Partial purification and photoaffinity labelling of sunflower acyl-CoA:lysophosphatidylcholine acyltransferase

Previous attempts to purify acyl-CoA:1-acyl-lysophosphatidylcholine acyltransferase (EC 2.3.1.23) have been frustrated by difficulties in solubilizing the enzyme without inactivation. Microsomal preparations, from the developing cotyledons of sunflower, in high concentrations of urea retain activity. Gel-filtration liquid chromatography followed by trypsin treatment (minus urea) resulted in the removal of many contaminating proteins without loss of enzyme activity. SDS/PAGE showed the presence of two major peptides with apparent molecular masses of 52 and 59 kDa. These polypeptides cross-reacted with the radiolabelled photoreactive substrate 1-azido-oleoyl-sn-lysophosphatidyl-[N-methyl-(3)H]choline.     

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     

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.     

Lysophosphatidylcholine acyltransferase and lysophosphatidylcholine: lysophosphatidylcholine acyltransferase in alveolar type II cells from fetal rat lung

The specific activity of lysophosphatidylcholine acyltransferase in sonicated fetal rat lung type II cells was found to be an order of magnitude greater than that of lysophosphatidylcholine:lysophosphatidylcholine acyltransferase. The specific activity of lysophosphatidylcholine acyltransferase in sonicated fetal rat lung type II cells increases towards the end of gestation, whereas that of lysophosphatidylcholine:lysophosphatidylcholine acyltransferase does not show a change. While lysophosphatidylcholine acyltransferase in whole fetal lung homogenate is more active towards oleoyl-CoA than towards palmitoyl-CoA, the enzyme in sonicated fetal type II cells is more active towards palmitoyl-CoA. If measured with palmitoyl-CoA as acyl donor, the specific activity of lysophosphatidylcholine acyltransferase in type II cells is higher than that in whole lung during late gestation. In contrast, the specific activity of lysophosphatidylcholine:lysophosphatidylcholine acyltransferase in type II cells is lower than that in whole lung. These observations indicate that in fetal rat type II cells the deacylation-reacylation cycle is more important for the formation of dipalmitoylphosphatidylcholine than the deacylation-transacylation process.     

Effect of albumin on acyl-CoA: lysolecithin acyltransferase,lysolecithin : lysolecithin acyltransferase and acyl-CoA hydrolase from rabbit lung

Acyl-CoA : lysolecithin and lysolecithin : lysolecithin acyltransferases, as well as acyl-CoA hydrolase are important enzymes in lung lipid metabolism. They use amphiphylic lipids as substrates and differ in subcellular localization. In this sense, lipid-protein interactions can be an essential factor in their activity. We have studied the effect of albumin, as lipid-binding protein model, in the activities of these enzymes. Acyl-CoA hydrolase was inhibited in the presence of albumin, whereas acyl-CoA : lysolecithin acyltransferase showed a complex effect of activation depending on both albumin concentration and palmitoyl-CoA/lysolecithin molar ratio. Lysolecithin : lysolecithin acyltransferase was affected differentially on its two activities. Hydrolysis remained unaffected and transacylation was inhibited by albumin. These results are consequence of the interaction of albumin with both lipidic substrates that changes their critical micellar concentration.Abbreviations TNS 6-(p-toluidino)-2-naphthalene-sulfonic acid - CMC Critical Micellar Concentration - LP Lysolecithin (1-acyl-sn-glycero-3-phosphocholine) - PalmCoA palmitoyl-CoA     

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.     

Selective inhibitors of a PAF biosynthetic enzyme lysophosphatidylcholine acyltransferase 2

Platelet-activating factor (PAF) is a potent pro-inflammatory phospholipid mediator. In response to extracellular stimuli, PAF is rapidly biosynthesized by lyso-PAF acetyltransferase (lyso-PAFAT). Previously, we identified two types of lyso-PAFATs: lysophosphatidylcholine acyltransferase (LPCAT)1, mostly expressed in the lungs where it produces PAF and dipalmitoyl-phosphatidylcholine essential for respiration, and LPCAT2, which biosynthesizes PAF and phosphatidylcholine (PC) in the inflammatory cells. Under inflammatory conditions, LPCAT2, but not LPCAT1, is activated and upregulated to produce PAF. Thus, it is important to develop inhibitors specific for LPCAT2 in order to ameliorate PAF-related inflammatory diseases. Here, we report the first identification of LPCAT2-specific inhibitors, N-phenylmaleimide derivatives, selected from a 174,000-compound library using fluorescence-based high-throughput screening followed by the evaluation of the effects on LPCAT1 and LPCAT2 activities, cell viability, and cellular PAF production. Selected compounds competed with acetyl-CoA for the inhibition of LPCAT2 lyso-PAFAT activity and suppressed PAF biosynthesis in mouse peritoneal macrophages stimulated with a calcium ionophore. These compounds had low inhibitory effects on LPCAT1 activity, indicating that adverse effects on respiratory functions may be avoided. The identified compounds and their derivatives will contribute to the development of novel drugs for PAF-related diseases and facilitate the analysis of LPCAT2 functions in phospholipid metabolism in vivo.     

Incorporation of cis-parinaric acid, a fluorescent fatty acid, into synaptosomal phospholipids by an acyl-CoA acyltransferase

The cis-isomer of parinaric acid, a naturally occurring C-18 polyene fatty acid, was incubated with brain subcellular fractions and the polarization of fluorescence increased in a time dependent manner. Greatest increases occurred in synaptosomal and microsomal membranes. This increase in polarization of fluorescence was found with the cis, but not the trans, isomer of parinaric acid and required Mg2+ or Ca2+ and was stimulated by coenzyme A and ATP. Synaptosomes were incubated with cis-parinaric acid and lipids were extracted and examined by high performance liquid chromatography. The highest incorporations of cis-parinaric acid were found in phosphatidylcholine (71%) and phosphatidylethanolamine (20%) while only traces were found in phosphatidylserine and phosphatidylinositol. [3H]Oleic acid was also incorporated into membrane phospholipids and unlabeled oleic acid blocked incorporation of cis-parinaric acid. It is proposed that cis-parinaric acid, like fatty acids normally found in brain, is incorporated into membrane phospholipids by an acyl-CoA acyltransferase. The presence of this enzyme in nervous tissue may make it possible to easily introduce fluorescent fatty acid probes into membrane phospholipids and to thereby facilitate study of membrane-mediated processes.     

Cellular pregnenolone esterification by acyl-CoA:cholesterol acyltransferase

Pregnenolone (PREG) can be converted to PREG esters (PE) by the plasma enzyme lecithin: cholesterol acyltransferase (LCAT), and by other enzyme(s) with unknown identity. Acyl-CoA:cholesterol acyltransferase 1 and 2 (ACAT1 and ACAT2) convert various sterols to steryl esters; their activities are activated by cholesterol. PREG is a sterol-like molecule, with 3-β-hydroxy moiety at steroid ring A, but with much shorter side chain at steroid ring D. Here we show that without cholesterol, PREG is a poor ACAT substrate; with cholesterol, the V(max) for PREG esterification increases by 100-fold. The binding affinity of ACAT1 for PREG is 30-50-fold stronger than that for cholesterol; however, PREG is only a substrate but not an activator, while cholesterol is both a substrate and an activator. These results indicate that the sterol substrate site in ACAT1 does not involve significant sterol-phospholipid interaction, while the sterol activator site does. Studies utilizing small molecule ACAT inhibitors show that ACAT plays a key role in PREG esterification in various cell types examined. Mice lacking ACAT1 or ACAT2 do not have decreased PREG ester contents in adrenals, nor do they have altered levels of the three major secreted adrenal steroids in serum. Mice lacking LCAT have decreased levels of PREG esters in the adrenals. These results suggest LCAT along with ACAT1/ACAT2 contribute to control pregnenolone ester content in different cell types and tissues.     

Chemical mechanism of lysophosphatidylcholine: lysophosphatidylcholine acyltransferase from rabbit lung. pH-dependence of kinetic parameters.

Lysophosphatidylcholine: lysophosphatidylcholine acyltransferase is an enzyme that catalyses two reactions: hydrolysis of lysophosphatidylcholine and transacylation between two molecules of lysophosphatidylcholine to give disaturated phosphatidylcholine. Following the kinetic model previously proposed for this enzyme [Martín, Pérez-Gil, Acebal & Arche (1990) Biochem. J. 266, 47-53], the values of essential pK values in free enzyme and substrate-enzyme complexes have now been determined. The chemical mechanism of catalysis was dependent on the deprotonation of a histidine residue with pK about 5.7. This result was supported by the perturbation of pK values by addition of organic solvent. Very high and exothermic enthalpy of ionization was measured, indicating that a conformational re-arrangement in the enzyme accompanies the ionization of the essential histidine residue. These results, as well as the results from previous studies, enabled the proposal of a chemical mechanism for the enzymic reactions catalysed by lysophosphatidylcholine: lysophosphatidylcholine acyltransferase from rabbit lung.     

Biosynthesis of phosphatidylcholine by human lysophosphatidylcholine acyltransferase 1

Pulmonary surfactant is a complex of phospholipids and proteins lining the alveolar walls of the lung. It reduces surface tension in the alveoli, and is critical for normal respiration. Pulmonary surfactant phospholipids consist mainly of phosphatidylcholine (PC) and phosphatidylglycerol (PG). Although the phospholipid composition of pulmonary surfactant is well known, the enzyme(s) involved in its biosynthesis have remained obscure. We previously reported the cloning of murine lysophosphatidylcholine acyltransferase 1 (mLPCAT1) as a potential biosynthetic enzyme of pulmonary surfactant phospholipids. mLPCAT1 exhibits lysophosphatidylcholine acyltransferase (LPCAT) and lysophosphatidylglycerol acyltransferase (LPGAT) activities, generating PC and PG, respectively. However, the enzymatic activity of human LPCAT1 (hLPCAT1) remains controversial. We report here that hLPCAT1 possesses LPCAT and LPGAT activities. The activity of hLPCAT1 was inhibited by N-ethylmaleimide, indicating the importance of some cysteine residue(s) for the catalysis. We found a conserved cysteine (Cys²¹¹) in hLPCAT1 that is crucial for its activity. Evolutionary analyses of the close homologs of LPCAT1 suggest that it appeared before the evolution of teleosts and indicate that LPCAT1 may have evolved along with the lung to facilitate respiration. hLPCAT1 mRNA is highly expressed in the human lung. We propose that hLPCAT1 is the biosynthetic enzyme of pulmonary surfactant phospholipids.     

Incorporation of cis-parinaric acid,a fluorescent fatty acid,into synaptosomal phospholipids by an acyl-CoA acyltransferase

The cis-isomer of parinaric acid, a naturally occurring C-18 polyene fatty acid, was incubated with brain subcellular fractions and the polarization of fluorescence increased in a time dependent manner. Greatest increases occurred in synaptosomal and microsomal membranes. This increase in polarization of fluorescence was found with the cis, but not the trans, isomer of parinaric acid and required Mg²⁺ or Ca²⁺ and was stimulated by coenzyme A and ATP. Synaptosomes were incubated with cis-parinaric acid and lipids were extracted and examined by high performance liquid chromatography. The highest incorporations of cis-parinaric acid were found in phosphatidylcholine (71%) and phosphatidylethanolamine (20%) while only traces were found in phosphatidylserine and phosphatidylinositol. [³H]Oleic acid was also incorporated into membrane phospholipids and unlabeled oleic acid blocked incorporation of cis-parinaric acid. It is proposed that cis-parinaric acid, like fatty acids normally found in brain, is incorporated into membrane phospholipids by an acyl-CoA acyltransferase. The presence of this enzyme in nervous tissue may make it possible to easily introduce fluorescent fatty acid probes into membrane phospholipids and to thereby facilitate study of membrane-mediated processes.     

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.     

Upregulation of acyl-CoA: cholesterol acyltransferase in chronic renal failure

Chronic renal failure (CRF) is associated with profound abnormalities of lipid metabolism and accelerated arteriosclerotic cardiovascular disease. In a recent study, we found marked downregulation of hepatic lecithin-cholesterol acyltransferase, or LCAT, expression, which can account for impaired HDL maturation and depressed HDL cholesterol concentration in CRF. Here, we report on the effect of CRF on acyl-CoA:cholesterol acyltransferase (ACAT) expression. ACAT is an intracellular enzyme that catalyzes esterification of free cholesterol to cholesterol ester for storage or secretion. ACAT plays a major role in hepatic production and release of VLDL, intestinal absorption of cholesterol, foam cell formation, and atherogenesis. We examined hepatic expression of ACAT-1 and ACAT-2 mRNA (Northern blot) and protein (Western blot) abundance and total ACAT activity in male CRF rats (6 wk after 5/6 nephrectomy) and sham-operated controls. The CRF animals showed a significant reduction in creatinine clearance, marked hypertriglyceridemia, modest hypercholesterolemia, and significant upregulation of hepatic tissue ACAT-2 protein and mRNA abundance. In contrast, hepatic ACAT-1 mRNA and protein abundance were unaffected by CRF. Upregulation of ACAT-2 expression was accompanied by a significant increase in hepatic ACAT activity and a significant decrease in hepatic microsomal and whole liver free cholesterol concentration. Thus CRF results in significant upregulation of hepatic ACAT-2 (but not ACAT-1) expression and ACAT activity, which may, in part, contribute to the associated lipid disorders.     

Characterization of acyl-CoA:cholesterol acyltransferase from neonatal chick liver

Endogenous cholesterol esterification in chick liver microsomes was catalyzed by acyl-CoA:cholesterol acyltransferase using palmitoyl-CoA as substrate. An acyl-CoA hydrolase activity was also found in our microsomal preparations. Acyltransferase activity was stable after microsomes storage at -40 degrees C for 6 weeks and increased linearly with the preincubation time between 0 and 45 min. In our assay conditions, cholesteryl ester formation was linear up to 0.3 mg of microsomal protein in the reaction vial and 10 min of incubation. Maximal activity was found in reactions carried out in the presence of 1-2 mM dithiothreitol and 1.2 mg of bovine serum albumin, while acyl-CoA hydrolase was clearly inhibited by increasing albumin amounts.     

Saucerneol B derivatives as human acyl-CoA: cholesterol acyltransferase inhibitors

A series of 2a-i were prepared from a lead compound, saucerneol B (1) for evaluating their acyl-CoA: cholesterol acyltransferase inhibitory activities. Compounds 2a-g exhibited the high specificity of hACAT-1 than hACAT-2, whereas 2h and 2i showed very weak inhibitory activities in both hACAT-1 and hACAT-2. Saucerneol B (1) exhibited strong cholesterol-lowering effect in high cholesterol-fed mice.     

The kinetic mechanism of acyl-CoA:lysolecithin acyltransferase from rabbit lung

Acyl-CoA:lysolecithin acyltransferase is a key enzyme in the deacylation-reacylation pathway of biosynthesis of molecular species of lecithin. However, the mechanism of the reaction has been little studied. In this paper, the kinetic mechanism of acyl-CoA:lysolecithin acyltransferase, partially purified from rabbit lung, is studied. The double-reciprocal plots of initial velocity vs substrate concentration gave two sets of parallel lines which fitted to a ping-pong equation with the following parameters: Km (palmitoyl-CoA) = 8.5 +/- 2 microM, Km (lysolecithin) = 61 +/- 16 microM, and V = 18 +/- 4 nmol/min/mg protein. Inhibition studies by substrates, alternate substrates, and products supported the ping-pong mechanism, although some nonclassical behavior was observed. Palmitoyl-CoA did not inhibit even at concentrations of 100 Km. In contrast, lysolecithin was a dead-end inhibitor with a dissociation constant of Ki = 930 +/- 40 microM. Alternate substrates and CoA showed alternate pathways for the reaction due to the formation of ternary complexes. Dipalmitoylphosphatidylcholine inhibition pointed to an isomerization of the free enzyme prior to the start of the reaction. From these results, an iso-ping-pong kinetic mechanism for lysolecithin acyltransferase is proposed. The kinetic steps of the reaction are correlated with previous chemical studies of the enzyme.     

Glibenclamide acts as an inhibitor of acyl-CoA:cholesterol acyltransferase enzyme

Sulfonylureas are used in the treatment of non-insulin-dependent diabetes mellitus. Little is known, however, about their effects on cholesterol metabolism. We tested in the present study the effects of glibenclamide (GB) on cholesterol esterification (CE) in macrophage-derived cells. GB inhibited intracellular accumulation of CE induced by acetylated LDL or oxidized LDL in J774 cells, but no such effect on total cholesterol, suggesting that the target of GB was acyl-CoA:cholesterol acyltransferase (ACAT). In the cell-free reconstitution ACAT assay, GB inhibited the ACAT activity with an IC(50) value of 20 microM. Furthermore, GB effectively inhibited the ACAT activity of PMA-stimulated THP-1 cells to the undifferentiated level of THP-1. In the whole-cell ACAT assay using CHO cells overexpressed with ACAT-1 or ACAT-2, GB inhibited the activity of both isozymes with similar potency. Our in vitro data suggest that sulfonylurea could be a potential seed for a new generation of ACAT inhibitors.