Phospholipase-mediated preparation of 1-ricinoleoyl-2-acyl-sn-glycero-3-phosphocholine from soya and egg phosphatidylcholine

1-Ricinoleoyl-2-acyl-sn-glycero-3-phosphocholine was prepared by incorporating ricinoleic acid completely in the sn-1 position of egg and soya phosphatidylcholine (PC) using immobilized phospholipase A(1) as the catalyst. The optimum reaction conditions for maximum incorporation of ricinoleic acid into PC through transesterification were 10% (w/w) immobilized enzyme (116 mg), a 1:5 mol ratio of PC (soya, 387 mg; egg, 384 mg) to methyl ricinoleate (780 mg) at 50 degrees C for 24 h in hexane.     

Characterization of the transacylase activity of rat liver 60-kDa lysophospholipase-transacylase. Acyl transfer from the sn-2 to the sn-1 position.

Rat liver 60-kDa lysophospholipase-transacylase catalyzes not only the hydrolysis of 1-acyl-sn-glycero-3-phosphocholine, but also the transfer of its acyl chain to a second molecule of 1-acyl-sn-glycero-3-phosphocholine to form phosphatidylcholine (H. Sugimoto, S. Yamashita, J. Biol. Chem. 269 (1994) 6252-6258). Here we report the detailed characterization of the transacylase activity of the enzyme. The enzyme mediated three types of acyl transfer between donor and acceptor lipids, transferring acyl residues from: (1) the sn-1 to -1(3); (2) sn-1 to -2; and (3) sn-2 to -1 positions. In the sn-1 to -1(3) transfer, the sn-1 acyl residue of 1-acyl-sn-glycero-3-phosphocholine was transferred to the sn-1(3) positions of glycerol and 2-acyl-sn-glycerol, producing 1(3)-acyl-sn-glycerol and 1,2-diacyl-sn-glycerol, respectively. In the sn-1 to -2 transfer, the sn-1 acyl residue of 1-acyl-sn-glycero-3-phosphocholine was transferred to not only the sn-2 positions of 1-acyl-sn-glycero-3-phosphocholine, but also 1-acyl-sn-glycero-3-phosphoethanolamine, producing phosphatidylcholine and phosphatidylethanolamine, respectively. 1-Acyl-sn-glycero-3-phospho-myo-inositol and 1-acyl-sn-glycero-3-phosphoserine were much less effectively transacylated by the enzyme. In the sn-2 to -1 transfer, the sn-2 acyl residue of 2-acyl-sn-glycero-3-phosphocholine was transferred to the sn-1 position of 2-acyl-sn-glycero-3-phosphocholine and 2-acyl-sn-glycero-3-phosphoethanolamine, producing phosphatidylcholine and phosphatidylethanolamine, respectively. Consistently, the enzyme hydrolyzed the sn-2 acyl residue from 2-acyl-sn-glycero-3-phosphocholine. By the sn-2 to -1 transfer activity, arachidonic acid was transferred from the sn-2 position of donor lipids to the sn-1 position of acceptor lipids, thus producing 1-arachidonoyl phosphatidylcholine. When 2-arachidonoyl-sn-glycero-3-phosphocholine was used as the sole substrate, diarachidonoyl phosphatidylcholine was synthesized at a rate of 0.23 micromol/min/mg protein. Thus, 60-kDa lysophospholipase-transacylase may play a role in the synthesis of 1-arachidonoyl phosphatidylcholine needed for important cell functions, such as anandamide synthesis.     

Synthesis of 1-palmitoyl and 1-stearoyl phosphatidylcholines from mixtures of acyl acceptors via acyl-CoA:1-acyl-sn-glycero-3-phosphorylcholine acyltransferase in liver microsomes.

The fatty acid selectivity of the acyl-CoA:1-acyl-sn-glycero-3-phosphorylcholine acyltransferase in rat liver microsomes was studied using a mixture of the [1-(3)H]palmitoyl plus [1-(14C)stearoyl molecular species of 1-acylglyceryl-phosphorylcholine. At a 1-acyl-sn-glycero-3-phosphorylcholine concentration of 0.16 mM, the enzyme exhibited a selectivity of 3.5-fold for the 1-palmitoyl over the 1-stearoyl species of the acyl acceptor and reaction velocities with linoleoyl- and arachidonoyl-CoA were 38--47% greater than with oleoyl-CoA. Lowering the acceptor concentration to 0.016 mM gave reaction rates with the polyenoic thiolesters which were 174--187% greater than with oleoyl-CoA and the 1-palmitoyl-sn-glycero-3-phosphorylcholine was preferred by 2.2, 1.6, and 1.6-fold with oleoyl-, linoleoyl- and arachidonoyl-CoA, respectively. The results support the potential importance of the fatty acid selectivities of the acyl-CoA:1-acyl-sn-glycero-3-phosphorylcholine acyltransferase towards both acyl acceptor and donor in regulating the phosphatidylcholine species formed by the reaction in vivo.     

Hydrolysis of 2-acyl-sn-glycero-3-phosphocholines in guinea pig heart mitochondria

Although both 2-acyl-sn-glycero-3-phosphocholine and 1-acyl-sn-glycero-3-phosphocholine may be produced from phosphatidylcholine hydrolysis, studies on the former have lagged behind that of the latter. In this study a lysophospholipase A2 that hydrolyses 2-acyl-sn-glycero-3-phosphocholine has been characterized in guinea pig heart mitochondria. The lysophospholipase A2 activity was not dependent on Ca2+ and was inhibited differentially by saturated and unsaturated fatty acids. This lysophospholipase A2 activity was able to discriminate among different molecular species of 2-acyl-sn-glycero-3-phosphocholines when they were presented individually or in pairs. The order of decreasing rates of hydrolysis of different molecular species of 2-lysophosphatidylcholines, when the substrates were presented singly, was 18:2 greater than 20:4 greater than 18:1 greater than 16:0. A differential inhibition of the rate of hydrolysis of the individual substrates was observed when the substrates were presented in pairs. The degree of inhibition was dependent on the molar ratio of the mixed substrates. The characteristics of the enzyme suggest that involvement in the selective release of fatty acids from mitochondrial phosphatidylcholine would depend on a high selectivity of phospholipase A1 for different molecular species of phosphatidylcholine. A lysophospholipase A1 activity was also characterized in the mitochondria with a distinct acyl specificity from the lysophospholipase A2. Other characteristics of the two lysophospholipases suggest that the two reactions are not catalysed by the same enzyme.     

The biosynthesis of phosphatidylserines by acylation of 1-acyl-sn-glycero-3-phosphoserine in rat liver

The conversion of 1-[14C]acyl-sn-glycero-3-phosphoserine into molecular species of [14C]phosphatidylserine was studied using rat liver homogenate and microsomal preparations in the absence of added fatty acyl moieties. In liver homogenates, 81% of the newly-formed phosphatidylserines were tetraenoic (arachidonoyl) species while saturated, monoenoic, dienoic, trienoic, pentaenoic, and hexaenoic (docosahexaenoyl) species each represented 2-5% of the total. A similar pattern of molecular species was produced in liver microsomes. The selectivity of the microsomal acyl-CoA:1-acyl-sn-glycero-3-phosphoserine acyltransferase towards different acyl-CoA derivatives was also investigated. The relative suitability of the various acyl-CoA esters as substrates was found to be of the following order:20:4 = 18:2 greater than 18:1 greater than 16:0 = 18:0. These results with endogenous acyl donors suggest that the acylation of 1-acyl-sn-glycero-3-phosphoserine may partly account for the enrichment of liver phosphatidylserine in arachidonic acid but does not appear to be primarily responsible for the preponderance of docosahexaenoic acid in this phospholipid. The fatty acid specificity of the acyl-CoA: 1-acyl-sn-glycero-3-phosphoserine acyltransferase may contribute to the preferential formation of arachidonoyl phosphatidylserine.     

Acyl specificity in triglyceride synthesis by lactating rat mammary gland.

We have studied the specificity of the acyl-CoA:diglyceride acyltransferase reaction in lactating rat mammary gland to provide a rational explanation at the enzyme level for the nonrandom distribution of fatty acids in milk fat triglycerides. Acyl-CoA:diglyceride acyltransferase activity was measured using various diglyceride and radioactive acyl-CoA substrates; products were identified as triglycerides by thin-layer and gas-liquid chromatography. Most of the enzymatic activity was located in the microsomal fraction and showed a broad specificity for the acyl donors tested C10, C12, C14, C16, C18, and C18:1 CoA esters). The acyltransferase activity was highly specific for sn-1,2-diglyceride enantiomers; rac-1,3- and sn-2,3-diglycerides were relatively inactive. The acyl-CoA specificity was not affected by the type of 1,2-diglyceride acceptor offered, although dilaurin was the best acceptor and sn-1,2-dilaurin greater than sn-1,2-dimyristin greater than sn-1,2-dipalmitin greater than sn-1,2-distearin. We have previously shown that in the microsomal fraction from lactating rat mammary gland, the acyltransferase activities concerned with the conversion of sn-glycero-3-phosphate to diacylglycerophosphate show a very marked specificity for long chain acyl-CoA's. Therefore, we conclude that the predominant localization of long chain fatty acids in the 1 and 2 positions, and of shorter chain fatty acids in the 3 position of the glycerol backbone, results at least in part from the specificities of the mammary gland acyltransferases.     

Expression in yeast of a novel phospholipase A1 cDNA from Arabidopsis thaliana.

During a search for cDNAs encoding plant sterol acyltransferases, we isolated four full-length cDNAs from Arabidopsis thaliana that encode proteins with substantial identity with animal lecithin : cholesterol acyltransferases (LCATs). The expression of one of these cDNAs, AtLCAT3 (At3g03310), in various yeast strains resulted in the doubling of the triacylglycerol content. Furthermore, a complete lipid analysis of the transformed wild-type yeast showed that its phospholipid content was lower than that of the control (void plasmid-transformed) yeast whereas lysophospholipids and free fatty acids increased. When microsomes from the AtLCAT3-transformed yeast were incubated with di-[1-14C]oleyl phosphatidylcholine, both the lysophospholipid and free fatty acid fractions were highly and similarly labelled, whereas the same incubation with microsomes from the control yeast produced a negligible labelling of these fractions. Moreover when microsomes from AtLCAT3-transformed yeast were incubated with either sn-1- or sn-2-[1-14C]acyl phosphatidylcholine, the distribution of the labelling between the free fatty acid and the lysophosphatidylcholine fractions strongly suggested a phospholipase A1 activity for AtLCAT3. The sn-1 specificity of this phospholipase was confirmed by gas chromatography analysis of the hydrolysis of 1-myristoyl, 2-oleyl phosphatidylcholine. Phosphatidylethanolamine and phosphatidic acid were shown to be also hydrolysed by AtLCAT3, although less efficiently than phosphatidylcholine. Lysophospatidylcholine was a weak substrate whereas tripalmitoylglycerol and cholesteryl oleate were not hydrolysed at all. This novel A. thaliana phospholipase A1 shows optimal activity at pH 6-6.5 and 60-65 degrees C and appears to be unaffected by Ca2+. Its sequence is unrelated to all other known phospholipases. Further studies are in progress to elucidate its physiological role.     

Enzymic synthesis of 1-alkyl-2-acyl-sn-glycero-3-phosphorylethanolamines by the CDP-ethanolamine: 1-radyl-2-acyl-sn-glycerol ethanolaminephosphotransferase from microsomal fraction of rat brain

The incorporation of radioactivity from cytidine-5'-phosphate-[(32)P]phosphorylethanolamine into 1-alkyl-2-acyl-sn-glycero-3-phosphorylethanolamines and 1,2-diacyl-sn-glycero-3-phosphorylethanolamines was stimulated more than fourfold by 1-alkyl-2-acyl-sn-glycerols and 1,2-diacyl-sn-glycerols, respectively, with an ethanolaminephosphotransferase (EC 2.7.8.1) present in the microsomal fraction from brains of mature rats. The K(m) values, 0.28 mm for CDP-ethanolamine and 1.9 mm for 1-alkyl-2-acyl-sn-glycerols, were similar to those obtained by other investigators with other 1-radyl-2-acyl-sn-glycerols. The formation of 1,2-diacyl-sn-glycero-3-phosphorylethanolamines from endogenous 1,2-diacyl-sn-glycerols was inhibited by 1-alkyl-2-acyl-sn-glycerols. These properties indicate that the ethanolaminephosphotransferase lacks specificity for the type of group at the 1-position of the lipid substrate. The synthesis of 1-alkyl-2-acyl-sn-glycero-3-phosphorylethanolamines from 1-alkyl-2-acyl-sn-glycerols and CDP-ethanolamine by an enzyme from rat brain supports the inclusion of this reaction in the metabolic pathway for the synthesis of 1-alk-1'-enyl-2-acyl-sn-glycero-3-phosphorylethanolamines.     

Triglyceride metabolism in 3T3-L1 cells. An in vivo 13C NMR study.

13C nuclear magnetic resonance spectroscopy has been used to study triglyceride metabolism in 3T3-L1 cells incubated with [1-13/14C] acetate, myristate, palmitate, stearate, or oleate. Labeled cells embedded in agarose filaments were perfused in a specially fitted NMR tube within the spectrometer magnet. Incubation of 3T3-L1 cells with a specific fatty acid enriched the cellular triglycerides with that fatty acid; the NMR signal observed in the carbonyl region of the cell spectrum was due in large part to that fatty acid. NMR data demonstrated that cellular enzymes preferentially esterified saturated fatty acids at the glyceride sn-1,3 position and unsaturated fatty acids at the sn-2 position. cellular triglyceride hydrolysis by hormone-sensitive lipase was monitored by measuring the decrease in the integrated intensities of resonances arising from fatty acyl carbonyls esterified at glycerol carbons sn-1,3 and sn-2. Under basal conditions, the time courses were first-order, and the average rates were 0.14% of signal/min at both carbonyl positions. Under isoproterenol stimulated conditions, these rates were still first-order and increased 6.4-fold at the sn-1,3 position and 2.4-fold at the sn-2 position. The observation that the hydrolysis time courses were first-order suggested that only a small amount of cellular triglyceride was available to hormone-sensitive lipase, supporting the view that lipolytic enzymes operate at lipid surfaces where only small amounts of neutral lipid may be soluble. Attempts to correlate the measured rates with the rates of hydrolysis at the sn-1,3 and sn-2 positions were hindered by the fact that the chemical shifts of the carbonyl carbons of the diglyceride hydrolysis product did not overlie those of the triglyceride. Analysis of hydrolysis kinetics revealed that hormone-sensitive lipase exhibited little preference for a particular esterified fatty acid under basal conditions; however, under stimulated conditions, the enzyme exhibited a preference for certain triglyceride species.     

The acylation of 1-acyl-sn-glycero-3-phosphate by neuronal nuclei and microsomal fractions of immature rabbit cerebral cortex

The acylation of 1-acyl-sn-glycero-3-phosphate to form phosphatidic acid was studied using a neuronal nuclear fraction N1 and microsomal fractions P3, R (rough), S (smooth), and P (neuronal microsomes from nerve cell bodies) isolated from cerebral cortices of 15-day-old rabbits. The assays contained this lysophospholipid, ATP, CoA, MgCl2, NaF, dithiothreitol, and radioactive palmitate, oleate, or arachidonate. Of the subfractions, N1 and R had the highest specific activities (expressed per micromole phospholipid in the fraction). The rates with oleate were two to four times the values seen for phosphatidic acid formation from sn-[3H]glycero-3-phosphate and oleoyl-CoA. Using oleate or palmitate, fraction R had superior specific rates to N1 at low lysophosphatidic acid concentrations. With increasing lysophospholipid concentrations the specific rates of N1 and R came closer together and maintained at least a twofold superiority over fraction P. Fraction S had the lowest specific rates of phosphatidic acid formation. Fractions N1, R, and P showed a preference for palmitate and oleate over arachidonate, particularly at low concentrations of lysophosphatidic acid. For N1 and R, the preference was also more marked at higher concentrations of fatty acid. Thus a selectivity for saturated and monounsaturated fatty acids was shown in the formation of phosphatidic acid, as was a concentration of acylating activity in the neuronal nucleus and the rough endoplasmic reticulum.     

Metabolism of exogenous long-chain fatty acids by spinach leaves

When applied in liquid paraffin to the upper surface of expanding spinach leaves, [1-14C]palmitic acid was efficiently and exclusively incorporated into the sn-1 position of cellular glycerolipids, principally phosphatidylcholine and triacylglycerol. A slow transfer of fatty acids from phosphatidylcholine to chloroplast glycolipids subsequently occurred with the positional specificity of the label remaining intact. Labeled palmitate at the sn-1 position of monogalactosyldiacylglycerol was desaturated to hexadecatrienoate so that 1-[14C]hexadecatrienoyl-2-linolenoyl-3-galactosoylglycerol became the major labeled species of the lipid between 8 and 24 h. There was no evidence of deacylation/reacylation reactions modifying the acyl compositions of spinach leaf glycerolipids for at least 48 h after labeling with [1-14C]palmitic acid; even the partially prokaryotic glycerolipids remained firmly labeled at the sn-1 position. Exogenous [1-14C]stearic acid was also incorporated into the sn-1 position of MGD, presumably by the same mechanism, and was there desaturated to [14C]linolenate. Exogenous [1-14C]oleic acid was initially incorporated equally into both sn-1 and sn-2 positions of phosphatidylcholine, and was desaturated to linoleate at both positions before the label was rapidly transferred to monogalactosyldiacylglycerol. There, desaturation of linoleate to linolenate took place. Galactolipids remained equally labeled at both positions throughout the 6 days of the experiment, but label was concentrated in the 1-saturated-2-[14C]linolenoyl molecular species of phosphatidylcholine as those species with two [14C]linoleoyl residues were drawn off for monogalactolipid synthesis. Glycerolipids synthesised from exogenous [1-14C]acetate by spinach leaves were labeled equally at both the sn-1 and the sn-2 positions. These results are interpreted as providing strong support for the two-pathway scheme of glycerolipid synthesis in plants.     

Ether phospholipids as inhibitors of the arachidonoyl-CoA: 1-acyl-sn-glycero-3-phosphocholine acyltransferase in macrophages

1-Alkylglycerophosphatide analogs which are known to activate macrophages to enhanced tumor cytotoxicity are structurally closely related to 1-acyl-sn-glycero-3-phosphocholine. In this study we have examined the influence of some of these compounds and of platelet-activating factor (PAF-acether, 1-0-alkyl-2-0-acetyl-sn-glycero-3-phosphocholine) on the arachidonoyl-CoA: 1-acyl-sn-glycero-3-phosphocholine acyltransferase (EC 2.3.1.23) in homogenate of bone-marrow-derived murine macrophages. This enzyme is suggested to be involved in the control of the availability of the icosanoid precursor, arachidonic acid. Kinetic experiments revealed apparent Km and V values for 1-palmitoyl-sn-glycero-3-phosphocholine of 6.0 microM and 16.10 nmol/mg protein per min, respectively. When the 1-palmitoyl-sn-glycero-3-phosphocholine concentration was equal to Km, the enzyme was dose-dependently inhibited by 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine with a 50% inhibition at 30 microM. The kinetic parameters in the presence of 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine (K'm = 10.0 microM, V' = 11.40 nmol X mg-1 X min-1) suggest that this alkyl phospholipid is a mixed-type inhibitor. All other alkyl analogs tested (1-O-methyl-2-O-octadecyl-rac-glycerol-3-phosphocholine, racemic PAF-acether, L-PAF-acether, D-1-O-hexadecyl-sn-glycero-3-phosphocholine, 1-O-octadecyl-rac-glycero-3-phosphocholine) inhibited the enzyme to various degrees. Arachidonic acid transfer to the 1-alkylglycerophosphatide analogs themselves could be ruled out under the assay conditions used. Therefore, we conclude that the arachidonoyl-CoA: 1-acyl-sn-glycero-3-phosphocholine acyltransferase can be inhibited by synthetic and naturally occurring ether phospholipids in homogenate of bone-marrow-derived murine macrophages.     

Topology of acyltransferase motifs and substrate specificity and accessibility in 1-acyl-sn-glycero-3-phosphate acyltransferase 1

1-acyl-sn-glycero-3-phosphate (AGP) acyltransferases (AGPAT) are involved in de novo biosynthesis of glycerolipids, such as phospholipids and triacylglycerol. Alignment of amino acid sequences from AGPAT, sn-glycerol-3-phosphate acyltransferase, and dihydroxyacetonephosphate acyltransferase reveals four regions with strong homology (acyltransferase motifs I-IV). The invariant amino acids within these regions may be part of a catalytically important site in this group of acyl-CoA acyltransferases. However, in human AGPAT1 a transmembrane domain is predicted to separate motif I on the cytosolic side from motifs II-III on the lumenal side, with motif IV near surface of the membrane. The topology of motifs I and III was confirmed by experiments with recombinant AGPAT1 containing potential glycosylation site near the motifs. This topology conflicts with the expectation that catalytically important sites are near one another, raising questions of whether the acyltransferase motifs really are important for AGPAT catalysis, and how substrates access motifs II-III on the lumenal side of the endoplasmic reticulum membrane. Using human AGPAT1 as a model, we have examined the catalytic roles of highly conserved residues in the four acyltransferase motifs by site-directed mutagenesis. Modifications of the sidechain structures of His104, Asp109, Phe146, Arg149, Glu178, Gly179, Thr180, Arg181 and Ile208 all affected AGPAT1 activity, indicating that the acyltransferase motifs indeed are important for AGPAT catalysis. In addition, we examined substrate accessibility to the catalytic domain of human AGPAT1 using a competition assay. Lysophosphatidic acid (LPA) with fatty acid chains shorter than 10 carbons did not access the catalytic domain, suggesting that LPA hydrophobicity is important. In contrast, short chain acyl-CoAs did access the catalytic domain but did not serve as the second substrate. These results suggest that motifs II and III are involved in LPA binding and motifs I and IV are involved in acyl-CoA binding.     

Substrate specificity of phospholipase B from guinea pig intestine. A glycerol ester lipase with broad specificity.

The substrate specificity of a calcium-independent, 97-kDa phospholipase B purified from guinea pig intestine was further investigated using various natural and synthetic lipids. The enzyme was equally active toward enantiomeric phosphatidylcholines under conditions allowing a strict phospholipase A activity. The lysophospholipase activity declined with the following substrates: 1-acyl-sn-glycero-3-phosphocholine greater than 1-palmitoyl-propanediol-3-phosphocholine greater than 1-palmitoyl-glycol-2-phosphocholine, suggesting some influence of the polar residue vicinal to the cleavage site. The enzyme also acted on various neutral lipids including triacylglycerol, diacylglycerol, and monoacylglycerol, whereas cholesteryl oleate remained refractory to enzymatic hydrolysis. The lipase hydrolyzed sequentially the sn-2 and sn-1 acyl ester bonds of diacylglycerol, although some direct cleavage of the external acyl ester bond could also occur, as shown with diacylglycerol analogues bearing a nonhydrolyzable alkyl ether or amide bond in the sn-1 or sn-2 position. The three main activities of the enzyme (phospholipase A2, lysophospholipase, and diacylglycerol lipase) were resistant to 4-bromophenacyl bromide, but they were inhibited by N-ethylmaleimide, 5,5'-dithiobis-(2-nitrobenzoic acid), and diisopropyl fluorophosphate, suggesting the possible involvement of both cysteine and serine residues in a single active site. It is concluded that guinea pig intestinal phospholipase B, which was also detected in rat and rabbit, is actually a glycerol ester lipase with broad substrate specificity and some unique enzymatic properties.     

Metabolism of 1-acyl-2-acetyl-sn-glycero-3-phosphocholine in the human neutrophil

The biosynthesis of 1-acyl-2-acetyl-sn-glycero-3-phosphocholine (1-acyl-2-acetyl-GPC) together with that of 1-alkyl-2-acetyl-GPC (platelet-activating factor) has been demonstrated in a variety of inflammatory cells and tissues. It has been hypothesized that the relative proportion of these phospholipids produced upon cell activation may be influenced by their rates of catabolism. We studied the catabolism of 1-acyl-2-acetyl-GPC in resting and activated human neutrophils and compared it to that of 1-alkyl-2-acetyl-GPC. Neutrophils rapidly catabolize both 1-alkyl-2-acetyl-GPC and 1-acyl-2-acetyl-GPC; however, the rate of catabolism of 1-acyl-2-acetyl-GPC is approximately 2-fold higher than that of 1-alkyl-2-acetyl-GPC. In addition, most of 1-acyl-2-acetyl-GPC is catabolized through a pathway different from that of 1-alkyl-2-acetyl-GPC. The main step in the catabolism of 1-acyl-2-acetyl-GPC is the removal of the long chain at the sn-1 position; the long chain residue is subsequently incorporated either into triglycerides or into phosphatidylcholine. The 1-lyso-2-acetyl-GPC formed in this reaction is then further degraded to glycerophosphocholine, choline, or phosphocholine. 1-Acyl-2-acetyl-GPC is also catabolized, to a lesser extent, through deacetylation at the sn-2 position and reacylation with a long chain fatty acid. Stimulation of neutrophils by A23187 results in a higher rate of catabolism of 1-acyl-2-acetyl-GPC by increasing both the removal of the long chain at the sn-1 position and the deacetylation-reacylation at the sn-2 position. In a broken cell preparation, the cytosolic fraction of the neutrophil was shown to contain an enzyme activity which cleaved the sn-1 position of 1-acyl-2-acetyl-GPC and 1-acyl-2-lyso-GPC but not of 1,2-diacyl-GPC. Taken together, these data demonstrate that the human neutrophil is able to catabolize 1-acyl-2-acetyl-GPC in a manner both quantitatively and qualitatively different from that of platelet-activating factor. The differential catabolism may regulate the relative proportion of these two bioactive phospholipids in the neutrophil.     

The effect of sn-2 fatty acid substitution on phospholipase C enzyme activities.

The human monocyte cell line U937 expresses phospholipase A2 and phospholipase C activities and produces eicosanoids. The phospholipase C (PLC) activity exhibits substrate preference for phosphatidyl-choline (PC), rather than phosphatidylinositol or phosphatidylethanolamine. In order to characterize the PLC activity found in these cells, the effects of substitution of the sn-2 fatty acid on this activity were examined. PC substrates with palmitic acid (PC-2P), oleic acid (PC-2O), arachidonic acid (PC-2A) and linoleic acid (PC-2L) at the sn-2 position were used. The sn-1 fatty acid was palmitic acid. PC-2L and PC-2A with the longer-chain less-saturated fatty acids linoleic acid and arachidonic acid esterified at sn-2 were found to be better substrates for PLC activity than PC-2P or PC-2O in these cells. This preference was maintained even when substrate phospholipid was solubilized in non-ionic, anionic, cationic and zwitterionic amphiphiles. Furthermore, when a 500-fold excess of 1,2-diolein or 1,2-dipalmitin was added to the reaction, the specificity of the PLC activity for PC-2A and PC-2L remained unchanged. When similar experiments were performed with phosphatidylinositol as a substrate, we did not observe any effect when the sn-2 position was altered. These data show that the fatty acid constituent at the sn-2 position affects the observed PLC activity when phosphatidylcholine, but not phosphatidylinositol, is used as a substrate by these cells.     

Relative amounts of 1-O-alkyl- and 1-acyl-2-acetyl-sn-glycero-3-phosphocholine in stimulated endothelial cells.

The specific precursor for platelet-activating factor, 1-O-alkyl-2-acyl-sn-glycero-3-phosphocholine, constitutes 10 per cent of the 1-radyl-2-acyl-sn-glycero-3-phosphocholines in endothelial cells. Stimulation of endothelial cells results in accumulation of PAF and its sn-1-acyl- analog (acylPAF), with acylPAF the predominant product. Mass spectrometry confirmed these relative amounts and confirmed that stimulated endothelial cells accumulate 1-3 ng PAF per million cells. These data suggest that stimulated endothelial cells accumulate both PAF and acylPAF and that the PAF synthetic pathway in endothelial cells is not highly selective for the specific PAF precursor (1-O-alkyl-2-acyl-sn-glycero-3-phosphocholine).     

A lecithin:retinol acyltransferase activity in human and rat liver

This report demonstrates that exogenous phosphatidylcholine will serve as an acyl donor for the esterification of retinol complexed to cellular retinol-binding protein (CRBP) by human and rat liver microsomal preparations. The retinyl ester synthases utilized phosphatidylcholine but had little or no ability to transfer acyl groups from lysophosphatidylcholine, phosphatidyl-ethanolamine, or phosphatidic acid to retinol-CRBP. The human and rat activities also demonstrated positional selectivity as only the fatty acyl group at the sn-1 position of phosphatidylcholine was transferred. This in vitro activity may have considerable physiological importance since the fatty acyl composition at the sn-1 position of phosphatidylcholine is remarkably similar to the hepatic retinyl esters observed in vivo.     

Positional specificity of lysosomal phospholipase A2

Lysosomal phospholipase A(2) (Lpla2) is highly expressed in alveolar macrophages and may mediate the phospholipid metabolism of surfactant. Studies on the properties of this phospholipase are consistent with the presence of both phospholipase A(1) and phospholipase A(2) activities. These activities were studied through the production of O-acyl compounds, produced by the transacylase activity of Lpla2. Liposomes containing POPC and N-acetylsphingosine (NAS) were incubated with the soluble fraction obtained from MDCK cells stably transfected with the mouse Lpla2 gene. Two 1-O-acyl-NASs, 1-O-palmitoyl-NAS and 1-O-oleoyl-NAS, were produced by Lpla2. The formation rate of 1-O-oleoyl-NAS was 2.5-fold that of 1-O-palmitoyl-NAS. When 1-oleoyl-2-palmitoyl-sn-glycero-3-phosphocholine (OPPC) was used, the formation rate of 1-O-oleoyl-NAS was 5-fold higher than that of 1-O-palmitoyl-NAS. Thus, Lpla2 can act on acyl groups at both sn-1 and sn-2 positions of POPC and OPPC. When 1-palmitoyl-2-unsaturated acyl-sn-glycero-3-phosphocholines were used as acyl donors, the transacylation of the acyl group from the sn-2 position to NAS was preferred to that of the palmitoyl group from the sn-1 position. An exception was observed for 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (PAPC), for which the formation rate of 1-O-palmitoyl-NAS from PAPC was 4-fold greater than that of 1-O-arachidonoyl-NAS. Thus, Lpla2 has broad positional specificity for the sn-1 and sn-2 acyl groups in phosphatidylcholine and phosphatidylethanolamine.