Purification and kinetic properties of lysophosphatidylinositol acyltransferase from bovine heart muscle microsomes and comparison with lysophosphatidylcholine acyltransferase

The enzyme acyl-CoA:1-acyl-sn-glycero-3-phosphoinositol acyltransferase (LPI acyltransferase, EC 2.3.1.23) was purified approximately 11,000-fold to near homogeneity from bovine heart muscle microsomes. The purification was effected by extraction with the detergent 3-((3-cholamidopropyl)dimethylammonio)-1-propanesulfonate, followed by chromatography on Cibacron blue agarose, DEAE-cellulose, and Matrex gel green A. The isolated enzyme was a single protein of 58,000 Da as measured by polyacrylamide gel electrophoresis in the presence of dodecyl sulfate. This purification procedure also allows isolation of the related enzyme lysophosphatidylcholine (LPC) acyltransferase, which was separated from LPI acyltransferase at the final chromatographic step. The purified LPI acyltransferase exhibits an absolute specificity for LPI as the acyl acceptor. Broader specificity was found for acyl-CoA derivatives as substrates, although the preferred substrates are long-chain, unsaturated derivatives: measured reactivities were in the order arachidonoyl-CoA greater than oleoyl-CoA greater than eicosadienoyl-CoA greater than linoleoyl-CoA. Little activity was found with palmitoyl-CoA or stearoyl-CoA as potential substrates. These properties are consistent with a role of the enzyme in controlling the acyl group composition of phosphoinositides. Comparison of LPC acyltransferase and LPI acyltransferase shows that these two enzymes have distinct kinetic and physical properties and are affected differently by local anesthetics, which are potent inhibitors.     

Acylation of lysophosphatidylcholine in bovine heart muscle microsomes: purification and kinetic properties of acyl-CoA:1-acyl-sn-glycero-3-phosphocholine O-acyltransferase

Bovine heart muscle microsomes rapidly convert lysophosphatidylcholine (LPC) into phosphatidylcholine (PC) in the presence of oleoyl-CoA. Both substrates are incorporated into the product, although the rate of incorporation of radiolabel into PC from 1-[14C]palmitoyl-LPC was approximately threefold higher than the rate of incorporation from [14C]oleoyl-CoA. Furthermore, the rate of incorporation of radiolabel from [14C]LPC was stimulated fivefold by the presence of oleoyl-CoA. These results demonstrate the presence of both acyl-CoA:1-acyl-sn-glycero-3-phosphocholine O-acyltransferase (EC 2.3.1.23) and an LPC:LPC transacylase (EC 3.1.1.5) in microsomes. Separation of the two enzymatic activities and purification of the acyltransferase was achieved by a procedure involving extraction with 3-[3-cholamidopropyl)dimethylammonio)-1-propanesulfonate detergent and chromatography on DEAE-cellulose, Reactive blue agarose, and Matrex gel green A. The isolated acyltransferase was a single species of 64,000 Da as judged by polyacrylamide gel electrophoresis in the presence of dodecyl sulfate. The substrate specificity of the enzyme was studied by using a series of lysophospholipids as acyl acceptors and acyl-CoA derivatives as acyl donors. The enzyme was catalytically active with LPC as acyl acceptor but displayed little or no activity with lysophosphatidylethanolamine, lysophosphatidylinositol, or lysophosphatidylserine. Of the LPC derivatives tested, the highest activity was obtained with 1-palmitoyl-LPC. Wider specificity was exhibited for the nature of the acyl donor, for which arachidonoyl-CoA, linoleoyl-CoA, and oleoyl-CoA were highly active substrates. These properties of the acyltransferase are in accord with a role of the enzyme in determining the composition of PC in myocardium.     

Acyltransferase activities in adult rat type II pneumocyte-derived subcellular fractions

Acyl-CoA: lysophosphatidylcholine, acyl-CoA: lysophosphatidylethanolamine, and lysophosphatidylcholine:lysophosphatidylcholine acyltransferases were investigated using subcellular fractions derived from adult rat type II pneumocytes in primary culture. Acyl-CoA:lysophospholipid acyltransferase activities were determined to be microsomal, while lysophosphatidylcholine:lysophosphatidylcholine acyltransferase activity was found to be cytosolic. Total palmitoyl CoA:lysophosphatidylcholine acyltransferase activity was 30-fold greater than lysophosphatidylcholine:lysophosphatidylcholine acyltransferase activity, indicating that the former enzyme is more important in the synthesis of dipalmitoyl phosphatidylcholine. Palmitoyl-CoA and oleoyl-CoA lysophosphatidylcholine acyltransferase activities were approximately equal under optimal substrate conditions. Specific activities of the enzyme using arachidoyl-CoA and arachidonoyl-CoA were 46% and 18%, respectively, of those with palmitoyl-CoA. Acyl-CoA:lysophosphatidylethanolamine acyltransferase showed a preference for palmitoyl-CoA as opposed to oleoyl-CoA under optimal conditions. However, when equimolar concentrations of either palmitoyl-CoA and oleoyl-CoA or palmitoyl-CoA and arachidoyl-CoA were assayed together, the relative utilization of the two substrates was found to be dependent on total acyl-CoA concentration. At higher concentrations, the incorporation of palmitoyl-CoA into phosphatidylcholine was less than other acyl-CoAs. However, at lower concentrations palmitoyl-CoA was utilized quite selectively. Whole lung microsomes did not show as marked a preference for palmitoyl-CoA as did type II pneumocyte microsomes under these same conditions. In similar experiments, low total acyl-CoA concentrations produced greater incorporation of oleoyl-CoA into phosphatidylethanolamine. For both enzymes total activity at the lowest concentrations used was at least 45% that at optimal conditions. This demonstrates that the type II pneumocyte acyltransferase system(s) can selectively utilize palmitoyl-CoA. No evidence for direct exchange of palmitoyl-CoA with 1-saturated-2-unsaturated phosphatidylcholine in subcellular fractions from type II pneumocytes was found.     

Identification, purification, and characterization of monoacylglycerol acyltransferase from developing peanut cotyledons

Biosynthesis of diacylglycerols in plants occurs mainly through the acylation of lysophosphatidic acid in the microsomal membranes. Here we describe the first identification of diacylglycerol biosynthetic activity in the soluble fraction of developing oilseeds. This activity was NaF-insensitive and acyl-CoA-dependent. Diacylglycerol formation was catalyzed by monoacylglycerol (MAG) acyltransferase (EC ) that transferred an acyl moiety from acyl-CoA to MAG. The enzyme was purified by successive chromatographic separations on octyl-Sepharose, blue-Sepharose, Superdex-75, and palmitoyl-CoA-agarose to apparent homogeneity from developing peanut (Arachis hypogaea) cotyledons. The enzyme was purified to 6,608-fold with the final specific activity of 15.86 nmol min(-1) mg(-1). The purified enzyme was electrophoretically homogeneous, and its molecular mass was 43,000 daltons. The purified MAG acyltransferase was specific for MAG and did not utilize any other acyl acceptor such as glycerol, glycerol-3-phosphate, lysophosphatidic acid, and lysophosphatidylcholine. The K(m) values for 1-palmitoylglycerol and 1-oleoylglycerol were 16.39 and 5.65 micrometer, respectively. The K(m) values for 2-monoacylglycerols were 2- to 4-fold higher than that of the corresponding 1-monoacylglycerol. The apparent K(m) values for palmitoyl-, stearoyl-, and oleoyl-CoAs were 17.54, 25.66, and 9.35 micrometer, respectively. Fatty acids, phospholipids, and sphingosine at low concentrations stimulated the enzyme activity. The identification of MAG acyltransferase in oilseeds suggests the presence of a regulatory link between signal transduction and synthesis of complex lipids in plants.     

Purified Rat Brain Myelin Contains Measurable Acyl-CoA:Lysophospholipid Acyltransferase(s) but Little, if Any, Glycerol-3-Phosphate Acyltransferase

Previous reports from several laboratories have demonstrated the presence of many lipid-metabolizing enzymes in myelin, including all the enzymes needed to convert diacylglycerol to phosphatidylcholine and phosphatidylethanolamine. Axonal transport studies had suggested the presence of additional enzymes which incorporate acyl chains into specific phospholipids of myelin. We report here evidence for one such group of enzymes, the acyl-CoA:lysophospholipid acyltransferases. At the same time, activity of acyl-CoA:sn-glycerol-3-phosphate acyltransferase was negligible in myelin. Oleoyl-CoA and arachidonoyl-CoA were both active substrates for transfer of acyl chains to lysophosphatidylcholine and lysophosphatidylinositol. Activity in myelin varied from 7 to 19% of microsomal activity, values well above the likely level of microsomal contamination as judged by microsomal markers. Additional evidence for a myelin locus came from assays at sequential stages of purification and from mixing experiments. Arachidonoyl-CoA was somewhat more reactive than oleoyl-CoA toward lysophosphatidylcholine; the myelin Km for these two CoA derivatives was 98 microM and 6.6 microM, respectively. Activity with lysophosphatidylinositol as substrate was approximately 40% of that with lysophosphatidylcholine in myelin, whereas activities with lysophosphatidylethanolamine and lysophosphatidylserine were considerably less.     

Lysophospholipid acyltransferases and arachidonate recycling in human neutrophils

The cycle of deacylation and reacylation of phospholipids plays a critical role in regulating availability of arachidonic acid for eicosanoid production. The major yeast lysophospholipid acyltransferase, Ale1p, is related to mammalian membrane-bound O-acyltransferase (MBOAT) proteins. We expressed four human MBOATs in yeast strains lacking Ale1p and studied their acyl-CoA and lysophospholipid specificities using novel mass spectrometry-based enzyme assays. MBOAT1 is a lysophosphatidylserine (lyso-PS) acyltransferase with preference for oleoyl-CoA. MBOAT2 also prefers oleoyl-CoA, using lysophosphatidic acid and lysophosphatidylethanolamine as acyl acceptors. MBOAT5 prefers lysophosphatidylcholine and lyso-PS to incorporate linoleoyl and arachidonoyl chains. MBOAT7 is a lysophosphatidylinositol acyltransferase with remarkable specificity for arachidonoyl-CoA. MBOAT5 and MBOAT7 are particularly susceptible to inhibition by thimerosal. Human neutrophils express mRNA for these four enzymes, and neutrophil microsomes incorporate arachidonoyl chains into phosphatidylinositol, phosphatidylcholine, PS, and phosphatidylethanolamine in a thimerosal-sensitive manner. These results strongly implicate MBOAT5 and MBOAT7 in arachidonate recycling, thus regulating free arachidonic acid levels and leukotriene synthesis in neutrophils.     

Purification of long-chain acyl-CoA hydrolase from bovine heart microsomes and regulation of activity by lysophospholipids

Long-chain acyl-CoA hydrolase (EC 3.1.2.2) has been purified 12,000-fold from bovine heart muscle microsomes by extraction with Miranol detergent, followed by column chromatography on Reactive Blue agarose and DEAE-cellulose. The purified enzyme was nearly homogeneous on polyacrylamide gel electrophoresis and had a molecular weight of 41,000 in the presence of dodecyl sulfate. The specificity and kinetic properties of the enzyme were studied using several acyl-CoA derivatives as potential substrates. The enzyme showed a wide degree of specificity with little dependence on either the fatty acyl chain length or the degree of unsaturation of the acyl group. The kinetic properties were in accord with the Michaelis-Menten equation under most conditions, although high concentrations of substrates generally inhibited the enzyme. Arachidonoyl-CoA, which was the most effective substrate, had a K_m value of 0.4 μm and a V_max value of 6.0 μmol min⁻¹ mg⁻¹. The enzyme was strongly and specifically inhibited by lysophosphatidylcholine and lysophosphatidylinositol with kinetic inhibition constants of 16 and 30 nm, respectively. Other lysolipids and detergents such as deoxycholate and Triton X-100 were weak inhibitors. These properties and others distinguish this enzyme from other acyl-CoA hydrolases and support the idea that lysophospholipids may be important in vivo in the regulation of lipid metabolism.     

Synthesis of azidophospholipids and labeling of lysophosphatidylcholine acyltransferase from developing soybean cotyledons.

A photoreactive substrate analog of lysophosphatidylcholine (LPC), 1-([(4-azidosalicyl)-12-amino)]dodecanoyl-sn-glycerol-3-phospho cholin e (azido-LPC) was synthesized. Fast atom bombardment mass spectrometry was employed to confirm the structures of azido-LPC and its intermediates. Azido-LPC was used to label putative acyl-CoA:LPC acyltransferase from microsomal membranes of developing soybean cotyledons. The synthesized substrate analog acts as a substrate for the target acyltransferases and phospholipases in the dark. When the microsomal membranes were incubated with the acyl acceptor analog and immediately photolyzed, LPC acyltransferase was irreversibly inhibited. Photoinactivation of the enzyme by the photoprobe decreased in the presence of LPC. Microsomal membranes were photolyzed with 125I-labeled azido-LPC and analyzed by SDS-PAGE followed by autoradiography. These revealed that the analog preferentially labeled 54- and 114-kDa polypeptides. Substrate protected the labeling of both the polypeptides. In our earlier report, the same polypeptides were also labeled with photoreactive acyl-CoA analogs, suggesting that these polypeptides could be putative LPC acyltransferase(s). These results demonstrated that the photoreactive phospholipid analog could be a powerful tool to label acyltransferases involved in lipid biosynthesis.     

Evidence for the reversibility of the acyl-CoA:lysophosphatidylcholine acyltransferase in microsomal preparations from developing safflower (Carthamus tinctorius L.) cotyledons and rat liver.

Acyl exchange between acyl-CoA and position 2 of sn-phosphatidylcholine occurs in the microsomal preparations of developing safflower cotyledons. Evidence is presented to show that the acyl exchange is catalysed by the combined back and forward reactions of an acyl-CoA:lysophosphatidylcholine acyltransferase (EC 2.3.1.23). The back reaction of the enzyme was demonstrated by the stimulation of the acyl exchange with free CoA and by the observation that the added CoA was acylated with acyl groups from position 2 of sn-phosphatidylcholine. Re-acylation of the, endogenously produced, lysophosphatidylcholine with added acyl-CoA occurred with the same specificity as that observed with added palmitoyl lysophosphatidylcholine. A similar acyl exchange, catalysed by an acyl-CoA:lysophosphatidylcholine acyltransferase, occurred in microsomal preparations of rat liver. The enzyme from safflower had a high specificity for oleate and linoleate, whereas arachidonate was the preferred acyl group in the rat liver microsomal preparations. The rate of the back reaction was 3-5% and 0.2-0.4% of the forward reaction in the microsomal preparations of safflower and rat liver respectively. Previous observations, that the acyl exchange in safflower microsomal preparations was stimulated by bovine serum albumin and sn-glycerol 3-phosphate, can now be explained by the lowered acyl-CoA concentrations in the incubation mixture with albumin and in the increase in free CoA in the presence of sn-glycerol 3-phosphate (by rapid acylation of sn-glycerol 3-phosphate with acyl groups from acyl-CoA to yield phosphatidic acid). Bovine serum albumin and sn-glycerol 3-phosphate, therefore, shift the equilibrium in acyl-CoA:lysophosphatidylcholine acyltransferase-catalysed reactions towards the rate-limiting step in the acyl exchange process, namely the removal of acyl groups from phosphatidylcholine. The possible role of the acyl exchange in the transfer of acyl groups between complex lipids is discussed.     

A single enzyme catalyzes both platelet-activating factor production and membrane biogenesis of inflammatory cells. Cloning and characterization of acetyl-CoA:LYSO-PAF acetyltransferase

Platelet-activating factor (PAF) is a potent proinflammatory lipid mediator eliciting a variety of cellular functions. Lipid mediators, including PAF are produced from membrane phospholipids by enzymatic cascades. Although a G protein-coupled PAF receptor and degradation enzymes have been cloned and characterized, the PAF biosynthetic enzyme, aceyl-CoA:lyso-PAF acetyltransferase, has not been identified. Here, we cloned lyso-PAF acetyltransferase, which is critical in stimulus-dependent formation of PAF. The enzyme is a 60-kDa microsomal protein with three putative membrane-spanning domains. The enzyme was induced by bacterial endotoxin (lipopolysaccharide), which was suppressed by dexamethasone treatment. Surprisingly, the enzyme catalyzed not only biosynthesis of PAF from lyso-PAF but also incorporation of arachidonoyl-CoA to produce PAF precursor membrane glycerophospholipids (lysophosphatidylcholine acyltransferase activity). Under resting conditions, the enzyme prefers arachidonoyl-CoA and contributes to membrane biogenesis. Upon acute inflammatory stimulation with lipopolysaccharide, the activated enzyme utilizes acetyl-CoA more efficiently and produces PAF. Thus, our findings provide a novel concept that a single enzyme catalyzes membrane biogenesis of inflammatory cells while producing a prophlogistic mediator in response to external stimuli.     

Solubilization and modulation of acyl-CoA:1-acyl-glycerophosphocholine acyltransferase activity in rat liver microsomes

The acylation of 1-acyl-glycerophosphocholine is an important mechanism for the maintenance of the asymmetrical distribution of acyl groups in phosphatidylcholine. The majority of acyl-CoA:1-acyl-glycerophosphocholine acyltransferase is located in the microsomal fraction. In this study, the rat liver microsomes were incubated with various detergents, and the solubilized enzyme was separated from the remainder by centrifugation. Sodium cholate, sodium deoxycholate and octylglucopyranoside caused the Solubilization of 14–25% of the enzyme activity. The acyl specificity of the solubilized enzyme was similar to the insoluble enzyme, indicating that there was no selective solubilization of any acyl specific acyltransferase. The solubilized enzyme did not display any lipid requirement, and its activity was inhibited by phosphatidylcholine, phosphatidylethanolamine and 1,2-diacylglycerol. Kinetic studies with varying concentrations of acyl-CoAs revealed that the inhibition by 1,2-diacylglycerol was essentially uncompetitive. The modulation of acyltransferase activity by 1,2-diacylglycerol may be an important mechanism for controlling the acylation of lysophosphatidylcholine.     

Acylation of 1-alkenylglycerophosphoethanolamine and 1-acylglycerophosphoethanolamine in guinea-pig heart microsomes

Although the acylation of 1-alkenylglycerophosphocholine in mammalian heart is well documented, the acylation of 1-alkenylglycerophosphoethanolamine in the heart was not reported. In this study, the presence of acyl CoA: 1-alkenylglycerophosphoethanolamine acyltransferase in the guinea pig heart microsomes was demonstrated. 1-Alkenylglycerophosphoethanolamine acyltransferase displayed a high degree of specificity towards acyl-CoA. The order of reactivity with acyl-CoA was found to be: linoleoyl much greater than arachidonyl greater than palmitoyl greater than stearoyl = oleoyl. 1-Acylglycerophosphoethanolamine acyltransferase in the microsomes also exhibited specificity towards acyl-CoA in the following manner: linoleoyl greater than arachidonyl much greater than palmitoyl greater than oleoyl greater than stearoyl. However, such specificity appeared to be dependent on acyl-CoA concentration. The acyl-CoA specificities of both enzymes did not correlate with the C-2 acyl distribution observed in the corresponding microsomal phospholipids. Our results suggest that in addition to the acyl specificity of the acyltransferases, intracellular concentrations of acyl-CoAs may also have an important role in determining the observed acyl patterns of the phospholipids. Based on the acyl specificities, pH profiles, and their responses to heat inactivation and thiol reagents, we conclude that 1-alkenylglycerophosphoethanolamine acyltransferase and 1-acylglycerophosphoethanolamine acyltransferase in guinea-pig heart microsomes are not the same enzyme.     

Substrate selectivity of acyl-CoA:lysolecithin acyltransferase from rabbit lung

The influence of both polar head and acyl chain of lysophospholipid on the activity of partially purified acyl-CoA:lysolecithin acyltransferase from rabbit lung was studied. It was concluded that the presence of methyl groups on the nitrogen of the base was essential for recognition of lysophospholipid as substrate by the enzyme. With respect to the acyl chain length and saturation, the activity followed the order: 16:0 approximately equal to 18:1 greater than 14:0 greater than greater than greater than 18:0 approximately equal to 12:0. Also, the effect on the activity of the acyl chain on acyl-CoA was studied. The activity showed great selectivity for saturated acyl-CoAs. The activity with polyunsaturated fatty acids was very low and in the case of arachidonoyl-CoA was almost negligible. The comparison between crude microsomal preparations and partially purified preparations allowed to suggest that it could exist two different acyl-CoA:lysolecithin acyltransferases differing in their selectivity towards saturated and unsaturated fatty acids.     

Purification of long-chain acyl-CoA hydrolase from bovine heart microsomes and regulation of activity by lysophospholipids

Long-chain acyl-CoA hydrolase (EC 3.1.2.2) has been purified 12,000-fold from bovine heart muscle microsomes by extraction with Miranol detergent, followed by column chromatography on Reactive Blue agarose and DEAE-cellulose. The purified enzyme was nearly homogeneous on polyacrylamide gel electrophoresis and had a molecular weight of 41,000 in the presence of dodecyl sulfate. The specificity and kinetic properties of the enzyme were studied using several acyl-CoA derivatives as potential substrates. The enzyme showed a wide degree of specificity with little dependence on either the fatty acyl chain length or the degree of unsaturation of the acyl group. The kinetic properties were in accord with the Michaelis-Menten equation under most conditions, although high concentrations of substrates generally inhibited the enzyme. Arachidonoyl-CoA, which was the most effective substrate, had a Km value of 0.4 microM and a Vmax value of 6.0 mumol min-1 mg-1. The enzyme was strongly and specifically inhibited by constants of 16 and 30 nM, respectively. Other lysolipids and detergents such as deoxycholate and Triton X-100 were weak inhibitors. These properties and others distinguish this enzyme from other acyl-CoA hydrolases and support the idea that lysophospholipids may be important in vivo in the regulation of lipid metabolism.     

Purification of acyl CoA:1-acyl-sn-glycero-3-phosphorylcholine acyltransferase

Acyl coenzyme A:1-acyl-sn-glycero-3-phosphorylcholine acyltransferase (EC 2.3.1.23) is capable of forming lipid bilayer vesicles from its soluble substrates lysophosphatidylcholine (LPC) and oleoyl CoA. This suggested a purification method in which rat liver microsomes are first washed with deoxycholate to increase specific activity of the endogenous acyltransferase approximately fivefold, then solubilized by the detergent effect of excess LPC and oleoyl CoA in 1:1 stoichiometric ratios. As the LPC is converted to phosphatidylcholine by acyl group transfer, the detergent effect is lost and lipid vesicles containing the enzyme activity are produced. Other microsomal proteins are excluded from the vesicles. The vesicles may be separated by density gradient flotation and are found to contain acyltransferase with a specific activity of 9–10 µmol/mg/min. This reflects a purification of approximately 140-fold, about ten times greater than achieved in previous studies.     

Arachidonoyl-CoA:lysophosphatidylcholine acyltransferase activity in ras-transformed NIH 3T3 fibroblasts depends on the membrane composition.

Investigations were performed on the influence of membrane lipids on arachidonoyl-CoA:lysophosphatidylcholine acyltransferase in microsomal membranes from control and ras-transformed NIH 3T3 fibroblasts. Of all the tested phospholipids only sphingomyelin induced activation of acyltransferase in membranes from ras-transformed cells. No specific phospholipid effect on the acyltransferase was observed in microsomal membranes from control fibroblasts. Diacylglycerol was found to inhibit acyltransferase in both cell lines, whereas ceramide accumulation induced inhibition only in membranes from the transformed cells. The effects of diacylglycerol, ceramide, sphingomyelin and sphingomyelinase are discussed with respect to their putative roles in the signal transduction pathways in oncogene-expressing cells.     

An enzymatic rationale for the randomization of the positional distribution of fatty acids in phospholipids of ascites hepatoma AH 130

Fatty acids present in glycerophospholipids isolated from Yoshida ascites hepatoma AH 130 are more randomly distributed among the 1- and 2-positions than are fatty acids of normal liver phospholipids. The relative abundance of unsaturated fatty acids at the 1-position was ascribed to the lower palmitate-specific glycerophosphate acyltransferase activity in mitochondria of the hepatoma cells, an observation supporting the conclusion put forward for the similar randomization observed in Ehrlich ascites cells (Haldar, D., Tso, W.-W. and Pullman, M.E. (1979) J. Biol. Chem. 254, 4502-4509). The relative abundance of saturated fatty acids at the 2-position could be ascribed to the relatively lower acyl-CoA:1-acyl-glycerophosphocholine acyltransferase activity and to the change in the selectivity of the hepatoma acyl-CoA:1-acyl-glycerophosphate acyltransferase system into the lung type. The relatively lower selectivity for arachidonoyl-CoA as compared with oleoyl-CoA of the 1-acyl-glycerophosphocholine acyltransferase system is consistent with the decrease in polyenoic fatty acid content at the 2-position of the hepatoma phospholipids.     

Intestinal Lipid Esterification and Aging in Mice and Rats

The effects of aging on lipid absorption, particularly on fatty acid glycerophospholipid and triacylglycerol esterification, were investigated in 2.5-,12- and 24-month-old mice and rats. Two intestinal mucosa microsomal enzymes, involved in the dietary fatty acid absorption, were assayed:acylCoA:2-monoacylglycerol acyltransferase and acylCoA:1-lysophosphatidylcholine acyltransferase. In both mice and rats, the activities of both enzymes varied with the nature of the acyl-CoA. Indeed acylCoa:2-monoacylglycerol acyltransferase activities were significantly higher with oleoyl-CoA and linoleoyl-CoA than with palmitoyl-CoA and arachidonoyl-CoA, while acylCoA:1-lysophosphatidylcholine acyltransferase activities were highest with arachidonoyl-CoA. AcylCoA:2-monoacylglycerol acyltransferase activity did not decrease significantly with aging in mice or rats, whatever the acyl-CoA used. In contrast, acylCoA:1-lysophosphatidylcholine acyltransferase activity in the 24-month-old rats was significantly lower (−47 %) than in 2.5-month-old rats, with oleoyl-CoA, linoleoyl-CoA and arachidonoyl-CoA. Simultaneously we observed that less glycerophospholipid esterification of oleic and linoleic acid occurs in older rats than in 2.5-month-old rats.     

The yeast acylglycerol acyltransferase LCA1 is a key component of Lands cycle for phosphatidylcholine turnover

Cellular phospholipids undergo deacylation and reacylation through a process known as Lands cycle. In this report, we provide evidence demonstrating that yeast YOR175c, herein designated as LCA1, encodes a key component of the Lands cycle, the acyl-CoA: lysophosphatidylcholine acyltransferase (LPCAT). Deletion of LCA1 resulted in a drastic reduction in LPCAT activity, while over expression led to a several fold increase in enzyme activity. We further show that disruption of LCA1 caused an enhanced production of glycerophosphorylcholine, a product of phosphatidylcholine (PC) deacylation and that the lysophosphatidic acid acyltransferase SLC1 was not involved in this process. Identification of LCA1 provides an essential molecular tool for further study of Lands cycle in PC turnover.     

Acyltransferase-catalyzed transfer of arachidonic acid to lysophospholipids in rat pancreatic acini

The conversion of 2-lysophospholipids into corresponding phospholipids via acyl-CoA acyltransferase was demonstrated in homogenates of rat pancreatic acini. Arachidonic acid was greatly preferred over stearic acid as the acyl donor. Lysophophosphatidylinositol and lysophosphatidylcholine acyltransferases were distributed in subcellular fractions of acinar homogenates with specific activity highest in the fractions known to contain secretory organelles and mitochondria. The distribution of lysophosphatidylinositol acyltransferase paralleled that of a mitochondrial marker (succinate cytochrome C reductase). These findings extend the evidence implicating arachidonate release and reincorporation into phospholipids as a link in the pathway that culminates in pancreatic secretion.