fract ion and manifest maximal
enzymic act iv ity in the presence of the calcium chelator
EGTA. No calcium-independent hydrolysis of plasmenylcho-
line substrate could be detected in homogenates of whole
blood or plasma. The release of fatty acid from the sn-2
position of plasmalogen substrate by the cytosolic enzyme
was catalyzed by phospholipase AZ since inclusion of an excess
of lysophospholipid, diacylglycerol, 1-0-alk-l-enyl-2-acyl-sn-
glycerol or phosphatidic acid did not significantly attenuate
the rate of fatty acid release from radiolabeled plasmenylcho-
line substrate. Kinetic analyses of the cytosolic fraction uti-
lizing several synthetic sn-2-radiolabeled diacyl, alkyl-acyl,
and vinyl-ether choline glycerophospholipid molecular species
(Table I) confirm and extend our previous report (15) that
the major phospholipase AZ activity in myocardium selectively
hydrolyzes ether-linked choline glycerophospholipids. Fur-
thermore, the present resu lts indicate that cytoso l contains a
calcium-independent phospholipase AZ activ ity which prefer-
entially hydrolyzes choline glycerophospholipids containing
arachidonic acid at the sn-2 position.
Purification of Canine Myocardial Cytosolic Calcium-inde-
pendent Phospholipase AZ-To characterize the polypep-
tide(s) responsible for the observed calcium-independent
phospholipase AZ activity, canine myocardial cytosolic phos-
pholipase A Z was purified to homogeneity by sequential anion
exchange, chromatofocusing, affinity, FPLC-anion exchange,
and HPLC-hydroxylapatite chromatographies. First, dialyzed
cytosol was applied to a DEAE-Sephace l column, and phos-
pholipase APactiv ity was quantitatively eluted by application
of a 100
mM
NaCl stepwise gradient. The active fractions
were pooled, dialyzed, and loaded onto a previously equili-
brated chromatofocusing column as described under Exper-
imental Procedures. Phospholipase AZ act iv ity w as eluted by
the generation of a shallow pH gradient which resulted in a
TABLE I
Choline glycerophospholipid subclass specificity of myocardial cytosolic
phospholipase AP actiuity
Myocardial cytosol was incubated with l-100 M M radiolabeled
phospho lipid in the presence of 4
mM
EGTA and fatty acid was
extracted with butanol, separated by thin layer chromatography, and
quantified by scintillation spectrometry as described under Experi-
mental Procedures. All substrates were examined at a minim um of
five concentrations each in duplicate from multip le preparations.
Molecular
Subclass
species
V
rax K,
SIL-1
sn-2
nmol /mg . m in
P M
Phosphatidylcholine
16:0 18:l 0.5 18
Plasmenylcholine
16:0 181 1.5 16
Phosphatidylch oline 16:0 20:4
1.1
7
Alkyl-ether choline
16:0 20:4 1.5 9
glycerophospholipid
Plasmenylcholine
16:0 20:4 4.6 8
sharp ly focused peak of activ ity with an apparent isoelectric
point of 7.55 (Fig. 1). This step typically resulted in a 70-100-
fold purification of myocard ial phospholipase AP activ ity
(Table II).
Since initial studies identified the potential association of
ATP with myocardia l phospholipase A2 (34), further purifi-
cation was accomplished by exploiting the interaction of
myocardial phospholipase AZ with an ATP-agarose affinity
matrix. When active fractions from the chromatofocusing
column were applied to an ATP-agarose affinity column,
phospholipase A, activity was quantitatively and selectively
adsorbed (over 99% of other proteins present in the load
eluted in the vo id volume which was devoid of phospholipase
activity). The spec ificity of the interaction between myocar-
dial phospholipase A, and the ATP matrix was further ex-
ploited through utilization of sequential washes of the affinity
matrix with 10 mM adenosine and 10 mM AMP (which re-
moved the majority of bound protein but did not elute sub-
stantive phospholipase A2 activity). Enzymic activity was
quantitatively eluted from the ATP-agarose matrix with 1
mM
ATP (Fig. 2). Use of this nucleotide affinity matrix
resulted in a 150-fold purification of myocardial phospholi-
pase AZ n quantitative yield accompanied by a 50-fold reduc-
tion in volume. Thus, this 3-day procedure resu lts in a 52,000-
fold purification of myocardial phospholipase A Y activity in
86% yield which is moderately stable when stored at O-4 C
(ts = 5-7 d).
FIG. 1. Chromatofocusing of myocardial cytosolic phospho-
lipase AZ. The eluate from the DEAE-Se phacel column was dialyzed,
applie d to a chromatofocusing column, and phospholipase A, activity
was focused by development of a shallow pH gradient as described
under Experimental Procedures. Aliquots of column eluates were
assayed by quantifying radiolabe led fatty acid release from l-O-(Z)-
hexadec-l-enyl-2-[9,10-3H]octadec-9-enoyl-GPC (0) as described
under Experimental Procedures. -,
ultraviolet absorbance at 280
nm; W, pH.
TABLE I I
Myocardial cytosolic phospholipase AZ purification table
Myocardial cytosol and eluates from DEAE-Sep hacel, chromato-
focusing, ATP-agarose, Mono Q, and HPLC-hydroxylapatite (HA)
columns were incubated with 75 pM l-O-(2)hexadec-l-enoyl-2-
[9,10-3H]octadec-9-enoyl-GPC in the presence of 4 mM EGTA.
Fatty acid was extracted with butanol, separated by thin layer chro-
matography, and quantifie d by scintillation spectrometry as described
under Experimental Procedures.
Protein
Total Speci f ic Puri f i -
activitv
activity cation
Yie ld
w
nmol/min nmolfmg . min fold %
cytoso1 2,430 3,570 1.5
1 100
DEAE-Sephacel 540 3,430 6.3 4.3 96
Chromatofocusing 6.0 3,110 515 350 87
ATP-agarose
0.04 3,070 76,500 52,040 86
Mono Q
0.006 1,540 256,700 174,600 43
HPLC-HA 0.003 680 226,700 154.200
19
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32
r
24-
c?
0
;
16-
B
a-
o-
0 10 20 30 40
VOLUME (m l )
FIG. 2. ATP affinity chromatography of myocardial phos-
pholipase As. Active fract ions from chromatofocusing were imme-
diately applied to a previously equilibrated ATP-agarose column .
After loading,
the column was washed w ith equilibr ation buffer con-
taining 10
mM
adenosine, buffer containing 10
m M
AMP, and buffer
alone for the indicated volumes. Phospho lipase activity was eluted
with buffer containing 1
m M
ATP as described under Experime ntal
Procedures. Aliquots of column elua tes were incubated with l-O-
(Z)-hexadec-l-enyl-2-[9,10-H ]octadec-9-enoyl-G PC and released
radiolabeled fatty a cid (0) was quantified as described under Exper-
imental Procedures.
0
E
u
i
7
L
5
FRACTION NUMBER
FIG. 3. FPLC-anion exchange chromatography of myocar-
dial phospholipase AZ. The active fractions from ATP aff inity
chromatography were loaded onto a previously equilibra ted HR5/5
Mono Q column , and phospholipase A: was eluted utilizing a nonlin-
ear NaC l gradient as described under Experimental Procedures.
Phosph olipase Ar activity was assayed utilizing 1-0-(Z)-hexadec-l-
enyl-2-[9,10-Hloctadec-9-en oyl-GPC as substra te and fatty acid
release (A) wa s quantified as described under Experimental Proce-
dures. Lysophospholipase and palmitoyl-Co A hydrolase activities
were assaye d by quantifying fatty acid release from l-[l-Clpalmitoyl
lysophosphatidylcho line (O), or [l- Clpalmitoyl-CoA (II), respec-
tively, as described under Experimental Procedures. Approxim ately
five times the substrate concentration and 20 times the amoun t of
enzyme were used for assays of lysophospholipase and palmitoyl-Co A
hydrolase activities in comparison with phospholipa se Al assays as
described under Experimental Procedures. -, ultraviolet absorb-
ance at 280 nm; - - -, NaCl gradient.
Phospholipase AZ was further purified by application of the
ATP-agarose eluate onto an FPLC -Mono & anion exchange
column which was subseque ntly eluted utilizing a shallow
discontinuous NaCl gradient (Fig. 3). Mono Q active fract ions
were directly loaded onto an HPLC-hydroxylapatite column,
and phospholipase Ar activi ty was eluted with a nonlinear
K[PO ,] gradient a s described under Experimental Proce-
dures (Fig. 4). Since the purified enzym e w as extrem ely labile
(t , , g 30 min at 4 C), ass ays of enzymic activity following
hydroxylap atite chromatogra phy were performed directly
after elution of each fraction. Collec tively, this series of col-
umn chromatogra phic steps resulted in a 154,000-fold purifi-
cation of canine myoc ardial cytos olic phospholipase AP to a
specif ic act ivity of 227 pmol/mg min with an overall yield of
19% (Table II).
Purity of Myocard ial Phospholipase AZ after Column Chio-
matography-To assess the purity of myocardial phospholi-
pase A2 after sequential column chromatogra phies, the active
fractions from the hydroxylap atite column were iodinated
with Bolton-Hunter reagent, separated on sodium dodecyl
sulfate-polyacrylam ide gel electrophoresis , and protein was
visualized by autoradiography. Only a single intense band at
40 kDa wa s observed in the most active fract ion (Fig. 5).
0
5 20 25
FRACC:?ON:MBER
FIG. 4. HPLC-hydroxylapatite chromatography of myocar-
dial phospholipa se An. The active fractions from Mono Q chro-
matography were immedia tely loade d onto a previously equilibr ated
HPLC hydroxylapatite column, and phospholipa se A, activity was
eluted with a nonline ar K[PO,J gradient as described under Exper-
imenta l Procedures. Lysophospholipase (0) and palmitoyl-Co A hy-
drolase (0) activities were assayed as described under Experim ental
Procedures with over five times the substrate concentration and 20
times the amount of enzyme in comparison to phospholipa se A, assays
(A).
-97kD
-6BkD
- 1BkD
- 14kD
10 11 I2 13 I4 15
FRACTION NUMBER
FIG. 5. Sodi um dodecyl sulfate-polyacrylamide gel electro-
phoresis of myocardial cytosolic phospholipa se A*. Aliquots of
the active fractions from HPLC-hydroxylapatite chromatography
were iodinated, boiled for 3 min in the presence of 100 mM 2-
mercaptoethanol and 10% SDS, loaded onto a 10% polyacrylamide
slab gel, electrophoresed, fixed, dried, and subsequently visualized by
autoradiography as described under Experimental Procedures.
Fraction numbers on the n axis correspond to fractions from the
hydroxylapatite column shown in Fig. 4.
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Furthermore, the relative in tensity of the 40-kDa band pre-
cise ly paralleled the elution profile of phospholipase A2 activ-
ity during hydroxylapatite chromatography (compare Figs. 4
and 5). In multiple preparations, the 40-kDa polypeptide was
I / /
the only protein whose intensity paralleled enzymic activity
30
(n > 10) and was the only band visualized after autoradiog-
t/
raphy of the most active hydroxylapatite fraction in three ,s,l II
other independent preparations. Attempts to recover any
is
20
phospholipase Al activ ity from multiple a&amide-based gel
electrophoresis systems utilizing either pulverized, extracted,
or electroeluted gel slices have failed. In fact, incubation of
enzyme with even minute amounts of polymerized acrylamide
results in complete and unrecoverable loss of all enzymic
activity.
Characterizat ion
of
Myocardial Phaspholipase AP Binding to
Nucleot ide Aff inity Matrices-The specificity of the interac-
tion responsible for the adsorption of calcium-independent
phospholipase A: to immobilized nucleotide affinity matrices
was examined to gain insight into the chemica l interactions
contributing to the association of ATP with this phospholi-
pase. Of the three ATP resins tested (see Experimental
Procedures), coupling via the M-amino group provided the
highest yield. Attachment through the C-8 or the ribose
hydroxyl groups resulted in recovery of 60-80% of loaded
enzymic activity in the ATP wash with the majority of re-
maining activity present in the void volume. Other matrices
such as GTP-agarose, UTP-agarose, ADP-agarose, CoA-aga-
rose as well as AMP-agarose all bound myocardial phospho-
lipase to varying extents in the specified rank order (strong-
est-weakest, 60-10% binding). In contrast, D-ribose-5-phos-
phate-agarose did not bind canine myocardial phospholipase
A2 activity. Although Blue Sepharose (CL-6B) quantitatively
adsorbed enzymic activity (no activity was present in the void
volume), recovery of phospholipase activ ity after elution with
buffer containing ATP, ATP and 1 M NaCl or ATP, and 1 M
K[POJ was poor (C5%). With the exception of Blue Sepha-
rose (which nonspecifically adsorbed approximately 50% of
the loaded proteins), greater than 99% of loaded proteins did
not bind to these affinity matrices under the conditions em-
ployed. Furthermore, although classic calcium-dependent, low
molecular weight phospholipases AZ are known to bind to the
nucleotide analog dye Cibacron Blue FBGA (35), none of the
phospholipases A, examined (i.e. Naja naja, pancreatic, bee
venom, platelet cytosolic) adsorbed to the ATP resins used.
FIG. 6. Concordant production of lysophospholipids and
fatty acids by purified myocardial cytosolic phospholipase AZ.
Three experiments are depicted. Purif ied myocardial cytosolic phos-
pholipase Az was incubated with the indicated concentrat ions of W-
sn-l- labeled DPPE and the amount of [ C]DPPE hydrolyzed (O), l-
[ l- *C]palmitoyl-LPE produced (m), and [ l :W]palmit ic acid released
(A) were determined as described under Exnerimental Procedures.
¶llel experiments, choline-N-methyl-] H]DPPC was incubated
with purif ied-enzyme and the amount of [%]DPP C hydrolyzed (O),
13H]LPC produced (0) and 13H]GPC produced (0) was determined.
Finally, purified enzyme was incubated with l-palmitoyl-2-]9,10-3H]
palmitoyl-GPC and the amount of radiolabeled fat ty acid (A) quan:
titated asdescribedunder Exuerimental Procedures.Data reoresent
the mean of duplicate determkat ions.
Kinet ic Analyses
of
Purified Myoca rdial Phosp holipase AZ--
The homogeneous polypeptide exhibited maximal enzymic
activity in the presence of EGTA and possesseda pH optimum
of 6.4 for each phospholipid substrate examined. Incubation
of the purified enzyme with sn-2-rad iolabeled phospholipid
(e.g. plasmenylcholine, phosphatidylcholine, or phosphatidyl-
ethanolamine molecular species) resulted in the release of
radiolabeled fatty acid with no observable radioactivity in
lysophospholipid, dirady lglycerol, or phosphatidic acid. The
possibility that the release of sn-2 fatty acid from diradyl
glycerophospholipids occurred by sequential phospholipase A1
and lysophospholipase activit ies was eliminated by multiple
independent techniques. First, myocard ial phospholipase AP
was incubated w ith l-30
gM
[3H-Me]choline-labeled DPPC,
and the reaction products were isolated and quant ified as
described under Experimental Procedures. For each concen-
tration of substrate examined, the loss of PC and the accu-
mulation of LPC were stoichiometric (Fig. 6) with no detect-
able radiolabel in GPC. Second, when sn-2-3H-labeled DPPC
was utilized as substrate under identical assay conditions, the
resultant increase in 3H-fatty acid equalled (*3%,
n = 2)
the
increase in t3H-Me]LPC at each concentration examined (Fig.
6). Third, incubation of l-[ l-4C]palmitoyl-2-palmitoyl-GPE
with purified enzyme resulted in the production of l-[l-i4C]
palmitoyl-LPE without measurable amounts of radiolabeled
palmitic acid, and the mass of phosphatidylethanolamine
hydrolyzed was quantitatively accounted for by the mass of
l-acyl LPE produced (Fig. 6). Furthermore, no [1-14C]palmi-
tate was released from l-[l-4C]palmitoyl-2-palmitoyl-GPE in
the presence of several detergents (i.e. Triton X-100, n-octyl
glucoside, Lubrol-PX, or Tween-20). Finally, loo-fold molar
excessesof LPC, diacylglycerol, and PA did not substantially
diminish release of 3H-fatty acid from l-O-(Z)-hexadec-l-
enyl-2-[9,10-3H]octadec-9-enoyl-GPC. Thus, myocardial cy-
tosolic phospholipase Az is specific for hydrolysis of the sn-2
ester linkage in choline and ethanolamine dirady l glycero-
phospholipids and is devoid of measurable phospholipase Al,
C, or D activities. Attempts to demonstrate significant re-
vers ibility of the reaction by incubation of purified enzyme
with lysophospholipid and radiolabeled fatty acid (in the
absence or presence of CoA) were unsuccessful.
Characterization of the phospholipid substrate specific ity
of purified myocard ial cytosolic phospholipase A: was per-
formed by kinetic analyses of the ATP eluent (52,000-fold
purified, spec ific activi ty = 76 pmol/mg min) since the marked
labil ity of Mono Q or hydroxylapatite eluents precluded their
use. Examination of the choline glycerophospholipid subclass
spec ificity of the 52,000-fold purified enzyme revealed that
hydrolysis of plasmenylcholine substrate was more rapid than
hydrolysis of alkyl-ether choline glycerophospholipid or phos-
phatidylcholine (Fig. 7, Table III). Comparisons of phospho-
lipase ASactivity utilizing phosphatidylcholine molecular spe-
cies containing palmitate at the sn-1 position and either
palmitic, oleic, or arachidonic acid at the sn-2 position as
substrates demonstrated a rank order preference for cleavage
of arachidonate > oleate > palmitate (Fig. 7, Table III).
Furthermore, substantial enzymic activ ity required the pres-
ence of a long chain acyl group at the sn-2 position since PAF
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FIG. 7. Lineweaver-Burk plot of purified phospholipase A, activity. Purified phospholipase Al was
incubated with the indicated concentrations of radiolabe led glycerophospholipids in the presence of 4
m M
EGTA
for 1 min at 37 C, and reaction products were extracted with butano l, separated by thin layer chromatography
and quantifie d by scintillation spectrometry as described under Experimental Procedures. Right panel, DPPC
(0); 1-palmitoyl-2-oleoyl-GPC (A); DPPE (V); l-O-(Z)-hexadec-1-enyl-2-octadec-9-enoyl-GPC (0). Left panel,
1-palmito yl-2-ara chidon yl-GPC (A); 1-O-hexadec yl-2-arachidony l-GPC (W); I-0-(Z)-hexadec-1-enyl-2-arac hi-
donyl-GPC (0). Data points represent the mean of duplicate determinations.
TABLE I I I
Purified myocardial phospholipase A, substrate specificity
Phospholipas e A, was incubated with l-100
f iM
radiolabeled phos-
pholip id in the presence of 4
mM
EGTA, and initia l reaction velocities
were quantifie d by fatty acid extraction, thin layer chromatography,
and scintillation spectrometry. All assays were performed at a mini-
mum of five concentrations for each substrate (each in duplicate) as
described under Experimental Procedures.
Molecular
Substrate
species
V
mex Km
SO-1
sn-2
~mol/mg~ min
pM
Phosphatidylch oline 16:0 16:0 23
2
Phosphatidylcholine 16:0 l&l
38 2
Plasmenylcholine 16:0 l&l
77 3
Phosphatidylcholine
16:0 20:4 73 13
Alkyl-ether choline glycero- 16:0 20:4 109 16
phospholipid
Plasmenylcholine 1610 20~4 157 16
Platelet-activating factor
Palmitoyl lysophosphatidyl-
choline
Palmitoyl-CoA
Acetyl-CoA
ND, not detected.
0.1
0.9
0.4
ND
was hydrolyzed three orders of magnitude more slowly than
1-0-hexadecyl-2-arachidonyl-GPC.
Since previous work has demonstrated that plasmenylcho-
l ine and phosphat idylcholine bi layers possess dist inct molec-
ular dynamics (36), addit ional experiments were performed
to examine the substrate specif ic ity of myocardial phospho-
l ipase A2 in system s which minimize dif ferences in the phys-
ical properties of aggregated subs trate. In initial expe rimen ts,
we prepared mixed micelles o f phospholipids with selected
detergents (e.g. Triton X-100, Tween-20, n-octyl glucoside,
Nonidet P-40, CHAP S, Lubrol-PX, Bri j-35, deoxycholate,
and taurocholate) to compare hydrolyt ic rates for each choline
glycerophospholipid subclass in ident ical microenvironmen ts.
Unfortunately, myocardial phospholipase A2 act iv ity was
completely abolished by each of these detergents. To circum-
vent this dif f iculty, addit ional experiments employing binary
mixtures of plasmenylcholine and phosphat idylcholine in
mixed bi layers were performed (Table IV). Incubat ion of
vesicles comprised of 50 mol% plasmenylcholine and 50 mol%
phosphat idylcholine with purified enzyme resulted in the
TABLE IV
Myocardial cytosolic phospholipase AS phospho lipid subclass
selectivity in mixed bilayers
Vesicles comprised of the indicated compositions were prepared by
a single injection of the appropriate mixtures of phospholipids pre-
viously codissolved in ethanol. Myocardial cytosolic phosph olipase AZ
was incubated with 80
pM
substrate (total lipid) and released [3H]
arachidonic acid was quantifie d by thin layer chromatography and
scintillation spectrometry as described under Experimental Proce-
dures.
Plasmenylcho line = 1-O-hexadec-l-enyl-2-arachidonyl-
GPC; [3H]plasmenylcholine = l-O-hexadec-1-enyl-2-[5,6,8,9,11,12,-
14,15-3H]arachidonyl-GPC; phosphatidylcholine = 1-palmitoyl-2-
arachidonyl-GPC; [3H]phosphatidylcholine = 1-palmitoyl-2-[5,6,
8,9,11,12,14,15-3H]arachidonyl-GPC.
Substrate
50 mol% [3H]plasmenylcholine
[3H]Arachidonic acid release
pOUJ1
+50 mol% phosphatidylcholine
50 mol% plasmenylcholine
+50 mol% [3H]phosphatidylcholine
1100
210
10 mol% [3H]plasmenylcholine
+90 mol% phosphatidylcholine
90 mol% plasmenylcholine
+lO mol% [3H]phosphatidylcholine
200
15
select ive hydrolysis of plasmenylcholine (Table IV) demon-
strat ing that the observed subclass select iv ity of myocardial
phospholipase AZ is independent of alterations in the physical
properties and interfacial char acteristics of aggregated sub -
strate. To compare hydrolysis of each phospholipid subclass
in a microenvironment possessing physical properties and
interfacial characterist ics of i ts phospholipid subclass coun-
terpart , binary m ixtures comprised of 10 mol% [3H]plasmen-
ylcholine in phosphat idylcholine bi layers or 10 mol% [3H]
phosph atidylcholine in plasme nylcholine bilayers were pre-
pared. Purified myocardial phospholipase AP eff ic ient ly cata-
lyzed the hydrolysis of plasmenylcholine when the physical
characterist ics of the vesicles w ere largely those of phospha-
t idylcholine. In contrast, phosphat idylcholine was not sub-
stant ial ly hydrolyzed even when present in vesicles possessing
the physical propert ies of the preferred substrate in homoge-
neous system s (i.e. plasmenylcholine) (Fig. 7). Since the pu-
rified enzyme select ively hydrolyzed plasmenylcholine in 1)
homogeneous system s, 2) equimolar mixtures of plasmenyl-
choline/phosphatidylcholine, and 3) vesicles whose physical
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propert ies resemble those of phosphat idylcholine, these re-
sults demonstrate that myocardial phospholipase AP selec-
t ively hydrolyzes arachidonylated plasmenylcholine in phys-
iologically relevan t matr ices.
To further invest igate the diversity of the substrate speci-
f ic ity of purified cytosolic myocardial phospholipase A*, a
battery of l ipids was examined. When palmitoylcarnit ine,
sphingo myelin, acetylcho line, acetyl-C oA, triolein, l-palmi-
toyl-2-arachidonyl-sn-glycerol, 1-0-hexadecyl-2-arachidonyl-
sn-glycerol, l-0-(Z)-hexadec-1-enyl-2-arachidonyl-sn-glyc-
erol, or l-palmitoyl-2-palm itoyl-sn-glycero-3-ph ospha te were
incubated with the purified myocardial phospholipase AZ, no
hydrolysis of these moiet ies was observed. Similarly, the pu-
rif ied enzyme did not catalyze the disproport ion of LPC to
PC and GPC . Remarkab ly, the purified enzyme hydrolyzed l-
[1-Clpalmitoyl lysophosphat idylcholine and 1-[1-14C]pal-
mitoyl-CoA (Fig. 8, Table I I I ) albeit at rates two to three
orders of magnitude less than that manifest for choline or
ethanolamine glycerophospholipids. Kinet ic analyses dem-
onstrated that monome ric lysophosphat idylcholine and pal-
mitoyl-CoA are both poor substrates and that the observed
discontinu ities in their substra te ac tivity p rofiles (Fig. 8)
closely correspond to the crit ical micellar concentrat ion of
each lipid (37, 38) underscoring the importan ce of the lipid-
aqueous interface as a determinant of enzym ic act iv ity.
To verify that phospholipase AZ, lysophospholipase, and
palmitoyl-CoA hydrolase act iv it ies were mediated by a single
polypept ide with mult iple catalyt ic act iv it ies, addit ional ex-
periments were performed. First , parallel assays of phospho-
l ipase AZ, lysophospholipase, and palmitoyl-CoA hydrolase
act iv it ies from each column fract ion during Mono Q and
hydroxylapat ite chromatographies demonstrated that each
act iv ity precisely cochromatographed (Figs. 3 and 4) (see
Experimental Procedures for detai ls). Second, maximal cat-
alyt ic act iv ity for al l three substrates was manifest in the
presence of EGTA and was reduced similarly in the presence
of 10
mM
CaC12(52 f 3 %, n = 3). Third, each act iv ity was
comp letely and irreversibly inhibited by 1 mM dithiobisnitro-
benzoic acid (n = 3) and each act iv ity was relat ively insensi-
tive to inhibition by either parabrom ophena cylbromide or
phen ylmethy lsulfonyl fluoride (11 f 3% and 4 f 3% inhibi-
t ion, respect ively, n = 3). Fourth, the thermal denaturat ion
prof i les of phospholipase AZ, lysophospholipase, and palmi-
toyl-CoA hydrolase act iv it ies were indistinguishable at both
37 and 60 C.
To examine the potent ial physiologic relevance of lyso-
phosphat idylcholine hydrolysis catalyzed by myocardial cy-
FIG.
8. Lineweaver-Burk plots of lysophosphat idylcholine
and
palmitoyl-CoA hydrolysis by purified myocardial phos-
pholipase AZ.
Myocardial cytosolic phospholipase As was incubated
with the indicated concentrat ions of [ l- 4C]palmitoyl lysophosphat i-
dylcholine (left panel) or (l-14C]palm itoyl-CoA (right panel), and
released radiolabeled fatty acid was quantified as described under
Experimental Procedures. Data points represent the mean of du-
plicate determinations.
tosolic phospholipase Aa, addit ional studies were performed.
When bilayers containing 9 mol% lysophosphat idylcholine (5
pM
l-[l-4C]palmitoyl-L PC in 50 pM unlabeled l-O-(Z)-hex-
adec-l-enyl-2-octade c-9-enoyl-GP C) were incubated with
purified myoca rdial cytos olic phospholipase AZ, no radiola-
beled fat ty acid was released from LPC even though over 10%
of plasmenylcholine was hydrolyzed. Similarly, s ince the loss
of DPPE and DPPC and the accumulat ion of LPE and LPC
were stoichiometric (Fig. 6), measurable amounts of lysophos-
pholipid hydrolysis did not occur. Thus, under physiologically
relevant condit ions, m yocardial cytosolic phospholipase A:
hydrolyzes endogenous phospholipids to 1-acyl lysophospho-
l ipids and does not act ef fect ively as a lysophospholipase.
DISCUSSION
The results contained herein const itute the f irst purif ica-
t ion of a calcium-independent phospholipase act iv ity which
has absolute regiospecif ic ity for cleavage of the sn-2 acyl
linkage in diradyl glyceroph ospholipids. Although other cal-
cium-independent phospholipases have previously been de-
scribed (e.g. Refs. 39-42), detai led kinet ic analyses have dem-
onstrated that these phospholipases either specif ical ly cata-
lyze hydrolysis at the sn-1 posit ion or indiscriminately
hydrolyze acyl groups at both the sn-1 and sn-2 posit ions.
Since phospholipase A, act iv ity was not present ut i liz ing
multiple diradyl glycerophosph olipid subs trates in different
physical states, these results demonstrate the absolute regios-
pecif ic ity of myocardial cytosolic phospholipase AP and iden-
t i fy this phospholipase as the f irst regiospecif ic calcium-in-
dependent phospholipase AP purified to date.
Myocardial cytosolic calcium-independent phospholipase
A2 is the major measurable phospholipase act iv ity in myocar-
dium and is a low abundance, high specif ic act iv ity polypep-
tide which required a 154,000-fold purification to reach ho-
mogene ity. This degree of purif icat ion was faci l itated by the
unique, highly se lect ive, and reversible adsorpt ion of myocar-
dial cytosolic phospholipase AP to ATP-agarose resin. The
purity of the preparation was demonstrated by the presence
of a single 40-kDa protein band visualized by the highly
sensit ive method of lZ51 autoradiography. Although attempts
at obtaining phospholipase act iv ity after polyacrylamide gel
electropho resis have failed (the enzy me is irreversibly inacti-
vated by acrylamide), the high sensit iv ity and dynamic range
of the visualizat ion method employed, the high specif ic act iv-
i ty of the purified polypept ide (230 wmol/mg .min), as well as
the concordant appearance and disappearance of 40-kDa mass
with phospholipase act iv ity, col lect ively demonstrate that the
40-kDa polypept ide catalyzes phospholipase Al act iv ity.
Kinet ic analyses demonstrated several novel features of the
purified protein. Myoca rdial phospho lipase AZ is the first
purified calcium-independent phospholipase AZ which selec-
t ively hydrolyzes plasmalogen substrate and arachidonylated
glycerophospholipids. Rema rkably, the purified polypept ide
also contained intrinsic lysophospholipase and palmitoyl-CoA
hydrolase act iv it ies, albeit at rates two to three orders o f
magnitude less than its phospholipase AZ act iv ity. The con-
clusion that phospholipase AS, lysophospholipase, and pal-
mitoyl-CoA hydrolase act iv it ies are catalyzed by a single
polypept ide is substant iated by the coelut ion of each act iv ity
through mult iple chromatographic steps to a single polypep-
t ide, similar sensit iv ities of each act iv ity to divalent cat ions
and thiol oxidizing agen ts, and identical therma l denaturation
prof i les of each act iv ity at dif ferent temperatures. The possi-
bi l i ty that phospholipase AZ, lysophospholipase, and palmi-
toyl-CoA hydrolase act iv it ies are catalyzed by highly hom ol-
ogous yet dist inct polypept ides of nearly ident ical molecular
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Myocardial Cytosolic Calcium-independent Phospholipase A2 10629
mass which copurify over 154,000-fold cannot be defin itively
excluded but seems unlike ly.
Parenthetically, we note that venom phospholipase AZ (the
paradigm of sn-2 regiospecificity) possesses minute levels of
lysophospholipase activity (12). The highly regiospecific phos-
pholipolys is catalyzed by the venom phospholipase A2 and
myocardial cytosolic phospholipase AZ are in stark contrast
to the lack of regiospecificity of the previously isolated 98
kDa calcium-independent phospholipase in guinea pig intes-
tinal mucosa which possessed nearly identical phospholipase
Al, API and lysophospholipase activities (40). Although the
40-kDa polypeptide is the major measurable phospholipase Az
in myocardium, its lysophospholipase and palmitoyl-CoA hy-
drolase activities comprise only a small fraction of the total
lysophospholipase and palmitoyl-CoA hydrolase activities in
myocardium (4, 19, 21, 43, 44). According ly, based upon
in
vitro kinet ic measurements with the purified protein as well
as measurements of activ ities present in myocardial homog-
enates, it appears like ly that this protein functions as a
phospholipase AZand does not make substantial contributions
to lysophospholipid or palmitoyl-CoA hydrolysis in intact
tissue.
The phospholipase AZ purified in the present study is easily
distinguished from other previously described myocardial cy-
tosolic phospholipase activities. A calcium-independent phos-
pholipase A1 activity is present in rat myocardial cytosol but
specifically cleaves the sn-1 acyl linkage (41). A phospholipase
B activity was reported in Syrian hamster myocardial cytoso l
(42) but differs from the enzyme purified in the present study
by the following features: 1) it does not hydrolyze plasmen-
ylcholine substrate; 2) its specific activity is three to four
orders of magnitude less than the polypeptide purified herein;
3) it has a molecular weight of only 14 kDa; and 4) the
regiospecificity of the hamster phospholipase B is predomi-
nantly directed toward the sn-1 position while the polypeptide
purified in this report has absolute specificity for hydrolysis
of the acyl group at the
sn-2
position. It is important to note
that these cytosolic phospholipase A, and B activities com-
prise less than 10% of the phospholipase AZ activ ity present
in myocardial cytosol (Table I) utilizing optimal homogeni-
zation methods and substrates for each activ ity (41,42). Thus,
cytosolic calcium-independent phospholipase AZ is the major
measurable phospholipase in myocardium and possessessep-
arate and distinct physical characteristics and kinetic prop-
erties from other myocardial cytosolic phospholipase activities
previously described.
Ear ly experiments demonstrated that calcium-independent
phospholipase AZ was not present in serum or whole blood
and that comparable levels of calcium-independent phospho-
lipase A2 activ ity were present in perfused and nonperfused
hearts. However, comparisons of other calcium-independent
lipases (which are predominantly localized in plasma) to the
myocardial enzyme merit brief consideration. First, P AF ace-
tyl-hydrolase possessesdifferent chromatographic character-
istics (binds to DEAE-Sephacel resin at pH 6.8), thermal
stab ility (stable overnight at room temperature), detergent
sensitivity (measurable activity in Triton
X-100 or Tween-
20), and a substantially different pH optimum (pH 7.8) (45)
than the myocardial enzyme. Most importantly, PAF acetyl-
hydrolase is highly specific for hydrolysis of alkyl-ether cho-
line glycerophospholipids containing acetyl groups at the
sn-
2 position (45). In contrast, myocardial phospholipase A2
hydrolyzes alkyl-ether choline glycerophospholipids with long
chain
sn-2
aliphatic constituents three orders of magnitude
more rapidly than PAF. Second, phospholipase activity me-
Hazen, S. L., and Gross, R. W., unpublished observation.
diated by 1ecithin:cholesterol acyltransferase is distinguished
from myocardial phospholipase AZsince cholesterol acyltrans-
ferase is catalyzed by a 68-kDa polypeptide, requires a serum
protein cofactor for expression of phospholipase activ ity (in
its pure form), and exhibits no strict regiospecificity for
phospholipid hydrolysis (46, 47). Third, endothelial cell-de-
rived lipoprotein lipase is easily distinguished from myocar-
dial phospholipase A2 since myocardial lipoprotein lipase is a
34-kDa polypeptide, avidly binds to Heparin-Sepharose resin
(unlike myocardial cytosolic phospholipase AZ), and tolerates
acetone precipitation as well as homogenization in detergents
(48), both of which complete ly ablate myocardial phospholi-
pase AZ activity. Fourth, plasma carboxylesterase possessesa
different substrate selec tivity, thermal stability profile, and
molecular weight than myocardial phospholipase AP (49).
Finally, cholesterol esterase has a different substrate specific-
ity, a larger molecular mass (68 kDa), and has an absolute
requirement for cofactors for lipo lysis (29). Taken together,
these results demonstrate that the cytoso lic calcium- inde-
pendent myocardial phospholipase AS purified in this report
has physical and kinetic characteristics which discriminate it
from other calcium-independent lipase activities previously
studied.
We have recent ly demonstrated that myocardial sa rco-
lemma (the electrophysiologically active membrane in myo-
cytes) is the primary target of accelerated phospholipid hy-
drolysis in myocytes subjected to simulated ischemia (14) and
that myocardial sarcolemma is predominantly comprised of
plasmenylcholine and plasmenylethanolamine molecular spe-
cies which are highly enriched in a rachidonic acid (12). Since
the myocardial phospholipase AP purified herein has direct
physical access o the sarcolemmal membrane and selectively
hydrolyzes both plasmalogen substrate and arachidonylated
glycerophospholipids, this phospholipase has the catalytic
potential to selective ly hydro lyze the predominant phospho-
lipid constituents present in myocardial sarcolemma (i.e. ar-
achidonylated plasmalogens). Accordingly, activation of this
polypeptide is anticipated to result in the selective release of
arachidonic acid and the catabolism of sarcolemmal mem-
brane phospholipids simi lar to that seen during myocardia l
ischemia (4, 14). Although regulation of intracellular phos-
pholipases activated by physiologic increments in calcium ion
is now accepted (e.g. Refs. 50 and 51), the biochemical mech-
anisms responsible for regulation of calcium-independent
phospholipases AZ are unknown. Accordingly , future efforts
directed toward identification of the molecular mechanisms
responsible for the activation of this calcium-independent
phospholipase AZ should provide direct insight into the bio-
chemical mechanisms precipitating electrophysiologic dys-
function during myocardial ischemia.
1.
2.
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8.
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10.
REFERENCES
Katz, A. M., and Messineo, F. C. (1981) Circ. Res. 48, l-16
Gross, R. W., and Sobe l, B. E. (1983) J. Biol. Ch em. 258, 5221-
5226
Corr, P. B., Gross, R. W., and Sobel, B. E. (1984) Circ. Res. 55,
135-154
Chien, K. R., Han. A., Sen, A., Buia. L. M.. and Willerson. J. T.
(1984) Circ. Res. 54, 313-322
Scherrer. L. A.. and Gross. R. W. (1989) Mol. Cell. Biochem. 88.
97-1oi
Shaikh, N. A., and Downar, E. (1981) Circ. Res. 49, 316-325
Corr, P. B., Snyder, D. W., Lee, B. I., Gross, R. W., Keim, C. R.,
and Sobel, B. E. (1982) Am. J. Physiol. 12, H187-H195
van der Vusse, G. J., Roemen, T. H. M., Prinzen, F. W., Coumans.
W. A., and Reneman, R. S. (1982) Circ. Res. 50,538~54 6
Fink, K. L., and Gross, R. W. (1984) Circ. Res. 55, 585-594
Corr, P. B., Snyder, D. W., Cain, M. E., Crafford, W. A., Jr.,
Gross, R. W., and Sob el, B. E. (1981) Circ. Rex 49 , 354-363
bygu
estonOctober2,2014
http://www.jbc.org/
Downloadedfrom
http://www.jbc.org/http://www.jbc.org/http://www.jbc.org/http://www.jbc.org/http://www.jbc.org/http://www.jbc.org/http://www.jbc.org/http://www.jbc.org/http://www.jbc.org/http://www.jbc.org/http://www.jbc.org/http://www.jbc.org/http://www.jbc.org/http://www.jbc.org/http://www.jbc.org/http://www.jbc.org/http://www.jbc.org/http://www.jbc.org/http://www.jbc.org/http://www.jbc.org/http://www.jbc.org/ -
8/11/2019 Isolation of CA Independent Cytosolic PLA2
10/10
10630 Myocard ial Cytosolic Calcium-independent Phospholipase A2
11.
12.
13.
14.
15.
Gross, R. W., Corr, P. B., Lee, B. I., Saffitz, J. E., Crafford, W.
A., Jr., and Sobel, B. E. (1982) Circ. Res. 51,27-36
Gross, R. W. (1984) Biochemistry 23, 158-165
Gross, R. W. (1985) Biochemisrry 24, 1662-1668
Miyazaki, Y., Gross, R. W., Sobel, B. E., and Saffitz, J. E. (1990)
Am. J. Physiol., in press
Wolf, R. A., and Gross, R. W. (1985) J. Biol. Chem. 260, 7295-
7303
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
Creer, M. H., and Gross, R. W. (1985) J. Chromutogr. 338, 61-
69
Wolf, R. A., and Gross, R. W. (1985) J. Lipid Res. 26,629-633
Gross, R. W., and S obel, B. E. (1980) J. Chromatogr.
197, 79-85
Gross, R. W., and Sobel , B. E. (1982) J. Biol. Che m. 257, 6702-
6708
Gross, R. W., Ahumada, G. G., and Sobel, B. E. (1984) Am. J.
Physiol. 246, C266-C270
Gross, R. W. (1983) Biochemistry 22,5641-5646
Gatt, S., Dinur, T., and Kopolovic, J. (1978) J. Neurochem.
31,
547-550
Majerus, P. W., and Prescott, S. M. (1982) Methods Enzymol.
86,11-17
Billah , M. M., Lapetina, E. G., and Cuatrecasas, P. (1981) J. Biol.
Chem.
256,5399-5403
Kates. M. (1986) in Techniaues of LiDidohxv: Isolation. Analvsis
1 I . 1 I
and Zdentificakon of Lipids. (Burdon, R. H., and van Knippen-
berg, P. H., eds) p. 327, Elsevier Science Publishers, New York
26.
Blank, M. L., Snyder, F., Byers, L. W., Brooks, B., and Muirhead,
E. E. (1979) Biochem. BioDhvs. Res. Commun. 90. 1194-1200
27.
Mueller, H. W., OFlaherty, J. T., and Wykle, R. .L. (1983) J.
Biol. Chem. 258,6213-6218
28.
Potter, L. T. (1967) J. Pharmacol. Exp. Zher.
156, 500-506
29. Hvun. J.. Steinberg. M.. Treadwell. C. R.. and Vahoune . G. V.
30.
31.
i19k) kochem. kophys. Res. Cokmun. a4, 819-825 -
Breckenridge, W. C., and Kuksis, A. (1968) Lipids 3,291-300
Rider, L. G., Dougherty, R. W., and Niedel, J. E. (1988) J.
Zmmunol.
140,200-207
32. Bolton, A. E., and Hunter, W. M. (1973) Biochem. J.
133, 529-
539
33. Laemm li. U. K. (1970) Nature 227, 680-685
34. Stuppy, R. J., and Gross, R. W. (1987) Circulation 76, 4-11 339
35. Barden. R. E.. Darke. P. L.. Deems. R. A.. and Dennis, E. A.
(1986) BiochemistrylS,
1621-1625
36.
Pak, J. H., Bork, V. P., Norberg, R. E., Creer, M. H., Wolf, R.
A., and Gross, R. W. (1987) Biochem istry 26,4824-4830
37.
Constantinides, P. P., and Steim, J. M. (1985) J. Biol. Chem.
260,7573-7580
38.
Weltzien, H. U. (1979) Biochim. Biophys. Acta 559, 259-287
39. Kawasaki, N., Sugata ni, J., and Saito, K. (1975) J. Biochem.
(Tokyo) 77,1233-1241
40. Gassama-Diagne, A., Fauvel, J., and Chap, H. (1989) J. Biol.
Chem. 264,9470-9475
41. Nalbone, G., Hostetler, K. Y., Leonardi, J., Trotz, M., and Lafont,
H. (1986) Biochim. Biophys. Acta 877, 8 8-95
42. Cao. Y.. Tam. S. W.. Arthur. G.. Chen, H.. and Chov. P. C. (1987)
J.Bik. Chem. 26b, 16927-16935 -
43. Gross, R. W., Drisdel, R. C., and Sob el, B. E. (1983) J. Biol.
Chem.258,15165-15172
44.
Gross, R. W., and Sobel, B. E. (1982) J. Biol. Chem. 257,6702-
6708
45.
Stafforini, D. M., Prescott, S. M., and McIntyre, T. M. (1987) J.
Biol. Chem. 262,4223-4230
46. Jauhiainen, M., and Dolphin, P. J. (1986) J. Biol. Chem. 261,
7032-7043
47.
Aron, L., Jones, S., and Fielding , C. J. (1978) J. Biol. Chem. 253,
7220-7226
48.
Chung, J., and Scanu, A. M. (1977) J. Biol. Chem. 252, 4202-
4209
49. Shirai, K., Ohsawa, I., Ishikawa, Y., Saito, Y ., and Yoshida, S.
(1985) J. Biol. Chem. 260. 5225-5227
50. Loeb, L. A., and Gross, R. W. (1986) J. Biol. Chem.
261,10467-
10470
51.
Leslie, C. C., Voelker, D. R., Channon, J. Y., Wall, M. M., and
Zelarney, P. T. (1988) Biochim. Biophys. Acta 963,476-492
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