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.     

Plant Acyl-CoA:Lysophosphatidylcholine Acyltransferases (LPCATs) Have Different Specificities in Their Forward and Reverse Reactions

Acyl-CoA:lysophosphatidylcholine acyltransferase (LPCAT) enzymes have central roles in acyl editing of phosphatidylcholine (PC). Plant LPCAT genes were expressed in yeast and characterized biochemically in microsomal preparations of the cells. Specificities for different acyl-CoAs were similar for seven LPCATs from five different species, including species accumulating hydroxylated acyl groups in their seed oil, with a preference for C₁₈-unsaturated acyl-CoA and low activity with palmitoyl-CoA and ricinoleoyl (12-hydroxyoctadec-9-enoyl)-CoA. We showed that Arabidopsis LPCAT1 and LPCAT2 enzymes catalyzed the acylation and de-acylation of both sn positions of PC, with a preference for the sn-2 position. When acyl specificities of the Arabidopsis LPCATs were measured in the reverse reaction, sn-2-bound oleoyl, linoleoyl, and linolenoyl groups from PC were transferred to acyl-CoA to a similar extent. However, a ricinoleoyl group at the sn-2-position of PC was removed 4–6-fold faster than an oleoyl group in the reverse reaction, despite poor utilization in the forward reaction. The data presented, taken together with earlier published reports on in vivo lipid metabolism, support the hypothesis that plant LPCAT enzymes play an important role in regulating the acyl-CoA composition in plant cells by transferring polyunsaturated and hydroxy fatty acids produced on PC directly to the acyl-CoA pool for further metabolism or catabolism.     

Cloning and characterization of mouse lung-type acyl-CoA:lysophosphatidylcholine acyltransferase 1 (LPCAT1). Expression in alveolar type II cells and possible involvement in surfactant production

Phosphatidylcholine (1,2-diacyl-sn-glycero-3-phosphocholine, PC), is an important constituent of biological membranes. It is also the major component of serum lipoproteins and pulmonary surfactant. In the remodeling pathway of PC biosynthesis, 1-acyl-sn-glycero-3-phosphocholine (LPC) is converted to PC by acyl-CoA:lysophosphatidylcholine acyltransferase (LPCAT, EC 2.3.1.23). Whereas LPCAT activity has been detected in several tissues, the structure and detailed biochemical information on the enzyme have not yet been reported. Here, we present the cloning and characterization of a cDNA for mouse lung-type LPCAT (LPCAT1). The cDNA encodes an enzyme of 60 kDa, with three putative transmembrane domains. When expressed in Chinese hamster ovary cells, mouse LPCAT1 exhibited Ca(2+)-independent activity with a pH optimum between 7.4 and 10. LPCAT1 demonstrated a clear preference for saturated fatty acyl-CoAs, and 1-myristoyl- or 1-palmitoyl-LPC as acyl donors and acceptors, respectively. Furthermore, the enzyme was predominantly expressed in the lung, in particular in alveolar type II cells. Thus, the enzyme might synthesize phosphatidylcholine in pulmonary surfactant and play a pivotal role in respiratory physiology.     

Properties of microsomal acyl-CoA: lysophosphatidylcholine acyltransferase from liver of thermally-acclimated rainbow trout,Salmo gairdneri

Summary Acyl-CoA: lysophosphatidylcholine acyltransferase (LPCAT) (EC 2.3.1.23) activity was assayed in liver microsomes from rainbow trout,Salmo gairdneri, acclimated to 5°C and 20°C to assess its contribution to the temperature-induced restructuring of phospholipid acyl chain composition. The synthesis of phosphatidylcholine (PC) (from lyso-PC) was threefold the synthesis of phosphatidylethanolamine (PE) (from lyso-PE) under similar assay conditions. LPCAT activity (i) displayed an absolute requirement for lysophosphatidylcholine (LPC) and was enhanced by the presence of ATP, MgCl₂ and CoA (which reduced the impact of endogenous acyl-CoA hydrolase activity by regenerating the acyl-CoA substrate) in the assay medium; (ii) remained linear with time up to 30 min; and (iii) increased linearly with microsomal protein concentration up to 0.2 mg/ml for the 20°C assay and 0.4 mg/ml for the 5°C assay. There was no difference in K_m or V_max values due to the acclimation history of the fish, but there were obvious differences due to assay temperature. The apparent K_m values for LPC were 58.54±7.24 M and 12.26±2.14 M when assayed at 5°C and 20°C respectively; values for oleoyl-CoA were 9.11±0.78 M and 1.23±0.25 M under the same assay conditions. Activity was 1.99±0.31 nmol min^–1 mg protein^–1 when assayed at 5°C, and 3.8±0.45 nmol min^–1 mg protein^–1 when assayed at 20°C. These findings indicate that adjustments in the activity of LPCAT play no significant role in the temperature-induced restructuring of PC molecular species composition. However, the marked temperature dependence of the K_m values for LPC and oleoyl CoA suggest that patterns of fatty acid incorporation (i.e. substrate preference) may vary with assay temperature, and in this way LPCAT could contribute to the restructuring response.Abbreviations PC phosphatidylcholine - PE phosphatidylethanolamine - LPCAT acyl-CoA: lysophosphatidylcholine acyltransferase - LPEAT acyl-CoA: lysophosphatidylethanolamine acyltransferase - LPC 1-palmitoyl,2-lysophosphatidylcholine     

Modeling and optimization of phospholipase A1‐catalyzed hydrolysis of phosphatidylcholine using response surface methodology for lysophosphatidylcholine production

Modeling the phospholipase A₁ (PLA₁)‐catalyzed partial hydrolysis of soy phosphatidylcholine (PC) in hexane for the production of lysophosphatidylcholine (LPC) and optimizing the reaction conditions using response surface methodology were described. The reaction was performed with 4 g of PC in a stirred batch reactor using a commercial PLA₁ (Lecitase Ultra) as the biocatalyst. The effects of temperature, reaction time, water content, and enzyme loading on LPC and glycerylphosphorylcholine (GPC) content in the reaction products were elucidated using the models established. Optimal reaction conditions for maximizing the LPC content while suppressing acyl migration, which causes GPC formation, were as follows: temperature, 60°C; reaction time, 3 h; water content, 10% of PC; and enzyme loading, 1% of PC. When the reaction was conducted with 40 g of PC under these conditions, the reaction products contained 83.7 mol % LPC and were free of GPC. LPC had a higher total unsaturated fatty acid content than original PC had and was mainly composed of linoleic acid (78.0 mol % of the total fatty acids). © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 31:35–41, 2015     

Identification of Brassica napus lysophosphatidylcholine acyltransferase genes through yeast functional screening

Acyl-CoA:lysophosphatidylcholine acyltransferase (LPCAT), which acylates lysophosphatidylcholine (LPC) to produce phosphatidylcholine (PC), is a key enzyme in the Lands cycle. There is evidence that acyl exchange involving LPCAT is a prevailing metabolic process during triacylglycerol (TAG) synthesis in seeds. In this study, by complementing the yeast lca1Δ mutant deficient in LPCAT activity with an Arabidopsis seedling cDNA library, it was found that the previously reported lysophospholipid acyltransferases (LPLATs), At1g12640 and At1g63050, were the only two acyltransferase genes that restored hyposensitivity of the lca1Δ mutant to lyso-platelet-activating factor (lyso-PAF). A developing seed cDNA library from Brassica napus L. cv Hero was constructed to further explore the heterologous yeast complementation approach. Three B. napus LPCAT homologs were identified, of which BnLPCAT1-1 and BnLPCAT1-2 are orthologous to Arabidopsis AtLPLAT1 (At1g12640) while BnLPCAT2 is an ortholog of AtLPLAT2 (At1g63050). The proteins encoded by BnLPCAT1-1 and BnLPCAT2 were chosen for further study. Enzymatic assays demonstrated that both proteins exhibited a substrate preference for LPCs and unsaturated fatty acyl-CoAs. In addition to the enzymatic properties of plant lysophosphatidylcholine acyltransferases uncovered in this study, this report describes a useful technique that facilitates subsequent analyses into the role of LPCATs in PC turnover and seed oil biosynthesis.     

Deciphering the roles of Arabidopsis LPCAT and PAH in phosphatidylcholine homeostasis and pathway coordination for chloroplast lipid synthesis

Phosphatidylcholine (PC) is a key intermediate in the metabolic network of glycerolipid biosynthesis. Lysophosphatidylcholine acyltransferase (LPCAT) and phosphatidic acid phosphatase (PAH) are two key enzymes of PC homeostasis. We report that LPCAT activity is markedly induced in the Arabidopsis pah mutant. The quadruple pah lpcat mutant, with dual defects in PAH and LPCAT, had a level of lysophosphatidylcholine (LPC) that was much higher than that in the lpcat mutants and a PC content that was higher than that in the pah mutant. Comparative molecular profile analysis of monogalactosyldiacylglycerol and digalactosyldiacylglycerol revealed that both the pah and pah lpcat mutants had increased proportions of 34:6 from the prokaryotic pathway despite differing levels of LPCAT activity. We show that a decreased representation of the C_16:0C_18:2 diacylglycerol moiety in PC was a shared feature of pah and pah lpcat, and that this change in PC metabolic profile correlated with the increased prokaryotic contribution to chloroplast lipid synthesis. We detected increased PC deacylation in the pah lpcat mutant that was attributable at least in part to the induced phospholipases. Increased LPC generation was also evident in the pah mutant, but the phospholipases were not induced, raising the possibility that PC deacylation is mediated by the reverse reaction of LPCAT. We discuss possible roles of LPCAT and PAH in PC turnover that impacts lipid pathway coordination for chloroplast lipid synthesis.     

Activation of fluoride-stimulated adenylate cyclase by phospholipase A2 in the caudate nucleus of the rat brain

Phospholipase A₂ (PLA₂) increases adenylate cyclase (AC) activity in the rat caudate nucleus in a dose-dependent manner. After maximal stimulation by fluoride, PLA₂ treatment further increases AC activity 2.4 fold. Adenylate cyclase activity is maximal after 45% hydrolysis of the phospholipids. Of the products of PLA₂ treatment only lysophosphatidylcholine (LPC) produces such an increase in AC activity. In contrast to PLA₂ treatment, LPC solubilizes the enzyme, decreases the Km value for ATP, and requires much larger amounts of LPC than that produced by lipase treatment. After maximal stimulation with fluoride and PLA₂, removal of most of the LPC does not reduce the activity of adenylate cyclase. These findings suggest that removal of membrane lipid rather than generation of LPC is responsible for the activation of brain adenylate cyclase by phospholipase A₂.     

Binding of peroxiredoxin 6 to substrate determines differential phospholipid hydroperoxide peroxidase and phospholipase A2 activities

Peroxiredoxin 6 (Prdx6) differs from other mammalian peroxiredoxins both in its ability to reduce phospholipid hydroperoxides at neutral pH and in having phospholipase A₂ (PLA₂) activity that is maximal at acidic pH. We previously showed an active site C47 for peroxidase activity and a catalytic triad S32-H26-D140 necessary for binding of phospholipid and PLA₂ activity. This study evaluated binding of reduced and oxidized phospholipid hydroperoxide to Prdx6 at cytosolic pH. Incubation of recombinant Prdx6 with 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphocholine hydroperoxide (PLPCOOH) resulted in peroxidase activity, cys47 oxidation as detected with Prdx6-SO₂₍₃₎ antibody, and a marked shift in the Prdx6 melting temperature by circular dichroism analysis indicating that PLPCOOH is a specific substrate for Prdx6. Preferential Prdx6 binding to oxidized liposomes was detected by changes in DNS-PE or bis-Pyr fluorescence and by ultrafiltration. Site-specific mutation of S32 or H26 in Prdx6 abolished binding while D140 mutation had no effect. Treatment of A549 cells with peroxides led to lipid peroxidation and translocation of Prdx6 from the cytosol to the cell membrane. Thus, the pH specificity for the two enzymatic activities of Prdx6 can be explained by the differential binding kinetics of the protein; Prdx6 binds to reduced phospholipid at acidic pH but at cytosolic pH binds only phospholipid that is oxidized compatible with a role for Prdx6 in the repair of peroxidized cell membranes.     

Cross-talk between Remodeling and de Novo Pathways Maintains Phospholipid Balance through Ubiquitination

Phosphatidylcholine (PtdCho), the major phospholipid of animal membranes, is generated by its remodeling and de novo synthesis. Overexpression of the remodeling enzyme, LPCAT1 (acyl-CoA:lysophosphatidylcholine acyltransferase) in epithelia decreased de novo PtdCho synthesis without significantly altering cellular PtdCho mass. Overexpression of LPCAT1 increased degradation of CPT1 (cholinephosphotransferase), a resident Golgi enzyme that catalyzes the terminal step for de novo PtdCho synthesis. CPT1 degradation involved its multiubiquitination and processing via the lysosomal pathway. CPT1 mutants harboring arginine substitutions at multiple carboxyl-terminal lysines exhibited proteolytic resistance to effects of LPCAT1 overexpression in cells and restored de novo PtdCho synthesis. Thus, cross-talk between phospholipid remodeling and de novo pathways involves ubiquitin-lysosomal processing of a key molecular target that mechanistically provides homeostatic control of cellular PtdCho content.     

Identification and characterization of a major liver lysophosphatidylcholine acyltransferase

Phosphatidylcholine (PC) is synthesized through the Kennedy pathway, but more than 50% of PC is remodeled through the Lands cycle, i.e. the deacylation and reacylation of PC to attain the final and proper fatty acids within PC. The reacylation step is catalyzed by lysophosphatidylcholine acyltransferase (LPCAT), and we report here the identification of a novel LPCAT, which we named LPCAT3. LPCAT3 belongs to the membrane-bound O-acyltransferase (MBOAT) family and encodes a protein of 487 amino acids with a calculated molecular mass of 56 kDa. Membranes from HEK293 cells overexpressing LPCAT3 showed significantly increased LPCAT activity as assessed by thin layer chromatography analysis with substrate preference toward unsaturated fatty acids. LPCAT3 is localized within the endoplasmic reticulum and is primarily expressed in metabolic tissues including liver, adipose, and pancreas. In a human hepatoma Huh7 cells, RNA interference-mediated knockdown of LPCAT3 resulted in virtually complete loss of membrane LPCAT activity, suggesting that LPCAT3 is primarily responsible for hepatic LPCAT activity. Furthermore, peroxisome proliferator-activated receptor alpha agonists dose-dependently regulated LPCAT3 in liver in a peroxisome proliferator-activated receptor alpha-dependent fashion, implicating a role of LPCAT3 in lipid homeostasis. Our studies identify a long-sought enzyme that plays a critical role in PC remodeling in metabolic tissues and provide an invaluable tool for future investigations on how PC remodeling may potentially impact glucose and lipid homeostasis.     

Solubilization and partial purification of dihydroxyacetone-phosphate acyltransferase from guinea pig liver

Dihydroxyacetone-phosphate:acyl coenzyme A acyltransferase (EC 2.3.1.42) was solubilized and partially purified from guinea pig liver crude peroxisomal fraction. The peroxisomal membrane was isolated after osmotic shock treatment and the bound dihydroxyacetone-phosphate acyltransferase was solubilized by treatment with a mixture of KCl-sodium cholate. The solubilized enzyme was partially purified by ammonium sulfate fractionation followed by Sepharose 6B gel filtration. The enzyme was purified 1200-fold relative to the guinea pig liver homogenate and 80- to 100-fold from the crude peroxisomal fraction, with an overall yield of 25–30% from peroxisomes. The partially purified enzyme was stimulated two- to fourfold by Asolectin (a soybean phospholipid preparation), and also by individual classes of phospholipid such as phosphatidylcholine and phosphatidylglycerol. The kinetic properties of the enzyme showed that in the absence of Asolectin there was a discontinuity in the reciprocal plot indicating two different apparent K_m values (0.1 and 0.5 mm) for dihydroxyacetone phosphate. The V_max was 333 nmol/min/mg protein. In the presence of Asolectin the reciprocal plot was linear, with a K_m = 0.1 mm and no change in V_max. The enzyme catalyzed both an exchange of acyl groups between dihydroxyacetone phosphate and palmitoyl dihydroxyacetone phosphate in the presence of CoA and the formation of palmitoyl [³H]coenzyme A from palmitoyl dihydroxyacetone phosphate and [³H]coenzyme A, indicating that the reaction is reversible. The partially purified enzyme preparation had negligible glycerol-3-phosphate acyltransferase (EC 2.3.1.15) activity.     

Stimulation of metamorphosis in Hydractinia echinata involves generation of lysophosphatidylcholine

Summary Whilst the significance of the phosphoinositide cycle in the activation of developmental events by extra-cellular signals is well established, the involvement of the phosphatidylcholine (PC) cycle is a matter just emerging. In the present study, the metabolism of phosphatidylcholine in early metamorphosis of Hydractinia echinata (Coelenterata; Hydrozoa) was investigated by incubation of planula larvae with ³H-choline, extraction of the metabolites and isolation of the metabolites by thin-layer chromatography (TLC). Phosphatidylcholine (PC), lysophosphatidylcholine (LPC), acetylcholine and glycerophosphocholine were the labelled metabolites. Induction of metamorphosis did not stimulate an increased incorporation of choline into PC. In larvae preincubated with ³H-choline to a steady state level of incorporation, a significant transient elevation of the radioactive label in LPC was observed 90 min after addition of metamorphosis stimulating agents. LPC probably derived from PC by the action of a phospholipase A₂ (PLA₂). LPCs from bovine and soybean origin as well as isolated larval LPC did not influence metamorphosis. PLA₂ from bee venom promoted Cs⁺-induced metamorphosis but did not influence phorbol ester-induced metamorphosis. The data suggest that a PLA₂ is activated during metamorphosis. This PLA₂ activation does not occur in those putative receptor cells which receive the primary external inducing stimulus but in the many larval cells which resume proliferation or differentiation in response to a second, internally propagated signal. Offprint requests to: T. Leitz     

The signal molecule lysophosphatidylcholine in Eschscholzia californica is rapidly metabolized by reacylation

In cultured cells of California poppy (Eschscholzia californica), lysophosphatidylcholine (LPC) triggers a signal path that finally induces alkaloid biosynthesis. LPC is transiently generated by elicitor-activated phospholipase A(2) of the plasma membrane. Externally added LPC is rapidly acylated by a membrane-bound enzyme that shows the highest specific activity in the purified plasma membrane. The fatty acid incorporated into the sn-2 position of LPC is preferentially linoleic (18:2), which is the most abundant acyl component in the PC species of Eschscholzia cells, but a minor component of the pool of free fatty acids. The fatty acid at the sn-1 position of LPC is less important for substrate specificity. The capacity of LPC acylation by intact cells or isolated plasma membranes by far exceeds the rate of LPC generation by activated phospholipase A(2) and is not limited by the availability of acyl donors. Metabolites other than phosphatidylcholine (PC) were not significantly produced from labeled LPC within 20 min, indicating that lysophospholipases are not significantly contributing to the short-time metabolism of LPC. It is concluded that reacylation to PC is the dominating process in the detoxication of LPC and ensures the transient character of its steady state concentrations, even at maximum phospholipase A(2) activities.     

Critical role of peroxiredoxin 6 in the repair of peroxidized cell membranes following oxidative stress

Phospholipids are a major structural component of all cell membranes; their peroxidation represents a severe threat to cellular integrity and their repair is important to prevent cell death. Peroxiredoxin 6 (Prdx6), a protein with both GSH peroxidase and phospholipase A₂ (PLA₂) activity, plays a critical role in antioxidant defense of the lung and other organs. We investigated the role of Prdx6 in the repair of peroxidized cell membranes in pulmonary microvascular endothelial cells (PMVEC) and isolated mouse lungs treated with tert-butyl hydroperoxide and lungs from mice exposed to hyperoxia (100% O₂). Lipid peroxidation was evaluated by measurement of thiobarbituric acid reactive substances, oxidation of diphenyl-1-pyrenylphosphine, or ferrous xylenol orange assay. The exposure dose was varied to give a similar degree of lipid peroxidation at the end of exposure in the different models. Values for lipid peroxidation returned to control levels within 2 h after oxidant removal in wild-type PMVEC and perfused lungs but were unchanged in Pxdx6 null preparations. An intermediate degree of repair was observed with PMVEC and lungs that expressed only C47S or D140A mutant Prdx6; the former mutant does not have peroxidase activity, while the latter loses its PLA₂ activity. Prdx6 null mice showed markedly delayed recovery from lipid peroxidation during 20 h observation following exposure to hyperoxia. Thus, Prdx6 plays a critical role in the repair of peroxidized phospholipids in cell membranes and the recovery of lung cells from peroxidative stress; the peroxidase and PLA₂ activity each contribute to the recovery process.     

Lysophosphatidate Acyltransferase in the Microsomes from Maturing Seeds of Meadowfoam (Limnanthes alba)

Lysophosphatidate (LPA) acyltransferase (EC 2.3. 1.51) in the microsomes from the maturing seeds of meadowfoam (Limnanthes alba), nasturtium (Tropaeolum majus), palm (Syagrus cocoides), castor bean (Ricinus communis), soybean (Glycine max), maize (Zea mays), and rapeseed (Brassica napus) were tested for their specificities toward 1-oleoyl-LPA or 1-erucoyl-LPA, and oleoyl coenzyme A (CoA) or erucoyl CoA. All the enzymes could use either of the two acyl acceptors and oleoyl CoA, but only the meadowfoam enzyme could use erucoyl CoA as the acyl donor to produce dierucoyl phosphatidic acid (PA). The meadowfoam enzyme was studied further. It had an optimal activity at pH 7 to 8, and its activity was inhibited by 1 millimolar MnCl₂, ZnCl₂, or p-chloromercuribenzoate. In a test of substrate specificity using increasing concentrations of either 1-oleoyl-LPA or 1-erucoyl-LPA, and either oleoyl CoA or erucoyl CoA, the enzyme activity in producing PA was highest for dioleoyl-PA, followed successively by 1-oleoyl-2-erucoyl-PA, dierucoyl-PA, and 1-erucoyl-2-oleoyl-PA. In a test of substrate selectivity using a fixed combined concentration, but varying proportions, of 1-oleoyl-LPA and 1-erucoyl-LPA, and of oleoyl CoA and erucoyl CoA, the enzyme showed a pattern of acyl preference similar to that observed in the test of substrate specificity, but the preference toward oleoyl moiety in the substrates was slightly stronger. The meadowfoam microsomes could convert [¹⁴C]glycerol-3-phosphate to diacylglycerols and triacylglycerols in the presence of erucoyl CoA. The meadowfoam LPA acyltransferase is unique in its ability to produce dierucoyl-PA, and should be a prime candidate for use in the production of trierucin oils in rapeseed via genetic engineering.     

Alterations in cerebrospinal fluid glycerophospholipids and phospholipase A2 activity in Alzheimer's disease

Our aim is to study selected cerebrospinal fluid (CSF) glycerophospholipids (GP) that are important in brain pathophysiology. We recruited cognitively healthy (CH), minimally cognitively impaired (MCI), and late onset Alzheimer''s disease (LOAD) study participants and collected their CSF. After fractionation into nanometer particles (NP) and supernatant fluids (SF), we studied the lipid composition of these compartments. LC-MS/MS studies reveal that both CSF fractions from CH subjects have N-acyl phosphatidylethanolamine, 1-radyl-2-acyl-sn-glycerophosphoethanolamine (PE), 1-radyl-2-acyl-sn-glycerophosphocholine (PC), 1,2-diacyl-sn-glycerophosphoserine (PS), platelet-activating factor-like lipids, and lysophosphatidylcholine (LPC). In the NP fraction, GPs are enriched with a mixture of saturated, monounsaturated, and polyunsaturated fatty acid species, while PE and PS in the SF fractions are enriched with PUFA-containing molecular species. PC, PE, and PS levels in CSF fractions decrease progressively in participants from CH to MCI, and then to LOAD. Whereas most PC species decrease equally in LOAD, plasmalogen species account for most of the decrease in PE. A significant increase in the LPC-to-PC ratio and PLA₂ activity accompanies the GP decrease in LOAD. These studies reveal that CSF supernatant fluid and nanometer particles have different GP composition, and that PLA₂ activity accounts for altered GPs in these fractions as neurodegeneration progresses.     

Lysophosphatidylcholine acyltransferase 3 knockdown-mediated liver lysophosphatidylcholine accumulation promotes very low density lipoprotein production by enhancing microsomal triglyceride transfer protein expression

After de novo biosynthesis phospholipids undergo extensive remodeling by the Lands' cycle. Enzymes involved in phospholipid biosynthesis have been studied extensively but not those involved in reacylation of lysophosphopholipids. One key enzyme in the Lands' cycle is fatty acyl-CoA:lysophosphatidylcholine acyltransferase (LPCAT), which utilizes lysophosphatidylcholine (LysoPC) and fatty acyl-CoA to produce various phosphatidylcholine (PC) species. Four isoforms of LPCAT have been identified. In this study we found that LPCAT3 is the major hepatic isoform, and its knockdown significantly reduces hepatic LPCAT activity. Moreover, we report that hepatic LPCAT3 knockdown increases certain species of LysoPCs and decreases certain species of PC. A surprising observation was that LPCAT3 knockdown significantly reduces hepatic triglycerides. Despite this, these mice had higher plasma triglyceride and apoB levels. Lipoprotein production studies indicated that reductions in LPCAT3 enhanced assembly and secretion of triglyceride-rich apoB-containing lipoproteins. Furthermore, these mice had higher microsomal triglyceride transfer protein (MTP) mRNA and protein levels. Mechanistic studies in hepatoma cells revealed that LysoPC enhances secretion of apoB but not apoA-I in a concentration-dependent manner. Moreover, LysoPC increased MTP mRNA, protein, and activity. In short, these results indicate that hepatic LPCAT3 modulates VLDL production by regulating LysoPC levels and MTP expression.     

Identification and Characterization of Mitochondrial Acetyl-Coenzyme A Hydrolase from Pisum sativum L. Seedlings

Mitochondria from Pisum sativum seedlings purified free of peroxisomal and chlorophyll contamination were examined for acetyl-coenzyme A (CoA) hydrolase activity. Acetyl-CoA hydrolase activity was latent when assayed in isotonic media. The majority of the enzyme activity was found in the soluble matrix of the mitochondria. The products, acetate and CoA, were quantified by two independent methods and verified that the observed activity was an acetyl-CoA hydrolase. The pea mitochondrial acetyl-CoA hydrolase showed a K_m for acetyl-CoA of 74 micromolar and a V_max of 6.1 nanomoles per minute per milligram protein. CoA was a linear competitive inhibitor of the enzyme with a K_is of 16 micromolar. The sensitivity of the enzyme to changes in mole fraction of acetyl-CoA suggested that the changes in the intramitochondrial acetyl-CoA/CoA ratio may be an effective mechanism of control. The widespread distribution of mitochondrial acetyl-CoA hydrolase activity among different plant species indicated that this may be a general mechanism in plants for synthesizing acetate.     

Characterization of long-chain fatty acid uptake in <Emphasis Type="Italic">Caulobacter crescentus</Emphasis>

Studies evaluating the uptake of long-chain fatty acids in Caulobacter crescentus are consistent with a protein-mediated process. Using oleic acid (C18:1) as a substrate, fatty acid uptake was linear for up to 15 min. This process was saturable giving apparent V_max and K_m values of 374 pmol oleate transported/min/mg total protein and 61 μM oleate, respectively, consistent with the notion that one or more proteins are likely involved. The rates of fatty acid uptake in C. crescentus were comparable to those defined in Escherichia coli. Uncoupling the electron transport chain inhibited oleic acid uptake, indicating that like the long-chain fatty acid uptake systems defined in other gram-negative bacteria, this process is energy-dependent in C. crescentus. Long-chain acyl CoA synthetase activities were also evaluated to address whether vectorial acylation represented a likely mechanism driving fatty acid uptake in C. crescentus. These gram-negative bacteria have considerable long-chain acyl CoA synthetase activity (940 pmol oleoyl CoA formed/min/mg total protein), consistent with the notion that the formation of acyl CoA is coincident with uptake. These results suggest that long-chain fatty acid uptake in C. crescentus proceeds through a mechanism that is likely to involve one or more proteins.