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.     

The effect of methyl lidocaine on lysophospholipid metabolism in hamster heart

An important feature in the remodelling of fatty acyl chains in cellular phospholipids is the acylation of lysophospholipids. Since lysophospholipids are cytolytic at high concentrations, the acylation reaction may provide an alternate pathway for the removal of cellular lysophospholipids. However, the physiological role of the acylation process in the maintenance of lysophospholipid levels in mammalian tissues has not been clearly defined. In this study, methyl lidocaine was found to inhibit both lysophosphatidylcholine:acyl-CoA and lysophosphatidylethanolamine:acyl-CoA acyltransferase activities in the hamster heart, but the drug had no effect on the other lysophospholipid metabolic enzymes. When the heart was perfused with 0.5 mg methyl lidocaine/mL, acyltransferase activities were attenuated, but there was no change in the activities of phospholipase A or lysophospholipase. The levels of the major lysophospholipids in the heart were not altered by methyl lidocaine perfusion. When the hearts were perfused with labelled lysophospholipid in the presence of methyl lidocaine, there was a reduction in the formation of the phospholipid and an increase in the release of the free fatty acid. However, the labelling of lysophospholipid in the heart was not altered by methyl lidocaine. We postulate that the acylation reaction has no direct contribution to the maintenance of the lysophospholipid levels in the heart.     

Substrate selectivity of lysophosphatidylcholine: lysophosphatidylcholine acyltransferase from rabbit lung

The influence of both polar group and acyl chain of lysophospholipids on the lysophosphatidylcholine: lysophosphatidylcholine acyltransferase from rabbit lung was studied. Both, transacylase and hydrolase activities of this enzyme, utilize selectively 1-[1-14C]palmitoyl-sn-glycero-3-phosphocholine when compared with 1-[9,10-3H2]palmitoyl-sn-glycero-3-phosphoethanolamine. Transacylase activity is more selective for lysophosphatidylcholine as acyl acceptor than as acyl donor. The amount of dipalmitoylphosphatidylcholine/min/mg protein synthesized from mixed lysophosphatidylcholine/lysophosphatidylethanolamine micelles does not change with increasing molar percentages of lysophosphatidylethanolamine in the mixture and is similar to that formed with pure lysophosphatidylcholine micelles. Transacylation reaction takes place preferentially with long and saturated acyl chains whereas hydrolysis reaction does more efficiently with longer acyl chains, independently of their insaturation degree.     

Acyltransferases and transacylases that determine the fatty acid composition of glycerolipids and the metabolism of bioactive lipid mediators in mammalian cells and model organisms

Over one hundred different phospholipid molecular species are known to be present in mammalian cells and tissues. Fatty acid remodeling systems for phospholipids including acyl-CoA:lysophospholipid acyltransferases, CoA-dependent and CoA-independent transacylation systems, are involved in the biosynthesis of these molecular species. Acyl-CoA:lysophospholipid acyltransferase system is involved in the synthesis of phospholipid molecular species containing sn-1 saturated and sn-2 unsaturated fatty acids. The CoA-dependent transacylation system catalyzes the transfer of fatty acids esterified in phospholipids to lysophospholipids in the presence of CoA without the generation of free fatty acids. The CoA-dependent transacylation reaction in the rat liver exhibits strict fatty acid specificity, i.e., three types of fatty acids (20:4, 18:2 and 18:0) are transferred. On the other hand, CoA-independent transacylase catalyzes the transfer of C20 and C22 polyunsaturated fatty acids from diacyl phospholipids to various lysophospholipids, especially ether-containing lysophospholipids, in the absence of any cofactors. CoA-independent transacylase is assumed to be involved in the accumulation of PUFA in ether-containing phospholipids. These enzymes are involved in not only the remodeling of fatty acids, but also the synthesis and degradation of some bioactive lipids and their precursors. In this review, recent progresses in acyltransferase research including the identification of the enzyme’s genes are described.     

Lysophospholipase and transacylase activities of rat gastric mucosa.

A transacylase that converts 1-palmitoyl lysophosphatidylcholine to dipalmitoyl phosphatidylcholine was demonstrated in the rat gastric mucosa. This enzyme required neither ATP or CoA nor bile salt and detergent for its activity. The enzyme preparation also exhibited powerful lysophospholipase activity. The transacylase and lysophospholipase were both located in the cytosol fraction, and their activities remained associated at a constant ratio throughout the purification steps, including the isoelectrofocusing procedure. They responded similarly with respect to the addition of metal ions, bile salt, detergent, and heat treatment. Both enzyme activities also exhibited similar apparent K_m values for lysophosphatidylcholine. These observations suggest that both the lysophospholipase and transacylase activities may reside in the same enzyme.     

Subcellular localization and lysophospholipase/transacylation activities of human group IVC phospholipase A2 (cPLA2γ)

cPLA2γ was identified as an ortholog of cPLA2α, which is a key enzyme in eicosanoid production. cPLA2γ was reported to be located in endoplasmic reticulum (ER) and mitochondria and to have lysophospholipase activity beside phospholipase A2 (PLA2) activity. However, subcellular localization, mechanism of membrane binding, regulation and physiological function have not been fully established. In the present study, we examined the subcellular localization and enzymatic properties of cPLA2γ with C-terminal FLAG-tag. We found that cPLA2γ was located not only in ER but also mitochondria even in the absence of the prenylation. Purified recombinant cPLA2γ catalyzed an acyltransferase reaction from one molecule of lysophosphatidylcholine (LPC) to another, forming phosphatidylcholine (PC). LPC or lysophosphatidylethanolamine acted as acyl donor and acceptor, but lysophosphatidylserine, lysophosphatidylinositol and lysophosphatidic acid (LPA) did not. PC and phosphatidylethanolamine (PE) also acted as weak acyl donors. Reaction conditions changed the balance of lysophospholipase and transacylation activities, with addition of LPA/PA, pH > 8, and elevated temperature markedly increasing transacylation activity; this suggests that lysophospholipase/transacylation activities of cPLA2γ may be regulated by various factors. As lysophospholipids are known to accumulate in ischemia heart and to induce arryhthmia, the cPLA2γ that is abundant in heart may have a protective role through clearance of lysophospholipids by its transacylation activity.     

Substrate Selectivity of Lysophospholipid Transporter LplT Involved in Membrane Phospholipid Remodeling in Escherichia coli

Lysophospholipid transporter (LplT) was previously found to be primarily involved in 2-acyl lysophosphatidylethanolamine (lyso-PE) recycling in Gram-negative bacteria. This work identifies the potent role of LplT in maintaining membrane stability and integrity in the Escherichia coli envelope. Here we demonstrate the involvement of LplT in the recycling of three major bacterial phospholipids using a combination of an in vitro lysophospholipid binding assay using purified protein and transport assays with E. coli spheroplasts. Our results show that lyso-PE and lysophosphatidylglycerol, but not lysophosphatidylcholine, are taken up by LplT for reacylation by acyltransferase/acyl-acyl carrier protein synthetase on the inner leaflet of the membrane. We also found a novel cardiolipin hydrolysis reaction by phospholipase A₂ to form diacylated cardiolipin progressing to the completely deacylated headgroup. These two distinct cardiolipin derivatives were both translocated with comparable efficiency to generate triacylated cardiolipin by acyltransferase/acyl-acyl carrier protein synthetase, demonstrating the first evidence of cardiolipin remodeling in bacteria. These findings support that a fatty acid chain is not required for LplT transport. We found that LplT cannot transport lysophosphatidic acid, and its substrate binding was not inhibited by either orthophosphate or glycerol 3-phosphate, indicating that either a glycerol or ethanolamine headgroup is the chemical determinant for substrate recognition. Diacyl forms of PE, phosphatidylglycerol, or the tetra-acylated form of cardiolipin could not serve as a competitive inhibitor in vitro. Based on an evolutionary structural model, we propose a “sideways sliding” mechanism to explain how a conserved membrane-embedded α-helical interface excludes diacylphospholipids from the LplT binding site to facilitate efficient flipping of lysophospholipid across the cell membrane.     

Biological roles of lysophosphatidic acid signaling through its production by autotaxin

Lysophosphatidic acid (LPA) exhibits a wide variety of biological functions as a bio-active lysophospholipid through G-protein-coupled receptors specific to LPA. Currently at least six LPA receptors are identified, named LPA₁ to LPA₆, while the existence of other LPA receptors has been suggested. From studies on knockout mice and hereditary diseases of these LPA receptors, it is now clear that LPA is involved in various biological processes including brain development and embryo implantation, as well as patho-physiological conditions including neuropathic pain and pulmonary and renal fibrosis. Unlike sphingosine 1-phosphate, a structurally similar bio-active lysophospholipid to LPA and produced intracellularly, LPA is produced by multiple extracellular degradative routes. A plasma enzyme called autotaxin (ATX) is responsible for the most of LPA production in our bodies. ATX converts lysophospholipids such as lysophosphatidylcholine to LPA by its lysophospholipase D activity. Recent studies on ATX have revealed new aspects of LPA. In this review, we highlight recent advances in our understanding of LPA functions and several aspects of ATX, including its activity, expression, structure, biochemical properties, the mechanism by which it stimulates cell motility and its pahto-physiological function through LPA production.     

Possible implication of lysophosphatidylcholine in cell fusion accompanying implantation in rabbits.

Implantation in rabbits involves the cellular fusion of trophoblastic and uterine epithelial cells resulting in embryo penetration of the uterine endometrium. Since lysophospholipids, known to have fusigenic properties, could be responsible for this cell fusion, the metabolism of lysophospholipids was studied throughout gestation in blastocyst/yolk sac and extracoelic amnioallantoic fluids. Analysis of phospholipid composition revealed that lysophospholipids are present in blastocyst/yolk sac fluid. Their concentrations and haemolytic activity change during pregnancy. They increase and reach their highest values during days 7 to 9, the implantation days in rabbits. A clear correlation was observed between lysophosphatidylcholine concentrations in blastocyst/yolk sac fluid and haemolysis induced by this fluid. Phosphatidylcholine concentrations, phospholipase A2 activity, which generates lysophospholipids, and lysophospholipase A activity which hydrolyses lysophosphatidylcholine into fatty acid, were at their highest value at day 12. These data suggest that a transient accumulation of lysophospholipids could ensure local cell fusion. Moreover, we propose that the lysophospholipid concentrations in blastocyst/yolk sac fluid are dependent upon activities of phospholipase A2 and lysophospholipase.     

The PLB2 gene of Saccharomyces cerevisiae confers resistance to lysophosphatidylcholine and encodes a phospholipase B/lysophospholipase

The PLB1 gene of Saccharomyces cerevisiae encodes a protein that demonstrates phospholipase B, lysophospholipase, and transacylase activities. Several genes with significant homology to PLB1 exist in the S. cerevisiae genome, raising the possibility that other proteins may contribute to the total phospholipase B/lysophospholipase/transacylase activities of the cell. We report the isolation of a previously uncharacterized gene that is highly homologous to PLB1 and that, when overexpressed, confers resistance to 1-palmitoyllysophosphatidylcholine. This gene, which is located adjacent to the PLB1 gene on the left arm of chromosome XIII and which we refer to as PLB2, encodes a phospholipase B/lysophospholipase. Unlike PLB1, this gene product does not contain significant transacylase activity. The PLB2 gene product shows lysophospholipase activity toward lysophosphatidylcholine, lysophosphatidylserine, and lysophosphatidylethanolamine. Whereas deletion of either PLB1 or PLB2 resulted in the loss of 80% of cellular lysophospholipase activity, a plb1/plb2 double deletion mutant is completely devoid of lysophospholipase activity toward the preferred substrate lysophosphatidylcholine. Overexpression of PLB2 was associated with an increase in total cellular phospholipase B/lysophospholipase activity, as well as the appearance of significant lysophospholipase activity in the medium. Moreover, overexpression of PLB2 was associated with saturation at a higher cell density, and an increase in total cellular phospholipid content, but no change in phospholipid composition or fatty acid incorporation into cellular lipids. Deletion of PLB2 was not lethal and did not result in alteration of membrane phospholipid composition or content. PLB2 gene expression was found to be maximal during exponential growth conditions and was decreased in late phase, in a manner similar to other genes involved in phospholipid metabolism.     

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.     

Characterization of human lysophospholipid acyltransferase 3

Esterifying lysophospholipids may serve a variety of functions, including phospholipid remodeling and limiting the abundance of bioactive lipids. Recently, a yeast enzyme, Lpt1p, that esterifies an array of lysophospholipids was identified. Described here is the characterization of a human homolog of LPT1 that we have called lysophosphatidylcholine acyltransferase 3 (LPCAT3). Expression of LPCAT3 in Sf9 insect cells conferred robust esterification of lysophosphatidylcholine in vitro. Kinetic analysis found apparent cooperativity with a saturated acyl-CoA having the lowest K_0.5 (5 μM), a monounsaturated acyl-CoA having the highest apparent V_max (759 nmol/min/mg), and two polyunsaturated acyl-CoAs showing intermediate values. Lysophosphatidylethanolamine and lysophosphatidylserine were also utilized as substrates. Electrospray ionization mass spectrometric analysis of phospholipids in Sf9 cells expressing LPCAT3 showed a relative increase in phosphatidylcholine containing saturated acyl chains and a decrease in phosphatidylcholine containing unsaturated acyl chains. Targeted reduction of LPCAT3 expression in HEK293 cells had essentially an opposite effect, resulting in decreased abundance of saturated phospholipid species and more unsaturated species. Reduced LPCAT3 expression resulted in more apoptosis and distinctly fewer lamellipodia, suggesting a necessary role for lysophospholipid esterification in maintaining cellular function and structure.     

Effects of nucleotides and divalent cations on phospholipase activity in Saccharomyces cerevisiae

Divalent cations activate the lysophospholipase and transacylase reactions catalyzed by the same enzymes in the yeast Saccharomyces cerevisiae. The activation was observed at neutral pH, but not at the pH optimum of lysophospholipase/transacylase, near 3.5. Adenine nucleotides, especially AMP and ADP, are strong inhibitors of the same group of enzymes. Half maximal inhibition by AMP was found at a concentration of about 20 M. The inhibition by nucleotides in low concentrations is enhanced by divalent cations.     

Regulation of lysophosphatidylcholine-metabolizing enzymes in isolated myocardial cells from rat heart

Enzymatic pathways involved in the metabolism of lysophosphatidylcholine were investigated in rat heart myocardial cells. Acyl CoA-dependent acyltransferase activity was localized in microsomes, and was much greater than lysophospholipase activity in either cytosolic or microsomal fractions. The cytosolic lysophospholipase was more sensitive to inhibition by palmitylcarnitine in comparison to free fatty acids. In contrast, free fatty acids (oleate and palmitate) produced a greater inhibition of the microsomal acyltransferase and lysophospholipase than did palmitylcarnitine. A reduction in the assay pH to 6.5 resulted in an increase in microsomal acyltransferase and cytosolic lysophospholipase activities, but brought about a marked reduction in the microsomal lysophospholipase activity. At pH 6.5, the percentage inhibition of the microsomal acyltransferase by palmitylcarnitine was reduced, whereas the inhibition by palmitic acid was enhanced. The inhibition of the microsomal lysophospholipase by both palmitylcarnitine and palmitic acid was reduced at pH 6.5. With respect to myocardial ischemia, the inhibition of microsomal acyltransferase by free fatty acids and the reduction in microsomal lysophospholipase activity due to acidosis may contribute to the elevation of cellular lysophosphoglycerides which are arrhythmogenic.     

Isolation and characterization of three lysophospholipases from the murine macrophage cell line WEHI 265.1.

Anion exchange chromatography of WEHI 265.1 cell homogenates resolved the lysophospholipase activity into three peaks, when assayed using lysophosphatidylcholine as a substrate. Peaks 1 and 2 were purified by sequential hydrophobic interaction and gel filtration chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified peaks 1 and 2 indicated homogeneous proteins with apparent masses of 28 and 27 kDa, respectively. Peak 3 lysophospholipases was partially purified by hydrophobic, hydroxyapatite and gel filtration chromatography. Peak 3 lysophospholipase also had calcium-dependent phospholipase A2 activity, which further co-purified with the lysophospholipase activity. The three lysophospholipases were characterized with respect to substrate specificity, additional enzymatic activities and the effects of lipids, metal ions and other compounds on enzymatic activity. Peaks 1, 2 and 3 hydrolyzed lysophosphatidylcholine most readily, but lysophosphatidylethanolamine also served as substrate for each enzyme. Furthermore, all three enzymes hydrolyzed platelet activating factor and acetylated lysophosphatidylcholine. Each lysophospholipase was inhibited by free fatty acids and by palmitoyl carnitine, although the relative sensitivities to these agents differed among the enzymes. The lysophospholipase activities of peaks 1 and 2, but not peak 3, were inhibited by phenylmethylsulfonyl fluoride, diisopropyl fluorophosphate and N-ethylmaleimide. Although they had similar masses, the amino acid compositions of peaks 1 and 2 differed, indicating that these are distinct proteins rather than posttranslational modifications of the same gene product.     

Thioesterase activity and subcellular localization of acylprotein thioesterase 1/lysophospholipase 1

Acylprotein thioesterase 1 (APT1), also known as lysophospholipase 1, is an important enzyme responsible for depalmitoylation of palmitoyl proteins. To clarify the substrate selectivity and the intracellular function of APT1, we performed kinetic analyses and competition assays using a recombinant human APT1 (hAPT1) and investigated the subcellular localization. For this purpose, an assay for thioesterase activity against a synthetic palmitoyl peptide using liquid chromatography/mass spectrometry was established. The thioesterase activity of hAPT1 was most active at neutral pH, and did not require Ca²⁺ for its maximum activity. The K_M values for thioesterase and lysophospholipase (against lysophosphatidylcholine) activities were 3.49 and 27.3 μM, and the V_max values were 27.3 and 1.62 μmol/min/mg, respectively. Thus, hAPT1 revealed much higher thioesterase activity than lysophospholipase activity. One activity was competitively inhibited by another substrate in the presence of both substrates. Immunocytochemical and Western blot analyses revealed that endogenous and overexpressed hAPT1 were mainly localized in the cytosol, while some signals were detected in the plasma membrane, the nuclear membrane and ER in HEK293 cells. These results suggest that eliminating palmitoylated proteins and lysophospholipids from cytosol is one of the functions of hAPT1.     

Molecular identification of a novel mammalian brain isoform of acyl-CoA:lysophospholipid acyltransferase with prominent ethanolamine lysophospholipid acylating activity, LPEAT2

Acyl-CoA-dependent lysophospholipid acyltransferases play an important role in attaining the appropriate molecular species of phospholipids. A number of genes encoding these activities were recently identified. It has become clear that multiple genes can encode one enzymatic activity and that a given gene may encode multiple activities. Here we report the identification of a gene encoding a mammalian acyl-CoA-dependent lysophospholipid acyltransferase with prominent activity toward ethanolamine-containing lysophospholipids, which we termed acyl-CoA:lysophosphatidylethanolamine acyltransferase 2, LPEAT2 (previously annotated as AYTL3 or AGPAT7). LPEAT2 is predominantly expressed in brain, coinciding with an enrichment of phosphatidylethanolamine in this tissue. Ectopic expression of LPEAT2 in mammalian HEK293T cells led to a dramatic increase (up to 9-fold) in LPEAT activity when compared with cells transfected with empty vector or an unrelated acyltransferase. LPEAT2 also exhibited significant acyl-CoA-dependent acyltransferase activity toward 1-O-alkenyl-lysophosphatidylethanolamine, lysophosphatidylglycerol, 1-O-alkyl-lysophosphatidylcholine, lysophosphatidylserine, and lysophosphatidylcholine but lacked appreciable acylating activity toward glycerol 3-phosphate, lysophosphatidic acid, lysophosphatidylinositol, and diacylglycerol, demonstrating multiple but selective functions of LPEAT2 as an enzyme involved in phospholipid remodeling. LPEAT2 recognizes a broad range of medium and long chain fatty acyl-CoA, and its activity was not affected by Ca(2+). When overexpressed in mammalian cells, LPEAT2 is localized to the endoplasmic reticulum. siRNA-mediated knockdown of LPEAT2 in HEK293T cells significantly decreased LPEAT and 1-alkenyl-LPEAT activities but did not affect other lysophospholipid acylating activities. These findings identify LPEAT2 as an important enzyme in the biosynthesis of ethanolamine-containing phospholipids, especially in brain.     

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.     

Phospholipases of Leptospira. I. Presence of phospholipase A1 and lysophospholipase in Leptospira biflexa

The hydrolysis of phosphatidylethanolamine, phosphatidylcholine, lysophosphatidylcholine, and trioleoylglycerol by Leptospira biflexa strain Urawa was studied in vitro. Phospholipase A₁ was identified by the formation of ³²P- and ¹⁴C-labeled lyso-derivatives from ³²P-phosphatidylcholine, ³²P-phosphatidylethanolamine, or 1-acyl-2-[1-¹⁴C]oleoyl-sn-glycero-3-phosphorylcholine. Phospholipase A₁ activity was independent of lipase in the microorganism since ¹⁴C-labeled trioleoylglycerol was scarcely attacked under the same conditions in which the phospholipids were hydrolyzed. Lysophospholipase activity was also demonstrated using ³²P- and non-labeled lysophosphatidylcholine. The activity of phospholipase A₁ was found in a broad range of pH but no optimal pH was determined. The pH optimum of lysophospholipase was 8.0. Both enzymes were labile to heat. Phospholipase C activity, however, could not be detected because no radioactive di- and monoacylglycerol was found in the experiment with 1-acyl-2-[1-¹⁴C]-oleoyl-sn-glycero-3-phosphorylcholine as the substrate. It was inferred that phosphatidylethanolamine, which was the major component of phospholipids in leptospirae, was hydrolyzed serially by phospholipase A (A₁ and/or A₂?) and lysophospholipase to glycerophosphorylethanolamine via 2-acyl-type-lyso-derivative as one metabolic pathway of the substrate.