Substrate specificity of a CoA-dependent stearoyl transacylase from bovine testis membranes.

We identified a CoA-dependent stearoyl transacylase activity in bovine testis membranes, then examined the enzyme's specificity in mixed micelle systems containing the neutral detergent Triton X-100. The enzyme transferred stearoyl groups from a variety of phospholipids to sn-2-arachidonoyl lysophosphatidic acid (lysoPA), but showed very little palmitoyl transacylase activity. Its ability to transfer stearoyl groups was both donor- and acceptor-dependent. For example, it used weakly acidic phospholipids, such as sn-1-stearoyl-2-acyl species of phosphatidylinositol (PI), as donors, but did not use phosphatidylinositol-4,5-bisphosphate or sn-1-stearoyl-2-arachidonoyl phosphatidylcholine. Moreover, it used sn-2-acyl species of lysoPA and sn-2-arachidonoyl lysoPI as acceptors but did not use sn-2-arachidonoyl species of lysophosphatidylserine, lysophosphatidylethanolamine, or lysophosphatidylcholine. When taken together, our results raise the possibility that sn-1-stearoyl-2-acyl species of PI may be the primary acyl donors in the transacylase reaction in vivo, while sn-2-acyl species of lysoPA may be the primary acyl acceptors. Available evidence suggests that the PA that is formed may subsequently be converted into PI, but the metabolic fate of the other reaction product, sn-2-acyl lysoPI, remains to be determined.     

The lipid binding site of the phosphatidylcholine transfer protein from bovine liver

The phosphatidylcholine transfer protein (PC-TP) from bovine liver has a binding site for phosphatidylcholine (PC). Structural and molecular characteristics of this site were investigated by binding PC-analogues carrying photolabile, fluorescent and short-chain fatty acids. Analysis of the photolabeled PC/PC-TP adduct showed that the hydrophobic peptide segment Val171-Phe-Met-Tyr-Tyr-Phe-Asp177 is part of the lipid binding site for the 2-acyl chain. This site was further studied by binding PC carrying cis-parinaric acid at the sn-2-position. Time resolved fluorescence anisotropy measurements indicated that the 2-acyl chain was immobilized following the rotation of PC-TP. Similar experiments with PC carrying cis-parinaric acid at the sn-1-position demonstrated that the 1-acyl chain was immobilized as well but at a site distinctly different from that of the 2-acyl chain. Binding sites for the 1- and 2-acyl chain were then explored by use of PC-isomers carrying decanoic, lauric and myristic acid at the sn-1- (or sn-2-)-position and oleic acid at the sn-2- (or sn-1-)-position. Incubation with vesicles prepared of these PC-species indicated that binding to PC-TP diminished with decreasing acyl chain length but more so for species with short-chain fatty acids on the sn-2-position than on the sn-1-position. Transfer experiments confirmed that PC-TP discriminates between PC-isomers of apparently equal hydrophobicity favouring the transfer of these species carrying oleic acid at the sn-2-position.     

Synthetic branched-chain analogues of distearoylphosphatidylcholine: structure-activity relationship in inhibiting and activating protein kinase C

A series of distearoylphosphatidylcholine (DSPC) analogues having various branched alkyl chains were synthesized and tested for their abilities to regulate protein kinase C (PKC). The greatest improvement (about 3-fold) in the PKC inhibitory activity over that seen for the parental lipid (i.e., DSPC) was accomplished by substitution of 8-methylstearate at sn-2 and 16-methylstearate at both sn-1 and sn-2 positions of glycerol; substitutions at both sn-1 and sn-2 with 8-methylstearate, on the other hand, caused a decrease (about 4-fold) in its inhibitory activity. Introduction of butyl, phenyl, or keto functions to various positions in the fatty alkyl chain substituted at both sn-1- and sn-2 positions imparted upon the DSPC analogues an ability to potently stimulate PKC to an extent comparable to those attainable by diacylglycerol or phorbol ester; the analogues having substitution only at the sn-2 position, in comparison, had no or reduced stimulatory activity. The butyl, phenyl, and keto analogues of DSPC, as with DSPC itself and its methyl analogues, inhibited PKC at high concentrations. Kinetic analysis indicated that the methyl DSPC analogues inhibited the enzyme competitively with respect to phosphatidylserine (PS; a phospholipid cofactor) and Ca2+. The butyl analogues activated the enzyme without affecting its affinity for PS or Ca2+, indicating a mechanism different from that seen for diacylglycerol or phorbol ester. The inhibitory activity of the methyl DSPC analogues and the stimulatory activity of the butyl DSPC analogues were reduced when PKC was activated by phorbol ester. Both classes of the analogues were unable to compete for the binding of [3H]phorbol dibutyrate to PKC.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evidence for existence of various homologues and analogues of platelet activating factor in a lipid extract of bovine brain

Vasodepressor phospholipid with platelet-aggregating activity was highly purified from a lipid extract of bovine brain and subjected to field desorption-mass spectrometry. It was further analyzed by gas-liquid chromatography-mass spectrometry after hydrolysis with phospholipase C and conversion to tert-butyldimethylsilyl derivatives. Results indicated the presence of four species of platelet activating factor (1-0-alkyl-2-acetyl-sn-glycero-3-phosphocholine, PAF) and ten acyl analogues of PAF. The acyl analogues of PAF included species having an sn-2-propionyl or sn-2-butyryl group, which have not been previously detected in natural sources. The total amount of acyl analogues of PAF was much higher than that of PAF.     

Altered positional specificity of human plasma lecithin-cholesterol acyltransferase in the presence of sn-2 arachidonoyl phosphatidyl cholines. Mechanism of formation of saturated cholesteryl esters.

The positional specificity of purified human lecithin-cholesterol acyltransferase (LCAT) was studied by analyzing the labeled cholesteryl ester (CE) species formed in the presence of proteoliposome substrates containing mixed chain phosphatidylcholine (PC) species, labeled cholesterol and apoprotein A-I. Whereas over 90% of the acyl groups used for CE synthesis were derived from the sn-2 position of most of the naturally occurring PC substrates, about 75% of the CE species formed in the presence of sn-1-myristoyl 2-arachidonoyl PC, sn-1-palmitoyl-2-arachidonoyl (PAPC) and sn-1-palmitoyl 2-docosahexaenoyl PC were derived from the sn-1-position. On the other hand, rat LCAT utilized mostly sn-2-acyl group from either PAPC or from sn-1-palmitoyl 2-linoleoyl PC. The positional specificity of the human enzyme was not affected by the alteration in the matrix fluidity, type of the apoprotein activator used, or by the free cholesterol/PC ratio in the substrate. These results show that the positional specificity of human plasma LCAT is altered in the presence of sn-2-arachidonoyl PC, or sn-2-docosahexaenoyl PC, probably due to steric restrictions at the active site, and this may account for the formation of disproportionately high concentrations of saturated CE, and low concentrations of long-chain polyunsaturated CE in human plasma, relative to the composition of sn-2-acyl groups in plasma PC.     

Phospholipid remodeling in human neutrophils. Parallel activation of a deacylation/reacylation cycle and platelet-activating factor synthesis

A23187 stimulated two enzymatic activities of human neutrophils (polymorphonuclear leukocytes), phospholipase A2 and fatty acyl-CoA acyltransferase, which resulted in a stimulated deacylation/reacylation cycle. The incorporation of fatty acids, other than arachidonic or eicosapentaenoic acid, into diacyl and alkylacyl species of choline phosphoglycerides was stimulated by 10-fold by A23187. These fatty acids were exclusively incorporated into the sn-2 position, and [3H]glycerol labeling showed there was no stimulation of de novo synthesis. A23187 also stimulated fatty acid incorporation into other phospholipids, but de novo synthesis accounted for a portion of this uptake. Inhibitors of protein kinase C prevented the stimulated recycling of phosphatidylcholine, and the simultaneous induction of platelet-activating factor synthesis, by inhibiting phospholipase A2 activation. They inhibited [3H]arachidonate release from prelabeled polymorphonuclear leukocytes, but had no effect on in vitro fatty acyl-CoA acyltransferase or acetyl-CoA acetyltransferase activity. Extracts from A23187-treated cells contained a fatty acyl-CoA acyltransferase, which did not utilize arachidonoyl-CoA, that was 2.3-fold more active than that of control extracts. Phosphatase treatment decreased this stimulated activity by 66%. Thus, A23187 stimulated a phospholipase A2 activity that generated both 1-alkyl and 1-acyl lysophosphatidylcholines. A stimulated acetyltransferase used a portion of the alkyl species for platelet-activating factor synthesis, while the acyl species and residual alkyl species were rapidly reacylated to phosphatidylcholine by a stimulated acyl-transferase. Arachidonate, an eicosanoid precursor, was spared by this process.     

Substrate specificities and properties of human phospholipases A2 in a mixed vesicle model.

Studies of the specificity of phospholipases A2 (PLA2s) for different substrates have usually been carried out in vesicles or mixed micelles, where differences in shape, size, or charge of vesicles formed with different phospholipids may give misleading results. Another factor is binding of the enzyme to the phospholipid surface, which has recently been addressed using vesicles of an anionic phospholipid, dimyristoyl-sn-glycero-3-phosphomethanol (DMPM) to which some extracellular PLA2s were shown to bind with a very high affinity (Jain, M. K., and Berg, O. G. (1989) Biochem. Biophys. Acta 1002, 127-156). In the present report we have used a similar system to study the substrate preferences of two human PLA2s that are thought to be physiologically relevant in the metabolism of arachidonic acid: a recombinant form of the human synovial fluid (14 kDa) PLA2 and the cytosolic (85 kDa) PLA2 found in monocytic cells. It is shown that both human enzymes bind tightly to DMPM vesicles and follow the basic characteristics of processive hydrolysis in this model using analysis of progress curves and substrate competition experiments. Mixed vesicles containing DMPM with small amounts (3-5 mol%) of other phospholipids have been used to study the substrate selectivity of the two human isoenzymes. The synovial fluid PLA2 shows a clear preference (approximately 7-fold) for sn-glycero-3-phosphoethanolamine over sn-glycero-3-phosphocholine. Within glycerophosphocholines, this enzyme displays little preference for the sn-2 fatty acyl group, and a slight preference for phospholipids with sn-1-acyl versus sn-1-alkyl substituents. In contrast, the cytosolic PLA2 shows a marked selectivity for arachidonoyl in the sn-2 position and only minor differences in selectivity for the polar head group in the sn-3 position. This enzyme does not distinguish between sn-1-acyl and sn-1-alkyl subclasses of glycerophosphocholines.     

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.     

Swiss 3T3 cells preferentially incorporate sn-2-arachidonoyl monoacylglycerol into sn-1-stearoyl-2-arachidonoyl phosphatidylinositol.

The sn-1-stearoyl-2-arachidonoyl phospholipids of animal cells appear to be formed by special mechanisms. To determine whether monoacylglycerol (MG) incorporation pathways are involved we incubated quiescent Swiss 3T3 cells with [3H]glycerol-labeled sn-2-arachidonoyl MG, then analyzed the radioactive cell lipids that accumulated. We also examined cell homogenates to identify enzyme activities that might promote the incorporation of sn-2-arachidonoyl MG into other cell lipids. The cell incubation experiments demonstrated rapid labeling of several lipids, including diacylglycerol, lysophosphatidic acid, phosphatidic acid, and phosphatidylinositol. They also demonstrated selective labeling of sn-1-stearoyl-2-arachidonoyl species of phosphatidylinositol, phosphatidylethanolamine, and phosphatidylserine. The cell homogenate experiments identified an sn-2-acyl MG acyltransferase activity, an MG kinase activity that phosphorylates sn-2-arachidonoyl MG in preference to sn-2-oleoyl MG, and a stearoyl-specific acyl transferase activity that converts sn-2-arachidonoyl lysophosphatidic acid into sn-1-stearoyl-2-arachidonoyl phosphatidic acid. The results also showed that this stearoyl transferase could act with other enzymes to convert sn-2-arachidonoyl lysophosphatidic acid into sn-1-stearoyl-2-arachidonoyl phosphatidylinositol. The combined results indicate that Swiss 3T3 cells incorporate sn-2-arachidonoyl MG into phospholipids by at least two different pathways, including one that specifically forms sn-1-stearoyl-2-arachidonoyl phosphatidylinositol.     

Stereoselectivity of lipases. I. Hydrolysis of enantiomeric glyceride analogues by gastric and pancreatic lipases, a kinetic study using the monomolecular film technique

In the present study, porcine pancreatic lipase, rabbit gastric lipase, and human gastric lipase stereospecificity toward enantiomeric glyceride derivatives was kinetically investigated using the monomolecular film technique. Pseudoglycerides such as enantiomeric 1(3)-alkyl-2,3(1,2)-diacyl-sn-glycerol, enantiomeric 1(3)-alkyl-2-acyl-sn-glycerol, or enantiomeric 1(3)-acyl-2-acylamino-2-deoxy-sn-glycerol were synthesized in order to assess the lipase stereoselectivity during the hydrolysis of either the primary or the secondary ester position of these glycerides analogues. The cleaved acyl moiety was the same in both enantiomers, thereby excluding the possibility of effects occurring due to fatty acid specificity. We observed a porcine pancreatic lipase sn-3 stereoselectivity when using the enantiomeric 1(3)-alkyl-2-acylamino-2-deoxy-sn-glycerol (diglyceride analogue) which contrasted with the lack of stereoselectivity observed when using the enantiomeric 1(3)-alkyl-2,3(1,2)-diacyl-sn-glycerol (triglyceride analogue). The gastric lipases, in contrast to the pancreatic lipase, preferentially catalyze the hydrolysis of the primary sn-3 ester bond of the enantiomeric monoakyl-diacyl pair tested. From these kinetic data, high hydrolysis rates and no chiral discrimination were observed in the case of rabbit gastric lipase, whereas low rates and a clear chiral discrimination was noticed in the case of human gastric lipase during hydrolysis of the acyl chain from the secondary ester bond of 1(3)-alkyl-2-acyl enantiomers. It is particularly obvious that in the case of human gastric lipase decreasing the lipid packing increases the lipase sn-3 stereopreference during hydrolysis of the primary ester bond of the enantiomeric 2-acylamino derivatives (diglyceride analogue).     

Purification and regiospecificity of multiple enzyme activities of phospholipase A(1) from bonito muscle

Phospholipase A(1) (PLA(1)), which catalyzes the hydrolysis of the sn-1 ester bond of diacyl phospholipids, was purified from 100,000 x g supernatant of bonito muscle to homogeneity by ammonium-sulfate precipitation and four consecutive column chromatographies (DEAE anion-exchange, ether-Toyopeal, hydroxylapatite and Toyopeal HW 50S columns). The final preparation showed a single band above the 67-kDa molecular marker on SDS-PAGE, and the molecular mass was determined to be 71.5 kDa by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry using bovine serum albumin as a standard for calibration. The N-terminal 8 amino residues were determined to be Ala-Pro-Ala-Glu-Lys-Val-Lys-Try. Regiospecificity of multiple enzyme activities of the PLA(1) was examined using positionally defined synthetic phosphatidylcholine (PC) and lysophosphatidylcholines (LPC). An acyl ester bond at the sn-1 position of PC was exclusively hydrolyzed by phospholipase activity, and 1-acyl LPC was cleaved to fatty acid and glycerophosphocholine by lysophospholipase (LPL) activity. However, the positional isomer, 2-acyl LPC was a poor substrate for LPL activity. PC/transacylation activity was also observed when excess 2-acyl LPC was supplied in the reaction mixture, and fatty acid at the sn-1 position of donor PC was transferred to the sn-1 position of acceptor LPC. These results demonstrate that the multiple enzyme activities of PLA(1), this is lysophospholipase, transacylase as well as phospholipase, have a strict regiospecificity at the sn-1 position of substrates.     

Substrate specificity and partial characterization of the PAF-acylhydrolase in human serum that rapidly inactivates platelet-activating factor

The structure of the potent inflammatory mediator, platelet-activating factor, is 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine (AGEPC, PAF-acether). Human sera contain an acid labile factor (ALF) that is a Ca+2-independent 2-acylhydrolase-specific for AGEPC and AGEPC-like molecules. The enzyme functions by catalytically removing the sn-2 acetyl moiety from AGEPC, producing the biologically inactive sn-2 hydroxy form or 2-lyso-GEPC. Incubation of ALF with sn-2 acyl PAF analogs indicated that the enzyme hydrolyzes the sn-2 fatty acid only if the chain length is five carbons or less, the sn-1 position fatty acid length is greater than 10 carbon units, and at least one methyl group is present on the terminal amine of the choline group. The enzyme was active with either an ether or ester linkage at the sn-1 position. ALF is inactivated by heating to 65 degrees C for 30 min. It is pronase and trypsin sensitive but resistant to papain and papain with dithiothreitol. Further characteristics of human ALF indicated a broad pH range of activity with an optimum of pH 6.2 and an isoelectric point of 6.2 to 6.7. The specificity and Ca+2 independence of human ALF sets it apart from phospholipase A2. It is proposed that human ALF be called human serum PAF-acylhydrolase to distinguish it from other hydrolases currently known to exist.     

Desorption chemical ionization mass spectrometry of 1-O-alkyl-2-O-acetyl-sn-glycero-3-phosphocholines and analogues

Ammonia desorption chemical ionization of ether-linked phospholipids of the type 1-O-alkyl-2-O-acetyl-sn-glycero-3-phosphocholine (platelet-activating factors) and a series of analogues revealed a systematic fragmentation pattern that is characteristic for these compounds. The predominant ions included the protonated molecular ion and a series of fragments derived from the molecular ion having the following nominal mass losses: MH-14, MH-42, MH-59, and MH-183. Deuterated ammonia was used to elucidate the nature of several fragments. In addition, desorption chemical ionization was used to quantitate 1-O-hexadecyl-2-O-acetyl-sn-glycero-3-phosphocholine at the nanogram/sample level.     

Production of platelet-activating factor by washed rabbit platelets

Production of platelet-activating factor by washed rabbit platelets under stimulation with the ionophore A23187 was investigated utilizing two groups of platelet preparations. The first platelet preparation contained 0.03 +/- 0.02% contaminating white cells, while the second preparation contained 0.48 +/- 0.27% white cells. The latter preparation produced platelet-activating factor, mainly 1-hexadecyl-2-acetyl-sn-glycero-3-phosphocholine, 8.3 +/- 6.3 pmol (mean +/- standard deviation) with a range of 2.6 to 21.4 pmol (n = 9), followed by small quantities of 1-octadecenyl- and 1-octadecyl-2-acetyl-sn-glycero-3-phosphocholine. In contrast, there was no production of 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine by the former platelet preparation having 0.03% leukocytes. These quantitative analyses were carried out by the selected ion monitoring technique and it was concluded that it is necessary to consider the presence of contaminating white cells in studies on the production of platelet-activating factor by platelets.     

sn-1,2-diacylglycerol kinase of Escherichia coli. Diacylglycerol analogues define specificity and mechanism

A detailed structure/function analysis of the substrate specificity of Escherichia coli sn-1,2-diacylglycerol kinase was performed with three goals in mind: (a) to define the substrate specificity; (b) to discover inhibitors; and (c) to elucidate the specificity of diacylglycerol-dependent inactivation. Forty-seven structural analogues of sn-1,2-diacylglycerol were prepared and examined as substrates, inhibitors, and irreversible inactivators of the enzyme using mixed micellar assay methods. Modification of the acyl chains or the sn-2 ester affected the apparent Km but had only small effects on Vm; modifications of the sn-1 ester, sn-3 methylene, or sn-3 hydroxyl had large effects on the apparent Vm and smaller effects on Km. Consistent with these observations, diacylglycerol analogues modified only in the acyl chains or sn-2 ester were not diacylglycerol kinase inhibitors, whereas analogues with substitutions of the sn-1 ester or sn-3 hydroxyl frequently caused inhibition. A hydrogen bond-donating group was required for an analogue to be a diacylglycerol kinase inhibitor. Studies of diacylglycerol kinase inactivation by the various analogues were consistent with the previous conclusion that this process involves an interaction of diacylglycerols with an enzyme conformation different from that active in catalysis (Walsh, J. P., and Bell, R. M. (1986) J. Biol. Chem. 261, 15062-15069). Studies with a water-soluble diacylglycerol, sn-1,2-dibutyrylglycerol, allowed direct comparison of diacylglycerol kinase activity in mixed micelles with that in native membranes. The results are discussed in relation to the structural requirements of other diacylglycerol-dependent enzymes.     

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).     

Evidence that increasing the cellular content of eicosapentaenoic acid does not reduce the biosynthesis of platelet-activating factor

Our study has examined platelet-activating factor (PAF) biosynthesis in neutrophils from individuals on a fish oil-enriched diet and in mast cells enriched with eicosapentaenoic acid (EPA) in vitro. Neutrophils isolated from males who were fed fish oil supplement (EPA; 2.8 g/day) for 5 wk contained large quantities of eicosapentaenoate in phosphatidylcholine (PC) and phosphatidylethanolamine and less in phosphatidylinositol. The ratio arachidonate/eicosapentaenoate in PC and phosphatidylethanolamine decreased from greater than 10 before the enriched diet to approximately 3 after the diet. The putative precursor of PAF, 1-O-alkyl-2-acyl-sn-glycero-3-phosphocholine (1-O-alkyl-2-acyl-GPC) contained the bulk of eicosapentaenoate in PC subclasses with smaller quantities found in 1-acyl and 1-alk-1'-enyl linked species. Ionophore A23187-stimulated neutrophils produced similar quantities of PAF before and after enriched diet. Neutrophils during normal diet acylated 1-O-alkyl-2-lyso-GPC only with arachidonate whereas neutrophils from individuals on enriched diet transferred both arachidonate and eicosapentaenoate into exogenously-provided 1-O-alkyl-2-lyso-GPC. This allowed for the labeling of neutrophils with 1-O-[3H]-alkyl-2-arachidonoyl-GPC (before diet) as well as neutrophils with 1-O-[3H]-alkyl-2-eicosapentaenoyl-GPC and 1-O-[3H]-alkyl-2-arachidonoyl-GPC (after diet). Neutrophils after diet converted similar quantities of these labeled precursors to labeled PAF upon stimulation as those before the diet. Analysis of the nature of the long chain acyl residue remaining in the sn-2 position of 1-alkyl-2-acyl-GPC after cell stimulation indicated that arachidonate and eicosapentaenoate were both released from 1-O-alkyl-2-acyl-GPC at comparable rates. Finally, in vitro supplementation of murine mast cells (PT-18) with arachidonic acid or EPA caused a marked increase in the amount of PAF produced by the cell without having any effect on histamine release. Data from these experiments suggest that EPA is incorporated into a PAF precursor pool. However, this appears not to inhibit PAF production because phospholipase A2 can use eicosapentaenoate- as well as arachidonate-containing phospholipids in the initial step of PAF biosynthesis.     

Synthesis of platelet-activating factor by polymorphonuclear neutrophils stimulated with interleukin-8.

Human interleukin-8 (IL-8) was evaluated for its capability to induce the synthesis and release of platelet-activating factor (PAF) from human polymorphonuclear neutrophils (PMN). IL-8 promotes in a dose-dependent fashion (1-100 ng/ml) a rapid synthesis of PAF, which is only partially released. The synthesis of PAF is preceded by the activation of acetyl-CoA: 1-alkyl-2-lyso-sn-glycero-3-phosphocholine acetyl-transferase, suggesting that IL-8 activates the "remodeling pathway" of PAF synthesis. By thin layer chromatography and reverse-phase high pressure liquid chromatography, we demonstrated that PAF synthesized by human PMN stimulated with IL-8 is heterogeneous: the 2-acetylated phospholipids having the biological and physicochemical characteristics of PAF include the 1-O-alkyl form, which is produced in large extent (51%), and the 1-acyl form (20%). The analysis of the individual molecular species of radyl chain indicated nine peaks, 16:0 and 18:0 being the predominant forms. These results identify PAF as a direct product of IL-8 stimulation in PMN.     

Formation of 1-alkyl-2-acetyl-sn-glycerols via the de novo biosynthetic pathway for platelet-activating factor. Characterization of 1-alkyl-2-acetyl-sn-glycero-3-phosphate phosphohydrolase in rat spleens

1-Alkyl-2-acetyl-sn-glycerol (alkylacetyl-G) is an important intermediate in the biosynthesis of 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine (platelet-activating factor) from 1-alkyl-2-lyso-sn-glycero-3-phosphate (alkyllyso-GP) via the de novo pathway. In the present investigation, we have characterized a 1-alkyl-2-acetyl-sn-glycero-3-phosphate (alkylacetyl-GP) phosphohydrolase in rat spleens that catalyzes the conversion of alkylacetyl-GP to alkylacetyl-G. The bulk of the enzymatic activity (53%) is located in the microsomal fraction, whereas 28% of the activity is present in mitochondria. The microsomal enzyme has an optimal pH of 7.0-7.4, an "apparent" Km of 31.8 microM for alkylacetyl-GP, and is widely distributed in various rat tissues. Studies of alkylacetyl-GP phosphohydrolase with respect to substrate specificity, pH profiles, sensitivities to temperature, and effects of detergent, ethanol, or cations indicate the activity of this enzyme can be distinguished from the activities of a nonspecific phosphomonoesterase or phosphatidate phosphohydrolase. Like alkyllyso-GP:acetyl-CoA acetyltransferase, the alkylacetyl-GP phosphohydrolase shows no notable substrate selectivities with regard to variations in alkyl chain length (C16:0 versus C18:0) at the sn-1 position or short chain acyl groups (C2:0 to C6:0, with the exception of C3:0) at the sn-2 position of the glycerol moiety. The enzymatic activity of alkylacetyl-GP phosphohydrolase is 30-90-fold higher than alkyllyso-GP:acetyl-CoA acetyltransferase in most tissues examined. Even though alkyllyso-GP is a substrate for alkyllyso-GP:acetyl-CoA acetyltransferase, it can also be degraded by alkylacetyl-GP phosphohydrolase. Thus, our findings coupled with earlier results imply that specificities of the molecular species of platelet-activating factor synthesized de novo are determined by the enzyme involved in the final step of this pathway, the dithiothreitol-insensitive alkylacetyl-G:CDP-choline cholinephosphotransferase. Furthermore, alkyl-lyso-GP:acetyl-CoA acetyltransferase appears to be the rate-limiting step in the de novo synthesis of alkylacetyl-G.     

Stimulated neutrophils produce an ethanolamine plasmalogen analog of platelet-activating factor

Human neutrophils stimulated by ionophore A23187 incorporate [3H]acetate into platelet-activating factor and an additional product which is chromatographically similar to phosphatidylethanolamine and accounts for approximately 25% of the [3H]acetate-containing lipids. Three general approaches indicated the sn-1 moiety of the unknown phospholipid is primarily alk-1'-enyl-linked: 1) approximately 80% of the intact phospholipid as well as its derivatives was highly sensitive to hydrolysis by HCl, 2) 80% of the product which resulted from treating the unknown with phospholipase C and acetylating the free hydroxyl group at the sn-3 position, chromatographed with authentic 1-O-alk-1'-enyl-2,3-diacetylglycerol, and 3) catalytic hydrogenation of the diacetylglycerol product described in 2) resulted in a product which chromatographed with alkyldiacetylglycerol and was not sensitive to strong acid. Treatment of the intact phospholipid with phospholipase A2 resulted in the release of 88% of the radiolabel into the acidified aqueous phase of the extraction mixture, indicating the moiety in the sn-2 position remained as acetate and had not been elongated to fatty acid. The head group was determined to be phosphoethanolamine based upon its complete conversion to the dinitro- and trinitrophenyl derivatives by the amine-derivatizing reagents fluorodinitrobenzene and trinitrobenzenesulfonic acid, respectively. From these data is was concluded that the unknown product is 1-O-alk-1'-enyl-2-acetyl-sn-glycero-3-phosphoethanolamine (80%), and 1-O-alkyl-2-acetyl-sn-glycero-3-phosphoethanolamine (10%). Sonicates prepared from neutrophils stimulated with ionophore A23187 contained an acetyltransferase activity capable of utilizing 1-O-alk-1'-enyl-2-lyso-sn-glycero-3-phosphoethanolamine and [14C]acetyl-CoA to produce the product identified as 1-O-alk-1'-enyl-2-acetyl-sn-glycero-3-phosphoethanolamine.