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Hydrolysis and Transesterification of Platelet-Activating Factor by Lecithin

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Hydrolysis and Transesterification of Platelet-Activating Factor by Lecithin Proc. Natd. Acad. Sci. USA Vol. 91, pp. 6035-6039, June 1994 Biochemistry Hydrolysis and transesterification of platelet-activating factor by lecithin-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 shown to be distinct from LCAT activity (10, 11), no studies (LCAT, EC 2.3.1.43) from human plaa was found to have been conducted to determine whether LCAT itself can hydrolyze platlet-activating factor (PAF) to Iyso-PAF and hydrolyze PAF. We have investigated the possible role of acetate. In addition, it catalyzed the transer of the acetate LCAT in the hydrolysis of PAF in plasma. Our results show group from PAF to lysophosphatidyicholine, fr i lyso-PAF that LCAT not only can hydrolyze PAF to lyso-PAFbut also and a 1-acyl analog of PAF. In contrast to the cholesterol- can transfer the acetyl group from PAF to lyso-PC, forming esterication reaction carried out by the enzyme, the hydrolysis a 1-acyl analog ofPAF. These results thus indicate a function and t etlation of PAF by LCAT did not require an for LCAT in the metabolism of PAF and identify a pathway apoprotein activator and were not inhibited by suhydryl for the inactivation of PAF in plasma. inhibitors but were inhibited by serum albumin. When added to a proteoliposome substrate of LCAT or to whole plasma, PAF inhibited Cholesterol esterification by LCAT competi- MATERIALS AND METHODS tively. PAF acetylhydrolase (EC 3.1.1.47), purified from hu- Materials. 1-[1-14C]Palmitoyl lyso-PC, [4-14C]cholesterol, man plasma, also catalyzed the transfer ofacetate from PAF to and 2-[3H]acetyl PAF were purchased from DuPont/NEN. lysophosphatidylcholine. However, the LCAT-catalyzed reac- Egg PC, unlabeled lyso-PC, and PAF were obtained from tions of PAF were not due to ontnation with PAF acetyl- Avanti Polar Lipids. Diisopropyl fluorophosphate and 5,5'- hydrolase, since the ratio of acetyl iraer to acetyl hydrolysis dithiobis(nitrobenzoic acid) (DTNB) were purchased from was 3 times greater for LCAT, when compared with PAF Sigma. acetylhydrolase under identical conditions. Furthermore, re- LCAT and apolipoprotein (apo) A-I were purified from combinant human LCAT secreted by baby hamser kidney cells normal human plasma (12, 13). The final preparations gave also catalyzed the hydrolysis and t ylation of PAF. single bands in SDS/polyacrylamide gels, corresponding to a These results demonstrate that LCAT can inactivate PAF in molecular weight of 67 kDa (for LCAT) or 28 kDa (for apo plasma by tracetlatio and suggest that it may have a role A-I). PAF-AH was purified from human plasma by the in the meta of PAF, and possibly of oxidized phospho- procedure of Stafforini et al. (10) up to the DEAE-Sepharose lipids, in plasa. column step. The enzyme was purified 1220-fold from the starting plasma and showed no LCAT activity when assayed Platelet-activating factor (PAF, 1-O-alkyl-2-acetyl-sn-glycer- with standard proteoliposome substrate (14). The final ol-3-phosphocholine) is a highly bioactive phospholipid pre- PAF-AH preparation hydrolyzed 39.8 pmol of PAF per hr sent in various tissues and plasma and has a broad range of per mg of protein. Recombinant human LCAT was a gener- biological effects, including stimulation of platelet aggrega- ous gift from P. H. Pritchard (University of British Colum- tion, neutrophil activation, smooth muscle contraction, vas- bia). This enzyme was secreted by stably transfected baby cular permeability, and hepatic glycogenolysis (1, 2). The hamster kidney (BHK) cells into serum-free medium and was half-life of PAF in plasma is very short (6-10 min), probably purified by chromatography on a phenyl-Sepharose column because it is rapidly hydrolyzed to lyso-PAF by a lipoprotein- (15). It had a specific activity of 9.3 nmol of cholesterol associated enzyme called PAF acetylhydrolase (PAF-AH; esterified per hr per ug of protein, in the proteoliposome EC 3.1.1.47). The activity of this enzyme is increased in assay (14). essential hypertension (3, 4) and other pathological condi- Enzyme Assays. PAF hydrolytic activity was determined tions (2). PAF-AH has also been shown to hydrolyze short- by the release of labeled acetate from labeled PAF (10). The chain analogs of phosphatidylcholine (PC) generated during reaction mixture (0.4 ml) contained 80 ,uM 3H-acetate-labeled the oxidation of lipoproteins (5). Another enzyme that can PAF (0.05 uCi; 1 pCi = 37 kBq) in 10 mM Tris'HCl (pH 7.4) potentially hydrolyze PAF as well as the oxidized phospho- and either purified LCAT (0.2-2 jug) or partially purified lipids in the plasma is lecithin-cholesterol acyltransferase PAF-AH (0.04-0.1 ptg). After incubation at 370C, the reaction (LCAT; PC-sterol O-acyltransferase, EC 2.3.1.43), whose mixture was extracted (16) and aliquots of the aqueous layer primary function is to synthesize cholesteryl esters by trans- and the chloroform layer were assayed in a liquid scintillation ferring a fatty acid from PC to a free cholesterol molecule (6). counter to determine the radioactivity in free acetate and This enzyme also exhibits phospholipase A activity in the unreacted PAF, respectively. The results obtained with this absence of an acyl acceptor (7) and can hydrolyze water- method corresponded closely to those obtained by the C18 soluble esters such as p-nitrophenyl esters of short-chain column method of Stafforini et al. (10). fatty acids (8). Earlier studies showed that LCAT can also The transfer of acetate from PAF to lyso-PC was deter- transfer an acyl group from PC to Iyso-PC, forming another mined in a reaction mixture (0.4 ml) which contained 1-[1- PC molecule (9). Although the PAF-AH activity has been Abbreviations: apo, apoplipoprotein; DTNB, 5,5'-dithiobis(ni- trobenzoic acid); LAT, lysolecithin acyltransferase; LCAT, lecithin- The publication costs ofthis article were defrayed in part by page charge cholesterol acyltransferase; PAF, platelet-activating factor; PAF- payment. This article must therefore be hereby marked "advertisement" AH, PAF acetylhydrolase; PC, phosphatidylcholine. in accordance with 18 U.S.C. §1734 solely to indicate this fact. *To whom reprint requests should be addressed. 6035 Downloaded by guest on October 3, 2021 6036 Biochemistry: Liu and Subbaiah Proc. Natl. Acad. Sci. USA 91 (1994) 14C]palmitoyl lyso-PC (12 nmol), 32 nmol of unlabeled PAF, fully activate cholesterol esterification by LCAT (22 pg/0.4 and 10 mM Tris HCl (pH 7.4). Where indicated, 22 pg ofapo ml) had no effect on PAF hydrolysis by the enzyme. Human A-I or 4 mg of human serum albumin was added. After serum albumin, another known activator of cholesterol es- incubation with purified LCAT or PAF-AH for various times terification, in contrast, inhibited the hydrolysis of PAF, at 370C, the reaction was stopped by the addition of 1 ml of presumably because PAF binds to albumin (17). These results methanol, and the lipids were extracted (16). Lyso-PC and show that LCAT can hydrolyze PAF to lyso-PAF and acetate PAF were separated on silica-gel TLC plates with the solvent and that this activity of LCAT does not require apoprotein system chloroform/methanol/water, 65:25:4 (vol/vol), and activators. their radioactivity was determined in a liquid scintillation Transfer of Acetate from PAF to Lyso-PC and Free Choles- counter. The labeled short-chain PC formed by the acetyla- terol by LCAT. To determine whether LCAT could transfer tion of labeled lyso-PC migrated with authentic PAF (Rf the acetyl group from PAF to free cholesterol or lyso-PC, we 0.089), which moved below normal PC (Rf0.189) but above incubated unlabeled PAF (80 pM) with purified LCAT and lyso-PC (Rf 0.053) in this solvent system. This reaction was either labeled lyso-PC or labeled cholesterol. There was termed as LAT-il to distinguish it from the formation of formation of labeled short-chain PC (LAT-Il activity) (Fig. labeled long-chain PC, which was called simply LAT (lyso- 2), but not cholesteryl ester (results not shown). The LAT-II lecithin acyltransferase). reaction was not activated by the presence ofapo A-I but was The assays of LCAT (cholesterol esterification) and LAT inhibited by albumin. The inhibition of transacetylation by (long-chain PC formation) activities were performed with a albumin was greater than that observed for the hydrolysis of proteoliposome substrate containing egg PC, 14C-labeled free PAF, probably because albumin sequesters both PAF and cholesterol, 14C-labeled lyso-PC, and apo A-I at molar ratios lyso-PC. There was no formation of labeled short-chain PC of 250:12.5:12.5:0.8 (13, 14). After incubation with the en- when labeled lyso-PC was incubated with the enzyme and zyme in the presence of5 mM 2-mercaptoethanol for 30 min, 80-320 mM sodium acetate instead of PAF (results not the reactions were stopped by the addition of methanol and shown). These results show that LCAT can transfer the the lipids were extracted (16). Aliquots of the lipid extracts acetyl group from PAF to lyso-PC, but not to cholesterol, were spotted on two separate silica-gel TLC plates. One plate under the conditions of the assay. was developed in chloroform/methanol/water, 65:25:4 (vol/ Effect ofPAF on LCAT and LAT Reactions Carried Out by vol), to separate lyso-PC and PC, and the second plate was LCAT Enzyme. If PAF binds to LCAT protein at the same developed in hexane/diethyl ether/acetic acid, 70:30:1 (vol/ site as PC, then it should inhibit the LCAT and LAT reactions vol), to separate free cholesterol and cholesteryl ester.
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