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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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Myocardial C ytosolic Calcium-independent Phospholipase AZ

10625

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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Myocard ial Cytosolic Calcium-independent Phospholipase A2

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.

&parallel 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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Myocardial Cytosolic Calcium-independent Phospholipase A2

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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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.

3.

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5.

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7.

8.

9.

10.

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