Packing and electrostatic behavior of sn-2-docosahexaenoyl and -arachidonoyl phosphoglycerides

Mammalian synaptic membranes appear to contain high proportions of specific, sn-1-stearoyl-2-docosahexaenoyl- and sn-1-stearoyl-2-arachidonoyl phosphoglycerides, but the structural significance of this is unclear. Here we used a standardized approach to compare the properties of homogeneous monolayers of the corresponding phosphatidylcholines, phosphatidylethanolamines, phosphatidylserines, and phosphatidic acids with those of control monolayers of sn-1-stearoyl-2-oleoyl- and sn-1-palmitoyl-2-oleoyl phosphoglycerides. Major findings were: 1), that the presence of an sn-2-docosahexaenoyl group or an sn-2-arachidonoyl group increases the molecular areas of phosphoglycerides by 3.8 A(2) (7%) relative to the presence of an sn-2-oleoyl group; 2), that the phosphorylcholine headgroup independently increases molecular areas by a larger amount, 7.1 A(2) (13%); and 3), that the dipole moments of species having an arachidonoyl moiety or an oleoyl moiety are 83 mD (19%) higher than those of comparable docosahexaenoic acid-containing phosphoglycerides. These and other results provide new information about the molecular packing properties of polyenoic phosphoglycerides and raise important questions about the role of these phosphoglycerides in synapses.     

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.     

Biosynthetic incorporation of cis-parinaric acid into radioactive sn-3-phosphatidic acid

Isolated guinea pig liver microsomal membranes catalyzed the incorporation of naturally occurring cis-parinaric acid into sn-3-[U-14C]glycerophosphate. This resulted in the formation of sn-3-[14C](parinaroyl)phosphatidic acid, which was isolated by Chelex-100 and DEAE-cellulose column chromatography and further purified by Sephadex-G 25. The sn-3-[14C](parinaroyl)phosphatidic acid thus obtained exhibited absorption and fluorescence spectra substantially different from the cis-parinaric acid. Distribution of the incorporated cis-parinaric acid between the hydroxyl groups of biosynthesized sn-3-[14C]phosphatidic acid was determined by degradation with Crotalus adamanteus venom. This established that the major portion of the incorporated cis-parinaric acid esterified the secondary hydroxyl group in the sn-3-[14C]phosphatidic acid, while the primary hydroxyl group was esterified to a significantly lesser degree. The similarity between the biochemical incorporation of isomeric doxyl stearic acids into lipids of biological membranes and that of cis-parinaric acid into sn-3-phosphatidic acid described in this communication are discussed in relation to the possible use of these probes in studies of intact biological membranes.     

Cell cycle-dependent changes in arachidonic acid and glycerol metabolism in Swiss 3T3 cells stimulated by platelet-derived growth factor

Quiescent Swiss 3T3 cells stimulated to divide by human platelet-derived growth factor (PDGF) were used to investigate cell cycle-dependent changes in arachidonic acid, stearic acid, and glycerol metabolism. PDGF at 12 ng/ml stimulated incorporation of labeled arachidonic and stearic acid into phosphatidic acid and phosphatidylinositol within 60 min. With similar kinetics PDGF stimulated glycerol incorporation into phosphatidic acid and phosphatidylinositol indicating early growth factor-dependent stimulation of de novo phosphatidylinositol synthesis. This early effect of PDGF was specific for the phosphatidylinositol synthesis pathway since no comparable changes were noted in other glycerolipids. After a lag of 4-6 h, PDGF strongly stimulated arachidonic acid incorporation into triacylglycerol: at 6 h, arachidonate radioactivity in triacylglycerol exceeded that in phosphatidylcholine, phosphatidylethanolamine, and phosphatidylinositol. This effect of PDGF was not associated with de novo triacylglycerol synthesis since no increase in the rate of glycerol incorporation into this lipid was noted. Finally, PDGF stimulated incorporation of glycerol into all major phospholipids and triacylglycerol during S-phase. These results disclose three novel effects of PDGF on glycerolipid metabolism in Swiss 3T3 cells: 1) early selective activation of the phosphatidylinositol synthesis pathway; 2) delayed strong stimulation of arachidonic acid incorporation into triacylglycerol; and 3) late induction of de novo phosphatidylcholine, phosphatidylethanolamine, and triacylglycerol synthesis. These PDGF effects are likely to play important roles in phosphatidylinositol metabolism, membrane biosynthesis, and fatty acid turnover in rapidly growing cells.     

Metabolic regulation during early frog development: flow of glycolytic carbon into phospholipids in Xenopus oocytes and fertilized eggs

32P-labeled glucose 6-phosphate, [32P]phosphoenolpyruvate, and [gamma-32P]ATP were injected into oocytes and fertilized eggs of Xenopus laevis, and the incorporation of the 32P label was followed into phospholipids. Several classes of phospholipids incorporated 32P label from the injected glycolytic intermediates, including lysophosphatidic acid, phosphatidic acid, phosphatidylinositol, and phosphatidylinositol phosphates, inferring de novo synthesis of these lipids from dihydroxyacetone phosphate or glycerol 3-phosphate. Injection of [gamma-32P]ATP into oocytes and fertilized eggs led to labeling of phosphatidylinositol phosphate and phosphatidylinositol bisphosphate, indicating an active phosphatidylinositol cycle in resting oocytes and fertilized eggs. Maturation and fertilization of the oocyte led to a qualitative change in phosphatidylinositol metabolism, increased labeling of phosphatidylinositol phosphate compared to phosphatidylinositol bisphosphate (either from glycerol 3-phosphate or from ATP). This change occurs late in the maturation process, and the new pattern of phosphatidylinositol metabolism is maintained during the rapid cleavage stages of early embryogenesis.     

In vivo studies on stereospecificity of the monoglyceride kinase, lysophosphatidic acid acyltransferase, phosphatidic acid phosphatase and CDP-diglyceride synthase of Streptococcus mutans BHT using the stereoisomers of the ether lipid, dodecylglycerol

Streptococcus mutans BHT metabolizes radioactive 3-dodecyl-sn-glycerol (sn-3-DDG) almost exclusively to lysophosphatidic acid, phosphatidic acid and 1,3-diradyl-sn-glycerol, whereas the cells of this organism metabolize 1-dodecyl-sn-glycerol (sn-1-DDG) to all of the glycerol lipids of S. mutans BHT, with the largest amounts incorporated into phosphatidylglycerol and diradylglycerol (mostly the 1,2- but also the 1,3-isomer). (The common names of lipids, such as phosphatidic acid, are used in the broader sense to mean that the lipid may contain alkyl as well as acyl groups.) The addition of an equivalent amount of nonradioactive sn-3-DDG to radioactive sn-1-DDG causes more of the radioactivity to accumulate at phosphatidic acid. These results indicate that the monoglyceride kinase (EC 2.7.1.94), lysophosphatidic acid acyltransferase (EC 2.3.1.40) and the monoglyceride acyltransferase (EC 2.3.1.22) enzymatic reactions are not stereospecific, and that the CDP-diglyceride synthase (EC 2.7.7.41) and phosphatidic acid phosphatase (EC 3.1.3.4) metabolic steps are stereospecific in S. mutans BHT. The synthesis of phosphatidic acid and lysophosphatidic acid from sn-3-DDG provides a unique method for synthesizing these glycerol lipids with the uncommon stereochemical configuration in which the phosphate moiety is in the sn-1 position.     

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.     

Bradykinin Stimulates Arachidonic Acid Release Through the Sequential Actions of an sn-1 Diacylglycerol Lipase and a Monoacylglycerol Lipase

In cultured dorsal root ganglion (DRG) neurons prelabeled with [3H]arachidonic acid [( 3H]AA), bradykinin (BK) stimulation resulted in increased levels of radioactive diacylglycerol, monoacylglycerol, and free AA. The transient increases in content of radioactive diacylglycerol and monoacylglycerol preceded the increase in level of free AA, suggesting the contribution of a diacylglycerol lipase pathway to AA release. An analysis of the molecular species of diacylglycerols in unstimulated cultures revealed the presence of two primary [3H]AA-containing species, 1-palmitoyl-2-arachidonoyl and 1-stearoyl-2-arachidonoyl diacylglycerol. BK stimulation resulted in a preferential increase in content of 1-stearoyl-2-arachidonoyl diacylglycerol. When DRG cultures were labeled with [3H]stearic acid, treatment with BK increased the amount of label in diacylglycerol and free stearic acid, but not in monoacylglycerol. This result suggested that AA release occurred through the successive actions of an sn-1 diacylglycerol lipase and monoacylglycerol lipase. Other data supporting a diacylglycerol lipase pathway was the significant inhibition of [3H]AA release and consequent accumulation of diacylglycerol by RG 80267, which preferentially inhibits diacylglycerol lipase. Analysis of the molecular species profiles of individual phospholipids in DRG neurons indicated that phosphoinositide hydrolysis may account for a significant portion of the rapid increase in content of 1-stearoyl-2-arachidonoyl diacylglycerol. We were unable to obtain evidence that the phospholipase A2 pathway makes a significant contribution to BK-stimulated AA release in DRG cultures. Under our assay conditions there were no BK-stimulated increases in levels of radioactive lysophosphatidylinositol, lysophosphatidylcholine, or lysophosphatidylethanolamine in cultures prelabeled with [3H]inositol, [3H]choline, or [3H]-ethanolamine, respectively.     

Pancreatic islets synthesize phospholipids de novo from glucose via acyl-dihydroxyacetone phosphate

There is considerable evidence that an increased turnover of phosphoinositides and phosphatidic acid accompanies stimulus-induced insulin release. As glucose metabolism via glycolysis produces precursors for phospholipid synthesis, the time course of incorporation of [U14C] labelled glucose was measured to determine the pathways of triose carbon incorporation into phospholipids in the islet. Cultured islets were stimulated with glucose 2.7 or 33 mM. The labelled phospholipids present after stimulation were acyldihydroxyacetone phosphate, lysophosphatidic acid, phosphatidic acid and phosphatidylinositol. Acyl-dihydroxyacetone phosphate rose promptly within 1 minute of raising the glucose concentration and was the primary acylated triose labelled during the first 15 minutes. It was possible to show in vitro conversion of [U14C] glucose-derived acyl-dihydroxyacetone phosphate to lysophosphatidic acid and phosphatidic acid in the presence of NADPH (100 microM), indicating the presence in the islet of acyl-dihydroxyacetone phosphate: NADP oxidoreductase and acyl CoA:1 acylglycerol-3-phosphate acyl transferase, respectively. This study suggests that de novo synthesis of phosphatidic acid provides a link between glucose metabolism and the release of insulin.     

Studies on the antibacterial activity of dodecylglycerol. Its limited metabolism and inhibition of glycerolipid and lipoteichoic acid biosynthesis in Streptococcus mutans BHT

Growth-inhibitory concentrations of racemic sn-1(3)-dodecylglycerol inhibit the incorporation of [14C] glycerol into lipids and lipoteichoic acid of Streptococcus mutans BHT and alter the per cent composition of the glycerolipids. Increases in phosphatidic acid and diphosphatidylglycerol (at the expense of phosphatidylglycerol) contribute the most to the change in lipid composition. No cellular lysis occurs under these conditions. Radioactive racemic sn-1(3)-dodecylglycerol is readily taken up by the cell and is metabolized primarily to lysophosphatidic acid and phosphatidic acid with smaller amounts converted to phosphatidylglycerol and diacylglycerol. The accumulation of phosphatidic acid and the loss of viability respond in parallel to different concentrations of dodecylglycerol. An increase in CTP is also observed which together with the increase in phosphatidic acid suggests a possible impairment in the synthesis of CDP-diacylglycerol.     

Substrate selectivity of plant and microbial lysophosphatidic acid acyltransferases

Linoleic acid (18:2) is found in a large variety of plant oils but to date there is limited knowledge about the substrate selectivity of acyltransferases required for its incorporation into storage triacylglycerols. We have compared the incorporation of oleoyl (18:1) and linoleoyl (18:2) acyl-CoAs onto lysophosphatidic acid acceptors by sub-cellular fractions prepared from a variety of plant and microbial species. Our assays demonstrated: (1). All lysophosphatidic acid acyltransferase (LPA-AT) enzymes tested incorporated 18:2 acyl groups when presented with an equimolar mix of 18:1 and 18:2 acyl-CoA substrates. The ratio of 18:1 to 18:2 incorporation into phosphatidic acid varied between 0.4 and 1.4, indicating low selectivity between these substrates. (2). The presence of either stearoyl (18:0) or oleoyl (18:1) groups at the sn-1 position of lysophosphatidic acid did not affect the selectivity of incorporation of 18:1 or 18:2 into the sn-2 position of phosphatidic acid. (3). All LPA-AT enzymes tested incorporated the saturated palmitoyl (16:0) acyl group from equimolar mixtures of 16:0- and 18:1-CoA. The ratios of 18:1 to 16:0 incorporation are generally much higher than those of 18:1 to 18:2 incorporation, varying between 2.1 and 8.6. (4). The LPA-AT from oil palm kernel is an exception as 18:1 and 16:0 are utilised at comparable rates. These results show that, in the majority of species examined, there is no correlation between the final sn-2 composition of oil or membrane lipids and the ability of an LPA-AT to use 18:2 as a substrate in in vitro assays.     

Metabolic fate of plasma membrane diacylglycerols in NIH 3T3 fibroblasts.

We have examined the metabolism of three radiolabeled 1,2-diacylglycerols (DGs) in NIH 3T3 fibroblasts. Since the lipids used are not appreciably taken up by the cells, we used a phosphatidylserine (PS)-based liposome fusion system to rapidly associate the lipid species with the plasma membrane. When 1,2-[1-14C]dioleoyl-sn-3-glycerol ([14C]DOG) is delivered in this way, it is rapidly converted predominantly to phosphatidylcholine (PC) and triacylglycerol (TG) and to a lesser extent, to monoacylglycerol (MG) and fatty acids (FA), as well as phosphatidic acid (PA) and phosphatidylinositol (PI). We present evidence that [14C] DOG is largely utilized as an intact molecule rather than being broken down to FA and then incorporated to cell lipids. Examination of the metabolism of 1-stearoyl-2-[1-14C]myristoyl-sn-3-glycerol ([14C]SMG) and 1-stearoyl-2-arachidonoyl-sn-3-glycerol ([14C]SAG) reveal important differences. Both produce substantial labeling of PC but [14C]SMG gives rise to the highest proportion of TG and the lowest of PA and PI, whereas [14C]SAG yields the opposite pattern. When phosphatidic acid labeled on its glycerol backbone (1,2-dioleoyl-sn-[U-14C] glycero-3-phosphate) was supplied to the cells via the liposomes, rapid appearance of labeled DG was found which then decreased with concomitant labeling of cellular PC and TG. Only small amounts of the glycerol backbone were recovered in PI. Our experiments identify three types of processes involved in the metabolism of plasma membrane DGs: (i) transferase-catalyzed conversions to PC and TG, (ii) lipolytic breakdown to MG and FA, and (iii) phosphorylation to PA and then conversion to PI. The relative proportions of each DG species converted to these different products are strongly dependent on the fatty acyl composition of the particular DG molecular species, even though formation of PC is the major event in all cases. Since DGs are important second messengers, our study supports the view that conversion to PC and TG can play a key role in DG signal attenuation.     

Biogenesis of mitochondria. Phospholipid synthesis in vitro by yeast mitochondrial and microsomal fractions

The ability in vitro of yeast mitochondrial and microsomal fractions to synthesize lipid de novo was measured. The major phospholipids synthesized from sn-[2-(3)H]glycerol 3-phosphate by the two microsomal fractions were phosphatidylserine, phosphatidylinositol and phosphatidic acid. The mitochondrial fraction, which had a higher specific activity for total glycerolipid synthesis, synthesized phosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine and phosphatidic acid, together with smaller amounts of neutral lipids and diphosphatidylglycerol. Phosphatidylcholine synthesis from both S-adenosyl[Me-(14)C]methionine and CDP-[Me-(14)C]choline appeared to be localized in the microsomal fraction.     

A novel phosphatidylcholine which contains pentadecanoic acid at sn-1 and docosahexaenoic acid at sn-2 in Schizochytrium sp. F26-b

Docosahexaenoic acid (DHA, 22:6n-3)-containing phospholipids are a ubiquitous component of the central nervous system and retina, however their physiological and pharmacological functions have not been fully elucidated. Here, we report a novel DHA-containing phosphatidylcholine (PC) in a marine single cell eukaryote, Schizochytrium sp. F26-b. Interestingly, 31.8% of all the fatty acid in F26-b is DHA, which is incorporated into triacylglycerols and various phospholipids. In phospholipids, DHA was found to make up about 50% of total fatty acid. To identify phospholipid species containing DHA, the fraction of phospholipids from strain F26-b was subjected to normal phase high-performance liquid chromatography (HPLC). It was found that DHA was incorporated into PC, lyso-PC, phosphatidylethanolamine, and phosphatidylinositol. The major DHA-containing phospholipid was PC in which 32.5% of the fatty acid was DHA. The structure of PC was analyzed further by phospholipase A2 treatment, fast atom bombardment mass spectrometry, and 1H- and 13C-NMR after purification of the PC with reverse phase HPLC. Collectively, it was clarified that the major PC contains pentadecanoic acid (C15:0) at sn-1 and DHA at sn-2; the systematic name of this novel PC is therefore "1-pentadecanoyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine."     

Rapid changes in phospholipid metabolism in the nuclei of Swiss 3T3 cells induced by treatment of the cells with insulin-like growth factor I

When highly-purified nuclei of Swiss-mouse 3T3 cells are incubated with [32P]-gamma ATP, radioactivity is incorporated into phosphatidic acid and the two polyphosphoinositol lipids, phosphatidylinositol(4)P and phosphatidylinositol(4,5)P2. If the cells are pre-treated with insulin-like growth factor I, this incorporation into the phospholipids is decreased. The effect is maximal by 2 minutes, is transient in that it has disappeared by one hour, and is increased markedly by the co-addition of bombesin, even though bombesin alone has no effect. We suggest that some aspect of polyphosphoinositide metabolism is altered in the nucleus (leading to a decreased radiolabelling) when the cells are treated with mitogenic growth factors, and that this change in inositide metabolism is a very early event in the sequence leading to cell division.     

The highly selective production of 2-arachidonoyl lysophosphatidylcholine catalyzed by purified calcium-independent phospholipase A2gamma: identification of a novel enzymatic mediator for the generation of a key branch point intermediate in eicosanoid signaling

Herein, we report the heterologous expression of the human peroxisomal 63-kDa calcium-independent phospholipase A2gamma (iPLA2gamma) isoform in Sf9 cells, purification of the N-terminal His-tagged enzyme by affinity chromatography, and the identification of its remarkable substrate selectivity that results in the highly selective generation of 2-arachidonoyl lysophosphatidylcholine. Mass spectrometric analyses demonstrated that purified iPLA2gamma hydrolyzed saturated or monounsaturated aliphatic groups readily from either the sn-1 or sn-2 positions of phospholipids. In addition, purified iPLA2gamma effectively liberated arachidonic acid from the sn-2 position of plasmenylcholine substrates. In contrast, incubation of iPLA2gamma with 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine resulted in the rapid release of palmitic acid and the selective accumulation of 2-arachidonoyl lysophosphatidylcholine (LPC), which was not metabolized further by iPLA2gamma. The putative regiospecificity of the 2-arachidonoyl LPC product was authenticated by its diagnostic fragmentation pattern during tandem mass spectrometric analysis. To identify the physiological relevance of iPLA2gamma-mediated 2-arachidonoyl LPC production utilizing naturally occurring membranes, we incubated purified rat hepatic peroxisomes with iPLA2gamma and similarly identified the selective accumulation of 2-arachidonoyl LPC. Furthermore, tandem mass spectrometric analysis demonstrated that 2-arachidonoyl LPC is a natural product in human myocardium, a tissue in which iPLA2gamma expression is robust. Because 2-arachidonoyl LPC represents a key branch point intermediate that can potentially lead to a variety of bioactive molecules in eicosanoid signaling (e.g. arachidonic acid, 2-arachidonoylglycerol), these results have uncovered a novel eicosanoid selective pathway through iPLA2gamma-mediated 2-arachidonoyl LPC production to amplify and diversify the repertoire of biologic lipid second messengers in response to cellular stimulation.     

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.     

Lipid composition of Trypanosoma cruzi.

1. Lipid composition of Trypanosoma cruzi epimastigote form in culture consist of 35% of phospholipids and 65% of neutral lipids. 2. Among the phospholipids, phosphatidylcholine is the more abundant (44%), followed by phosphatidylethanolamine (28%), phosphatidylinositol (12%), sphingomyelin (4%), and smaller amounts of cardiolipin, phosphatidic acid, lysolecithin, phosphatidylserine (traces), and an unidentified phospholipid (3%). 3. Pulse labeling with 32P showed highest specific incorporation in phosphatidylethanolamine, followed by phosphatidylinositol and phosphatidylcholine, suggesting a more active role for phosphatidylethanolamine in these organisms.     

Ca2+-translocation activities of phosphatidylinositol, diacylglycerol and phosphatidic acid inferred by quin-2 in artificial membrane systems

Ca2+-translocating activities of phosphatidylinositol, diacylglycerol and phosphatidic acid were investigated in phosphatidylcholine liposomes. Using a fluorescent indicator of Ca2+ concentration, quin-2, release of encapsulated Ca2+ from egg yolk phosphatidylcholine liposomes containing 2 mol% of one of these lipids was measured at 37 degrees C. The rate of Ca2+ translocation across the liposomal membrane mediated by phosphatidic acid was about 3-fold larger than those mediated by phosphatidylinositol and diacylglycerol. The result implies that phosphatidic acid has Ca2+-ionophore activity in the agonist dependent metabolism of inositol phospholipids. The ionophoretic activity depended on the degree of unsaturation of the fatty acyl chains. The Ca2+ translocation rate was smallest in dipalmitoylphosphatidic acid, and it increased in the order of dioleoyl-, dilinoleoyl- and dilinolenoyl-phosphatidic acid. Ca2+ mobilization of a stimulated cell is discussed in the light of Ca2+-ionophore activity of phosphatidic acid converted from inositol phospholipids.     

CGI-58/ABHD5 is a coenzyme A-dependent lysophosphatidic acid acyltransferase

Mutations in human CGI-58/ABHD5 cause Chanarin-Dorfman syndrome (CDS), characterized by excessive storage of triacylglycerol in tissues. CGI-58 is an α/β-hydrolase fold enzyme expressed in all vertebrates. The carboxyl terminus includes a highly conserved consensus sequence (HXXXXD) for acyltransferase activity. Mouse CGI-58 was expressed in Escherichia coli as a fusion protein with two amino terminal 6-histidine tags. Recombinant CGI-58 displayed acyl-CoA-dependent acyltransferase activity to lysophosphatidic acid, but not to other lysophospholipid or neutral glycerolipid acceptors. Production of phosphatidic acid increased with time and increasing concentrations of recombinant CGI-58 and was optimal between pH 7.0 and 8.5. The enzyme showed saturation kinetics with respect to 1-oleoyl-lysophosphatidic acid and oleoyl-CoA and preference for arachidonoyl-CoA and oleoyl-CoA. The enzyme showed slight preference for 1-oleoyl lysophosphatidic acid over 1-palmitoyl, 1-stearoyl, or 1-arachidonoyl lysophosphatidic acid. Recombinant CGI-58 showed intrinsic fluorescence for tryptophan that was quenched by the addition of 1-oleoyl-lysophosphatidic acid, oleoyl-CoA, arachidonoyl-CoA, and palmitoyl-CoA, but not by lysophosphatidyl choline. Expression of CGI-58 in fibroblasts from humans with CDS increased the incorporation of radiolabeled fatty acids released from the lipolysis of stored triacylglycerols into phospholipids. CGI-58 is a CoA-dependent lysophosphatidic acid acyltransferase that channels fatty acids released from the hydrolysis of stored triacylglycerols into phospholipids.