Proc. Natd. Acad. Sci. USAVol. 91, pp. 6035-6039, June 1994Biochemistry

Hydrolysis and transesterification of platelet-activating factor bylecithin-cholesterol acyltransferase

ysopbosatdyl coe/ l /W d perodaton/phosphoipase A)

MING LiU AND PAPASANI V. SUBBAIAH*Departments of Medicine and Biochemistry, Rush Medical College, Chicago, IL 60612

Communicated by John A. Glomset, March 14, 1994

AMBSRACT Purified lecithin-cholesterol acyltrnsferase(LCAT, EC 2.3.1.43) from human plaa was found tohydrolyze platlet-activating factor (PAF) to Iyso-PAF andacetate. In addition, it catalyzed the transer of the acetategroup from PAF to lysophosphatidyicholine, fr i lyso-PAFand a 1-acyl analog of PAF. In contrast to the cholesterol-esterication reaction carried out by the enzyme, the hydrolysisand t etlation of PAF by LCAT did not require anapoprotein activator and were not inhibited by suhydrylinhibitors but were inhibited by serum albumin. When addedto a proteoliposome substrate of LCAT or to whole plasma,PAF inhibited Cholesterol esterification by LCAT competi-tively. PAF acetylhydrolase (EC 3.1.1.47), purified from hu-man plasma, also catalyzed the transfer ofacetate from PAF tolysophosphatidylcholine. However, the LCAT-catalyzed reac-tions of PAF were not due to ontnation with PAF acetyl-hydrolase, since the ratio of acetyl iraer to acetyl hydrolysiswas 3 times greater for LCAT, when compared with PAFacetylhydrolase under identical conditions. Furthermore, re-combinanthuman LCAT secreted by baby hamser kidney cellsalso catalyzed the hydrolysis and t ylation of PAF.These results demonstrate that LCAT can inactivate PAF inplasma by tracetlatio and suggest that it may have a rolein the meta of PAF, and possibly of oxidized phospho-lipids, in plasa.

Platelet-activating factor (PAF, 1-O-alkyl-2-acetyl-sn-glycer-ol-3-phosphocholine) is a highly bioactive phospholipid pre-sent in various tissues and plasma and has a broad range ofbiological effects, including stimulation of platelet aggrega-tion, neutrophil activation, smooth muscle contraction, vas-cular permeability, and hepatic glycogenolysis (1, 2). Thehalf-life ofPAF in plasma is very short (6-10 min), probablybecause it is rapidly hydrolyzed to lyso-PAF by a lipoprotein-associated enzyme called PAF acetylhydrolase (PAF-AH;EC 3.1.1.47). The activity of this enzyme is increased inessential hypertension (3, 4) and other pathological condi-tions (2). PAF-AH has also been shown to hydrolyze short-chain analogs of phosphatidylcholine (PC) generated duringthe oxidation of lipoproteins (5). Another enzyme that canpotentially hydrolyze PAF as well as the oxidized phospho-lipids in the plasma is lecithin-cholesterol acyltransferase(LCAT; PC-sterol O-acyltransferase, EC 2.3.1.43), whoseprimary function is to synthesize cholesteryl esters by trans-ferring a fatty acid from PC to a free cholesterol molecule (6).This enzyme also exhibits phospholipase A activity in theabsence of an acyl acceptor (7) and can hydrolyze water-soluble esters such as p-nitrophenyl esters of short-chainfatty acids (8). Earlier studies showed that LCAT can alsotransfer an acyl group from PC to Iyso-PC, forming anotherPC molecule (9). Although the PAF-AH activity has been

shown to be distinct from LCAT activity (10, 11), no studieshave been conducted to determine whether LCAT itself canhydrolyze PAF. We have investigated the possible role ofLCAT in the hydrolysis of PAF in plasma. Our results showthat LCAT not only can hydrolyze PAF to lyso-PAF but alsocan transfer the acetyl group from PAF to lyso-PC, forminga 1-acyl analog ofPAF. These results thus indicate a functionfor LCAT in the metabolism ofPAF and identify a pathwayfor the inactivation of PAF in plasma.

MATERIALS AND METHODSMaterials. 1-[1-14C]Palmitoyl lyso-PC, [4-14C]cholesterol,

and 2-[3H]acetyl PAF were purchased from DuPont/NEN.Egg PC, unlabeled lyso-PC, and PAF were obtained fromAvanti Polar Lipids. Diisopropyl fluorophosphate and 5,5'-dithiobis(nitrobenzoic acid) (DTNB) were purchased fromSigma.LCAT and apolipoprotein (apo) A-I were purified from

normal human plasma (12, 13). The final preparations gavesingle bands in SDS/polyacrylamide gels, corresponding to amolecular weight of 67 kDa (for LCAT) or 28 kDa (for apoA-I). PAF-AH was purified from human plasma by theprocedure of Stafforini et al. (10) up to the DEAE-Sepharosecolumn step. The enzyme was purified 1220-fold from thestarting plasma and showed no LCAT activity when assayedwith standard proteoliposome substrate (14). The finalPAF-AH preparation hydrolyzed 39.8 pmol of PAF per hrper mg of protein. Recombinant human LCAT was a gener-ous gift from P. H. Pritchard (University of British Colum-bia). This enzyme was secreted by stably transfected babyhamster kidney (BHK) cells into serum-free medium and waspurified by chromatography on a phenyl-Sepharose column(15). It had a specific activity of 9.3 nmol of cholesterolesterified per hr per ug of protein, in the proteoliposomeassay (14).Enzyme Assays. PAF hydrolytic activity was determined

by the release of labeled acetate from labeled PAF (10). Thereaction mixture (0.4 ml) contained 80 ,uM 3H-acetate-labeledPAF (0.05 uCi; 1 pCi = 37 kBq) in 10 mM Tris'HCl (pH 7.4)and either purified LCAT (0.2-2 jug) or partially purifiedPAF-AH (0.04-0.1 ptg). After incubation at 370C, the reactionmixture was extracted (16) and aliquots of the aqueous layerand the chloroform layer were assayed in a liquid scintillationcounter to determine the radioactivity in free acetate andunreacted PAF, respectively. The results obtained with thismethod corresponded closely to those obtained by the C18column method of Stafforini et al. (10).The transfer of acetate from PAF to lyso-PC was deter-

mined in a reaction mixture (0.4 ml) which contained 1-[1-

Abbreviations: apo, apoplipoprotein; DTNB, 5,5'-dithiobis(ni-trobenzoic acid); LAT, lysolecithin acyltransferase; LCAT, lecithin-cholesterol acyltransferase; PAF, platelet-activating factor; PAF-AH, PAF acetylhydrolase; PC, phosphatidylcholine.*To whom reprint requests should be addressed.

6035

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Dow

nloa

ded

by g

uest

on

Apr

il 2,

202

1

6036 Biochemistry: Liu and Subbaiah

14C]palmitoyl lyso-PC (12 nmol), 32 nmol of unlabeled PAF,and 10mM Tris HCl (pH 7.4). Where indicated, 22 pg of apoA-I or 4 mg of human serum albumin was added. Afterincubation with purified LCAT or PAF-AH for various timesat 370C, the reaction was stopped by the addition of 1 ml ofmethanol, and the lipids were extracted (16). Lyso-PC andPAF were separated on silica-gel TLC plates with the solventsystem chloroform/methanol/water, 65:25:4 (vol/vol), andtheir radioactivity was determined in a liquid scintillationcounter. The labeled short-chain PC formed by the acetyla-tion of labeled lyso-PC migrated with authentic PAF (Rf0.089), which moved below normal PC (Rf 0.189) but abovelyso-PC (Rf 0.053) in this solvent system. This reaction wastermed as LAT-il to distinguish it from the formation oflabeled long-chain PC, which was called simply LAT (lyso-lecithin acyltransferase).The assays of LCAT (cholesterol esterification) and LAT

(long-chain PC formation) activities were performed with aproteoliposome substrate containing egg PC, 14C-labeled freecholesterol, 14C-labeled lyso-PC, and apo A-I at molar ratiosof 250:12.5:12.5:0.8 (13, 14). After incubation with the en-zyme in the presence of 5 mM 2-mercaptoethanol for 30 min,the reactions were stopped by the addition of methanol andthe lipids were extracted (16). Aliquots of the lipid extractswere spotted on two separate silica-gel TLC plates. One platewas developed in chloroform/methanol/water, 65:25:4 (vol/vol), to separate lyso-PC and PC, and the second plate wasdeveloped in hexane/diethyl ether/acetic acid, 70:30:1 (vol/vol), to separate free cholesterol and cholesteryl ester. Theradioactivity was determined in each of the spots and thepercent esterification of free cholesterol or lyso-PC wasdetermined. When PAF was included in the reaction mixture,the acetylation of lyso-PC (formation of labeled short-chainPC) was also determined. Unless otherwise mentioned, allthe values presented are averages of at least two experi-ments, each performed in duplicate. Variation between ex-periments was

Proc. Nail. Acad. Sci. USA 91 (1994) 6037

2.0

o 1.5 LCAT .- LAT-TI

1.0

0.5

/ LAT0.0

0 20 40 60 80 100 120

PAF (pM)

FiG. 3. Effect of PAF on LCAT and LAT reactions. Variousamounts of unlabeled PAF were added to a reaction mixture con-taining 2 eg of purified LCAT, 5 mM 2-mercaptoethanol, and aproteoliposome substrate containing egg PC, 14C-labeled lyso-PC,14C-labeled cholesterol, and apo A-I at molar ratios of250:12.5:12.5:0.8. The formation of labeled cholesteryl ester (LCATactivity), long-chain PC (LAT activity), and short-chain PC (LAT-ilactivity) was determined as described in the text.

of cholesteryl acetate, showing again that the acetyl group istransferred to lyso-PC, but not to cholesterol.To determine whether PAF could compete with PC in

intact plasma, we repeated the above experiment with freshlyprepared normal human plasma which has been prelabeledwith 14C-labeled cholesterol and "4C-labeled lyso-PC. Herealso we found an inhibition ofboth LCAT and LAT reactionswith simultaneous stimulation of the LAT-II reaction in thepresence of increasing amounts of PAF (Fig. 4). However,compared with the proteoliposome system, the concentrationof PAF required to inhibit LCAT activity was considerablyhigher, probably because of the presence of albumin andother factors in plasma which dampen PAF effects.

Hydrolysis and Transesterfication of PAF by RecombinantLCAT. Since human plasma contains a highly active PAF-AH, it was important to exclude the possibility that the

._4

04-1. 4

.. i

5.0

4.0

3.0

2.0

1.0

0.00 100 200 300 400 500 600 700

PAF (pM)

FIG. 4. Effect of PAF on LCAT and LAT reactions in wholeplasma. Freshly prepared human plasma was labeled with traceamounts of [14C]cholesterol and [14C]lyso-PC by overnight incuba-tion at 40C. Unlabeled PAF was added at the indicated concentra-tions and the mixture was incubated for 30 min at37C in the presenceof 5 mM 2-mercaptoethanol. Labeled cholesteryl ester and choles-terol were separated by TLC ofan aliquot ofthe lipid extract (solventsystem of hexane/diethyl ether/acetic acid, 70:30:1) and their ra-dioactivities were determined, to measure LCAT activity. Anotheraliquot of lipid extract was subjected to chromatography in chloro-form/methanol/water, 65:25:4, and the radioactivity in short-chainPC (LAT-II activity) and long-chain PC (LAT activity) was deter-mined.

activity observed with purified LCAT was due to contami-nation with small amounts of PAF-AH. For this purpose weobtained a recombinant LCAT secreted into serum-freemedium by BHK cells which have been stably transfectedwith human LCAT cDNA (15) and tested its ability tometabolize PAF. The recombinant human LCAT was able tohydrolyze PAF as well as transfer the acetate to lyso-PC (Fig.5). Similar results were obtained with a recombinant LCATsecreted by transiently transfected COS-1 cells (results notshown). In both cases, there was no activity in the mediumfrom control cells which had been transfected with "empty"expression vector (pNUT).Comparison of PAF-AH and LCAT Activities. To further

differentiate between the activities of PAF-AH and LCAT,we partially purified PAF-AH from human plasma and testedwhether it could transfer acetate from PAF to lyso-PC.PAF-AH was observed to transfer acetate from PAF tolyso-PC, in addition to releasing free acetate (Fig. 6). How-ever, it did not show any cholesterol-esterifying activity inthe presence of standard proteoliposome substrate (14) (re-sults not presented), showing that it was not contaminatedwith LCAT. The kinetic constants of purified LCAT andPAF-AH were determined in the presence of various con-centrations ofPAP. The apparent Km values calculated fromthe Lineweaver-Burk plots were 17.9 AM for PAF-AH and12.8 pM for LCAT. The V. value for pure LCAT was 0.2janol of PAF hydrolyzed per hr per mg, while for thePAF-AH preparation it was 41 umol per hr per mg. Incomparison, the cholesterol esterification rate by LCATunder optimal conditions was 12 pmol of cholesterol esteri-fied per hr per mg.The LCAT and LAT activities were inhibited 80-90%6 by

0.5 mM DTNB (Fig. 7). However, the inhibition of LAT-IIactivity was much lower even at higher DTNB concentra-tions. Therefore the sulflhydryl groups do not appear to beessential for the hydrolysis or transfer of acetate from PAF.The transfer and hydrolytic reactions of PAF-AH were notinhibited by DTNB, as reported by others (5, 17). Diisopropylfluorophosphate (1 mM) inhibited all the reactions of LCAT,although LAT-il appeared to be less sensitive than other

0.25

0.20 -0- Transfer'0~~~~~~~/x-- Hydrolysis / .,0.15

o 0o 0.100~~~~~1

0.05

0.00'0 30 60 90 120 150

Time (min)

FIG. 5. Hydrolysis and tranacetylation of PAF by recombinantLCAT. Recombinant human LCAT, expressed by transfected BHKcells (15), was obtained from P. H. Pritchard. The hydrolysis ofPAFwas determined with 80 AM [3H]acetate-labeled PAF, whereas thetransfer of acetate from PAF was determined in the presence of 80pM unlabeled PAF and 30 pM 1-[1-14C]palmitoyl lyso-PC. Bothreaction mixtures contained 5mM 2-mercaptoethanol and 0.22 jg ofpurified recombinant LCAT. The values shown are averages ofduplicate samples from one typical experiment. Similar results wereobtained with medium from transiently transfected COS-1 cells.Medium from control cells which had been transfected with vectorwithout cDNA insert did not show any enzyme activity (results notshown).

Biochemistry: Liu and Subbaiah

Dow

nloa

ded

by g

uest

on

Apr

il 2,

202

1

6038 Biochemistry: Liu and Subbaiah

5.0

10

4)

U-

4)

0

4.0

3.0

2.0

1.0

0.0O0 20 40 60 80 100

Time (min)

FiG. 6. Hydrolysis and transacetylation of PAF by PAF-AH.Hydrolysis was determined by the release of labeled acetate from[3H]acetate-labeled PAF (80 AM), and tranacetylation was measuredby the formation of labeled short chain-PC in the presence of1-[1-14C]palmitoyl lyso-PC and unlabeled PAF. All reaction mixturescontained 0.94 p&g of partially purified PAF-AH. Values shown areaverages of duplicate samples from a typical experiment.

reactions (results not shown). Diisopropyl fluorophosphatealso inhibited the PAF-AH activities, in agreement withprevious studies (5, 19).To compare the relative activities of acetyl transfer and

acetyl hydrolysis by LCAT and PAF-AH, we studied the tworeactions of each enzyme by employing identical amounts ofenzyme and PAF for both reactions. As expected, the ratioof acetyl transfer to acetyl hydrolysis increased for bothenzymes with increasing, concentrations of lyso-PC (Fig. 8).However, the ratio was about 3-fold higher forLCAT than forPAF-AH at all'lyso-PC concentrations. The ratio obtained forrecombinant LCAT was similar to that of purified plasmaLCAT (results not shown). These data indicate that it isunlikely that the transferase and hydrolase activities ofLCAT are due to contamination with PAF-AH.

DISCUSSIONThe results provide evidence for a function of LCAT in themetabolism ofPAF. Although the major function ofLCAT in

120

0 100'I-

a

a 800

t 60

.* 40

...

0 20

0

0.0 0.5 1.0 1.5 2.0 2.5 3.0

DTNB (mM)

FiG. 7. Effect of DTNB on the reactions carried out by LCATand PAF-AH. Various amounts of DTNB were added to reactionmixtures containing either 80 pM [3H]acetate-labeled PAF alone or80 pM unlabeled PAF and 30 pM labeled lyso-PC. Each reactionmixture contained either 1.5 pig of LCAT or 0.05 pg of PA-AH. a,Cholesterol esterification by LCAT; o, LAT reaction of LCAT(formation of long-chain PC); ,, LAT-I1 reaction by LCAT (forma-tion of short-chain PC); , hydrolysis of PAF by PAF-AH; A,formation of short-chain PC by PAF-AH.

2.00Cu*._94 1.6-4

~0.

0: 1.2

4)

.,

>b

Z 0.8

n 0.40.

0.00 10 20 30 40 50 60

LPC (pM)

FIG. 8. Ratio of acetyl transfer to acetyl hydrolysis reactions byLCAT and PAF-AH. Acetyl transfer (LAT-il reaction) was deter-mined in the presence of 15 IAM unlabeled PAF and the indicatedamounts of labeled lyso-PC (LPC), either with 1.5 pg of LCAT orwith 0.06 pg ofPAF-AH. Hydrolysis ofPAF was determined underidentical conditions, except that acetate-labeled PAF and unlabeledlyso-PC were employed.

plasma is to catalyze the esterification of free cholesterol, itis essentially a specialized phospholipase A which utilizescholesterol, instead of water, as the acyl acceptor. Theenzyme in fact exhibits phospholipase A activity in theabsence of an acyl acceptor (7). It has also been shown tohydrolyze short-chain acyl esters in vitro, and this reaction isneither apo A-I-dependent nor inhibited by sulflhydryl inhib-itors (8). The hydrolysis ofPAF by LCAT is therefore similarto its hydrolysis of other water-soluble esters, because it isalso not dependent upon the apoprotein activator and is notinhibited by DTNB. The apoprotein activatoris required onlyfor the interfacial activation of the enzyme, and DTNBinhibits the hydrolysis of only long-chainfatty acids from PC(8), presumably by steric interference at the active site (20).The'involvement of the same active site for cholesterolesterification and PAF hydrolysis is indicated because PAFinhibits cholesterol esterification competitively and PAFhydrolysis is inhibited by diisopropyl fluorophosphate. Al-though we have not been able to selectively inhibit PAF-AHactivity, it appears very unlikely that the hydrolysis ofPAFby LCAT is due to a contamination with PAF-AH because ofthe following. (i) All the PAF-AH in plasma is associated withlow density and high density lipoproteins (10), whereas ourstarting material for LCAT purification was lipoprotein-freeplasma (13). (ii) The purified LCAT gave a single proteinband at 67 kDa in SDS/polyacrylamide gels, whereasPAF-AH has a molecular mass of 43 kDa (10). (iii) Recom-binant LCAT secreted by two different cell lines also carriedout PAF hydrolysis. (iv) The ratio of acetyl transfer to acetylhydrolysis by LCAT was much higher than the ratio obtainedfor PAF-AH.

In addition to the hydrolysis of PAF to lyso-PAF and freeacetate, LCAT can transfer the acetate from PAF to lyso-PC.While Malone et al. (21) have reported that the acetate fromPAF is not directly transferred to other lipids in platelets, ourdata show that such a transfer does occur in plasma and thatit is carried out by both LCAT and PAF-AH. Since there isno acetyl-CoA-generating mechanism in plasma, the forma-tion of labeled short-chain PC from labeled lyso-PC couldhave taken place only by the direct transfer of acetate fromPAF. Lee et al. (22) reported the presence ofa similarenzymeactivity in HL-60 human promyelocytic leukemia cells', withbroad specificity toward acyl acceptors. However, this en-zyme activity differs from LCAT activity, because it ismembrane-bound, is activated by phenylmethanesulfonylfluoride, and does not transfer long-chain acyl groups.

LCAT

- /, PAF-AH0 o lo o o

. ,B°~~~~~~~~~1

Proc. Nad. Acad Sci. USA 91 (1994)

Dow

nloa

ded

by g

uest

on

Apr

il 2,

202

1

Proc. Nat!. Acad. Sci. USA 91 (1994) 6039

The possible physiological importance of LCAT in PAFmetabolism can only be speculative at present. Although thein vitro activity of PAF-AH (as assayed under optimalconditions) is in large excess relative to the PAF concentra-tion in the plasma (1), in vivo this activity is about 60 timeslower than expected (1, 23). It appears to be increased incertain pathological conditions (2) and is regulated by estro-gen (11). It is therefore possible that the PAF-AH activity islimiting in vivo and that LCAT may have a supplementaryrole in the degradation of PAF, especially in high densitylipoprotein and in the lipoprotein-free fraction of plasma,which contain most of the PAF (17) and LCAT (24) but littlePAF-AH (17). It is of interest that in the presence of phys-iological concentrations of lyso-PC, LCAT carries out pre-dominantly the transacetylation of PAF, whereas PAF-AHcatalyzes the hydrolysis (Fig. 8). It therefore appears thatPAF is converted to lyso-PAF by two different pathways inthe plasma; one by PAF-AH, which releases free acetatefrom PAF, and the other by LCAT, which predominantlytransfers the acetate to a lyso-PC acceptor. Since LCAT isalso capable of esterifying lyso-PAF with a long-chain fattyacid (25), the complete inactivation cycle ofPAF (hydrolysisfollowed by reacylation), which occurs intracellularly inseveral tissues (1, 2), can also take place in the plasma (Fig.9). Our preliminary results (not shown) suggest that LCATcan transfer the acetate group from 1-palmitoyl 2-acetyl PCto lyso-PAF, thus regenerating PAF.Another possible function ofthe LCAT-mediated reactions

described here is in the metabolism ofoxidized phospholipidsin plasma. The oxidation ofphospholipids is one of the initialevents in the oxidative modification of low density lipopro-teins, which leads to rapid uptake oflow density lipoproteinsthrough the scavenger-receptor pathway and the formation offoam cells (26). There is strong evidence that PAF-AH canhydrolyze the oxidatively modified short-chain residues (upto six carbons) from PC (5, 27), and this might be part of arepair mechanism following the oxidative damage. However,the oxidative cleavage of linoleate (9, 12-octadecadienoate),the major unsaturated fatty acid in plasma, leads to theformation of 9- or 12-carbon residues at the sn-2 position ofPC, which cannot be hydrolyzed by PAF-AH (5). Similarly,the hydroxy and hydroperoxy derivatives of the long-chainfatty acids cannot be cleaved from PC by PAF-AH (27). Ourrecent studies have shown that purified LCAT can hydrolyzemost of the oxidatively modified PCs, including 1-palmitoyl2-linoleoyl PC (unpublished results). Similar phospholipaseA activity associated with low density lipoproteins wasreported by Parthasarathy and Barnett (28), who suggested

1-alkyl-2-acetyl GPCPAF 1-acyl-2-lyso GPC

LCATPAF-AH LCAT

PAF-AH

acetateIC1-alkyl-2-lyso GPC 1-acyl-2-acetyl GPC1-alkyl-2-Iyso GPO

1,2-diacyl GPC `

LCAT

1-acyl-2-lyso GPCO*

1-alkyl-2-acyl GPC

FIG. 9. Possible pathways of PAF metabolism in plasma. GPC,sn-u1vceronhosnhochoine.

that this activity was intrinsic to apo B. Our results show thatpurified LCAT not only can hydrolyze the oxidized PCs butalso can transfer the modified acyl group to a lyso-PCacceptor. Further, we found that oxidation of whole plasmaby Cu2+ in the presence of labeled lyso-PC resulted in theformation of labeled "modified PC" which was more polarthan the normal PC. At the same time, the oxidation resultedin an inhibition of esterification of cholesterol and lyso-PCwith normal fatty acids. These results suggest that LCATmay play a role in the metabolism of oxidized phospholipids.

This work was supported by a Grant-in-Aid from the AmericanHeart Association, with funds contributed in part by the IllinoisAffiliate, and by Grant HL50495 from the National Institutes ofHealth.

1. Prescott, S., Zimmerman, G. A. & McIntyre, T. M. (1990) J.Biol. Chem. 26S, 17381-17384.

2. Snyder, F. (1987) Platelet-Activating Factor andRelated LipidMediators (Plenum, New York).

3. Blank, M. L., Hall, M. N., Cress, E. A. & Snyder, F. (1983)Biochem. Biophys. Res. Commun. 113, 666-671.

4. Satoh, K., Imaizumi, T., Kawamura, Y., Yoshida, H., Taka-matsu, S. & Takamatsu, M. (1989) Prostaglandins 37, 67-3-682.

5. Steinbrecher, U. P. & Pritchard, P. H. (1989) J. Lipid Res. 30,305-315.

6. Glomset, J. A. (1968) J. Lipid Res. 9, 155-167.7. Aron, L., Jones, S. & Fielding, C. J. (1978) J. Biol. Chem. 253,

7220-7226.8. Bonelli, F. S. & Jonas, A. (1989) J. Biol. Chem. 264, 14723-

14728.9. Subbaiah, P. V., Albers, J. J., Chen, C. H. & Bagdade, J. D.

(1980) J. Biol. Chem. 255, 9275-9280.10. Stafforini, D. M., Prescott, S. M. & McIntyre, T. M. (1987) J.

Biol. Chem. 262, 4223-4230.11. Pritchard, P. H. (1987) Biochem. J. 246, 791-794.12. Liu, M., Krul, E. S. & Subbaiah, P. V. (1992) J. Biol. Chem.

267, 5139-5147.13. Liu, M. & Subbaiah, P. V. (1993) Biochim. Biophys. Acta 1168,

144-152.14. Chen, C. H. & Albers, J. J. (1982) J. Lipid Res. 23, 680-691.15. Hill, J. S., 0, K., Wang, X., Paranjape, S., Dimitrijevich, D.,

Lacko, A. G. & Pritchard, P. H. (1993) J. Lipid Res. 34,1245-1251.

16. Bligh, E. G. & Dyer, W. J. (1959) Can. J. Biochem. Physiol. 37,911-917.

17. Stafforini, D. M., McIntyre, T. M., Carter, M. E. & Prescott,S. M. (1987) J. Biol. Chem. 262, 4215-4222.

18. Subbaiah, P. V., Liu, M., Bolan, P. J. & Paltaiif, F. (1992)Biochim. Biophys. Acta 1128, 83-92.

19. Alam, I., Smith, J. B. & Silver, M. J. (1983) Lipids 18,534-538.20. Francone, 0. L. & Fielding, C. J. (1991) Proc. Nat!. Acad. Sci.

USA 88, 1716-1720.21. Malone, B., Lee, T.-C. & Snyder, F. (1985)J. Biol. Chem. 260,

1531-1534.22. Lee, T.-C., Uemura, Y. & Snyder, F. (1992)J. Biol. Chem. 267,

19992-20001.23. Stafforini, D. M., Carter, M. E., Zimmerman, G. A., MlcIn-

tyre, T. M. & Prescott, S. M. (1989) Proc. Nat!. Acad. Sci.USA 86, 2393-2397.

24. Dobiasova, M. (1983) Adv. Lipid Res. 20, 107-194.25. Subbaiah, P. V., Chen, C. H., Bagdade, J. D. & Albers, J. J.

(1985) J. Biol. Chem. 260, 5308-5314.26. Steinberg, D., Parthasarathy, S., Carew, T. E., Khoo, J. C. &

Witztum, J. L. (1989) N. Engl. J. Med. 320, 915-924.27. Stremler, K. E., Stafforini, D. M., Prescott, S. M. & McIn-

tyre, T. M. (1991) J. Biol. Chem. 266, 11095-11103.28. Parthasarathy, S. & Barnett, J. (1990) Proc. Nat!. Acad. Sci.

USA 87, 9741-9745.

Biochemistry: Liu and Subbaiah

Dow

nloa

ded

by g

uest

on

Apr

il 2,

202

1