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At least 19 recordsLinked to original sources
PubMed2002

Fluorescently labelled bovine acyl-CoA-binding protein acting as an acyl-CoA sensor: interaction with CoA and acyl-CoA esters and its use in measuring free acyl-CoA esters and non-esterified fatty acids.

Long-chain acyl-CoA esters are key metabolites in lipid synthesis and beta-oxidation but, at the same time, are important regulators of intermediate metabolism, insulin secretion, vesicular trafficking and gene expression. Key tools in studying the regulatory functions of acyl-CoA esters are reliable methods for the determination of free acyl-CoA concentrations. No such method is presently available. In the present study, we describe the synthesis of two acyl-CoA sensors for measuring free acyl-CoA concentrations using acyl-CoA-binding protein as a scaffold. Met24 and Ala53 of bovine acyl-CoA-binding protein were replaced by cysteine residues, which were covalently modified with 6-bromoacetyl-2-dimethylaminonaphthalene to make the two fluorescent acyl-CoA indicators (FACIs) FACI-24 and FACI-53. FACI-24 and FACI-53 showed fluorescence emission maximum at 510 and 525 nm respectively, in the absence of ligand (excitation 387 nm). Titration of FACI-24 and FACI-53 with hexadecanoyl-CoA and dodecanoyl-CoA increased the fluorescence yield 5.5-and 4.7-fold at 460 and 495 nm respectively. FACI-24 exhibited a high, and similar increase in, fluorescence yield at 460 nm upon binding of C14-C20 saturated and unsaturated acyl-CoA esters. Both indicators bind long-chain (>C14) acyl-CoA esters with high specificity and affinity (K(d)=0.6-1.7 nM). FACI-53 showed a high fluorescence yield for C8-C12 acyl chains. It is shown that FACI-24 acts as a sensitive acyl-CoA sensor for measuring the concentration of free acyl-CoA, acyl-CoA synthetase activity and the concentrations of free fatty acids after conversion of the fatty acid into their respective acyl-CoA esters.

Acyl Coenzyme A↗
PubMed1980

Relative suitability of 1-palmitoyl and 1-stearoyl homologues of 1-acyl-sn-glycerylphosphorylcholine and different acyl donors for phosphatidylcholine synthesis via acyl-CoA:1-acyl-sn-glycero-3-phosphorylcholine acyltransferase in rat lung microsomes.

The relative suitability of the 1-palmitoyl and 1-stearoyl homologues of 1-acyl-sn-glyceryl-phosphorylcholine and different acyl donors were tested as substrates for phosphatidylcholine synthesis via the acyl-CoA:1-acyl-sn-glycero-3-phosphorylcholine acyltransferase in rat lung microsomes. The acyl acceptor was an almost equi-molar mixture of the [3H]palmitoyl plus [14C]stearoyl species of 1-acyl-sn-glycero-3-phosphorylcholine with palmitoyl-, stearoyl-, oleoyl-, linoleoyl-, or arachidonoyl-CoA serving as the acyl donor. At all concentrations of acyl acceptor, reaction velocities with 20:4-CoA greater than or equal to 18:2-CoA > 18:1-CoA > 16:0-CoA >18:0-CoA. Furthermore, the acyltransferase selectively utilized the 1-palmitoyl over the 1-stearoyl species of 1-acylglycerylphosphorylcholine by 4.2- to 5.7-fold under optimal assay conditions with the various acyl-CoA thiolesters. However, the degree of preference exhibited for the 1-palmitoyl-sn-glycero-3-phosphorylcholine, as acyl acceptor, versus the 1-stearoyl homologue with palmitoyl-CoA as the acyl donor was not significantly different from that obtained with the other acyl-CoA derivatives. Thus, the specificity of the acyl-CoA:1-acyl-sn-glycero-3-phosphorylcholine acyltransferase indicates an ability of this enzyme to produce dipalmitoyl phosphatidylcholine but it cannot independently explain the predominance of dipalmitoyl phosphatidylcholine in lung or the tendency of stearate at the 1-position to associate with fatty acids of increasing unsaturation at the 2-position.

1-Acylglycerophosphocholine O-Acyltransferase↗
PubMed1984

Acylation of plant acyl carrier proteins by acyl-acyl carrier protein synthetase from Escherichia coli.

The acyl-acyl carrier protein synthetase from Escherichia coli has been examined for its ability to specifically acylate acyl carrier protein (ACP) from higher plants in order to develop an assay for plant ACP, and to prepare labeled acyl-ACP of plant origin. It was found that the E. coli enzyme was able to acylate ACP from spinach, soybean, avocado, corn, and several other plants. The acylation was very specific because, in crude extracts of spinach leaves where ACP represented approximately 0.1% of the total soluble protein, ACP was shown to be the only protein acylated. In contrast to other E. coli enzymes that display 2- to 10-fold lower rates with plant versus bacterial ACP, the kinetic constants (Km and Vmax) for acyl-ACP synthetase were found to be essentially identical for spinach and E. coli ACP when acylated with palmitic acid. Palmitic, myristic, lauric, stearic, and oleic acid could all be esterified to both spinach and E. coli ACP with similar specificity. Procedures are described that allow the assay of ACP in plant extracts at the nanogram level.

Acylation↗
PubMed1995

Alteration of acyl-acyl carrier protein pools and acetyl-CoA carboxylase expression in Escherichia coli by a plant medium chain acyl-acyl carrier protein thioesterase.

Expression of a plant lauroyl-acyl carrier protein (ACP) thioesterase in an Escherichia coli strain deficient in beta oxidation results in the accumulation of free fatty acids in the culture. Overall fatty acid production by the cultures is increased severalfold, particularly in the late log and stationary stages of growth. In control E. coli cells, malonyl-ACP levels and rates of fatty acid synthesis are highest during rapid logarithmic growth and decline to undetectable levels in stationary stage. In contrast, in cells expressing plant acyl-ACP thioesterase, malonyl-ACP levels remain high in late log and stationary stage in association with the continued fatty acid production. In addition, the biotin carboxyl carrier protein component of acetyl-CoA carboxylase is expressed at higher levels in cultures expressing the acyl-ACP thioesterase. The data presented indicate that removal of the acyl-ACP products of fatty acid synthesis results in increased production of both malonyl-ACP and fatty acids, which may in turn result from higher activity and/or expression of acetyl-CoA carboxylase.

Acetyl-CoA Carboxylase↗
PubMed1984

Mechanism for adaptive modification during cold acclimation of phospholipid acyl chain composition in Tetrahymena. II. Activities of 2-acyl-sn-glycerol-3-phosphorylcholine and 2-acyl-sn-glycerol-3- phosphorylethanolamine acyltransferases involving the reacylation.

The deacylation-reacylation process is very important for the alteration of phospholipid fatty acyl composition on lowering of growth temperature in Tetrahymena pyriformis (Kameyama, Y., Yoshioka, S. and Nozawa, Y., (1984) Biochim. Biophys. Acta 793, 28-33). Microsomes isolated from Tetrahymena cells have reacylation activities not only for 1-acyl-sn-glycerol-3-phosphorylcholine (1-acyl-GPC) and 1-acyl-sn-glycerol-3-phosphorylethanolamine (1-acyl-GPE) but also for 2-acyl-GPC and 2-acyl-GPE. Unsaturated fatty acyl-CoAs were in general much better substrates than saturated fatty acyl-CoAs for acylations of 1-acyl-GPC and 1-acyl-GPE. The acylation rates for 1-acyl-GPE were almost the same in palmitoleoyl-CoA, oleoyl-CoA, linoleoyl-CoA and gamma-linoleoyl-CoA. However, the acylation activity for 1-acyl-GPC was more than 2-fold higher with palmitoleoyl-CoA than with any other unsaturated fatty acyl-CoAs. In contrast, both 2-acyl-GPC and 2-acyl-GPE acyltransferases did not show a distinct preference for various acyl-CoAs, although palmitoyl-CoA was incorporated into both 2-acylphospholipids at higher rates than into 1-acylphospholipids. These specificities for various acyl-CoAs of 1-acyl- and 2-acyl-GPC and 1-acyl- and 2-acyl-GPE acyltransferases were not changed in the microsomes isolated from cells grown isothermally at 39 degrees C and 15 degrees C and cells shifted from 39 degrees C to 15 degrees C. However, the acylating ratio of linoleoyl-CoA to palmitoyl-CoA, which were chosen as typical unsaturated and saturated fatty acyl-CoAs, in the microsomes from cells grown at 15 degrees C was 1.5-3.0-times higher than in the microsomes from 39 degrees C-grown cells in four acyltransferase activities. These results suggest that the changes of acyl-CoA specificities in reacylation enzyme activities during temperature down-shift would make little contribution to the increase in unsaturated fatty acids in phospholipids, although reacylating enzymes from isothermally grown cells at lower temperature would partially participate in those increases.

Acclimatization↗
PubMed1997

Acyl-CoA binding proteins inhibit the nonenzymic S-acylation of cysteinyl-containing peptide sequences by long-chain acyl-CoAs.

Acyl-CoA binding proteins (ACBPs) from rat and bovine liver were found to inhibit the nonenzymic S-acylation of two representative types of peptides by long-chain acyl-CoAs. As demonstrated previously [Quesnel, S. & Silvius, J. R. (1994) Biochemistry 33 13340-13348; Bharadwaj, M., & Bizzozero, O. A. (1995) J. Neurochem. 65, 1805-1815], peptides with the sequences myristoyl-GCG, myristoyl-GCV, and IRYCWLRR-NH2, all representing physiological S-acylation sites in mammalian proteins, become S-acylated at appreciable rates in the presence of long-chain acyl-CoAs and large unilamellar lipid vesicles. Addition of ACBP at physiological molar ratios with respect to long-chain acyl-CoAs strongly inhibits the spontaneous S-acylation reaction, in a manner that can be quantitatively described by assuming that the ACBP sequesters the acyl-CoA with nanomolar affinity in a complex unable to serve as an S-acyl donor. From these results, we calculate that at physiological (intracellular) concentrations of ACBP, long-chain acyl-CoAs, and membrane lipids the expected half-times for spontaneous S-acylation of such protein sequences by long-chain acyl-CoAs will lie in the range of several tens of hours. The nonenzymic reaction of protein cysteine residues with long-chain acyl-CoAs is thus unlikely to contribute significantly to the physiological modification of signaling and other proteins that show relatively rapid rates of S-acylation in mammalian cells. However, it cannot be excluded that a nonenzymic reaction with long-chain acyl-CoAs could contribute to the physiological S-acylation of certain membrane proteins if the latter exhibit very slow kinetics of S-acylation in vivo.

Acyl Coenzyme A↗
PubMed1991

Isolation and characterization of Escherichia coli K-12 mutants lacking both 2-acyl-glycerophosphoethanolamine acyltransferase and acyl-acyl carrier protein synthetase activity.

2-Acyl-glycerophosphoethanolamine (2-acyl-GPE) acyltransferase and acyl-acyl carrier protein (acyl-ACP) synthetase are thought to be dual catalytic activities of a single inner membrane enzyme. A filter disc replica print method for the detection of acyl-ACP synthetase activity by colony fluorography was used to screen a mutagenized population of cells for acyl-ACP synthetase mutants (aas). All aas mutants lacked both acyl-ACP synthetase and 2-acyl-GPE acyltransferase activities in vitro. There was no detectable acyl-CoA-independent incorporation of exogenous fatty acids into phosphatidylethanolamine or the major outer membrane lipoprotein in aas mutants. Exogenous lysophospholipid uptake and acylation was also lacking in aas mutants. Lipoprotein acylation by phospholipids synthesized by the de novo biosynthetic pathway was not affected in aas mutants showing that this gene product was not directly involved in lipoprotein biogenesis. The aas mutants had an altered membrane phospholipid composition and accumulated both 2-acyl-GPE and acylphosphatidylglycerol. Acylphosphatidylglycerol accumulation was due to the transacylase activity of lysophospholipase L2 (the pldB gene product) since aas pldB double mutants accumulated 2-acyl-GPE, but not acylphosphatidylglycerol. The aas allele was mapped to 61 min of the Escherichia coli chromosome, and the deduced gene order in this region was thyA-aas-lysA. The biochemical, physiological, and genetic analyses of aas mutants support the conclusion that 2-acyl-GPE acyltransferase and acyl-ACP synthetase are two activities of the same protein and confirm that this enzyme system participates in membrane phospholipid turnover and governs the acyl-CoA independent incorporation of exogenous fatty acids and lysophospholipids into the membrane.

Acylation↗
PubMed1995

Fatty acyl-CoA oxidase activity is induced before long-chain acyl-CoA hydrolase activity and acyl-CoA binding protein in liver of rat treated with peroxisome proliferating 3-thia fatty acids.

1. In this study we explored the relationship between specific acyl-CoA esters and induction of acyl-CoA binding protein (ACBP) and enzymes related to the proliferation of peroxisomes. Male Wistar rats were administered a single dose (150 mg/day/kg) of sulphur-substituted fatty acid analogues, and the effects of tetradecylthioacetic acid and 3-thiadicarboxylic acid, which both act as peroxisome proliferators, were compared with the effects of tetradecylthiopropionic acid and palmitic acid which do not induce peroxisome proliferation. 2. The hepatic level of total long-chain acyl-CoA was significantly increased within 12 h of feeding these fatty acids, except in rat fed tetradecylthioacetic acid. Hplc chromatograms of liver extracts prepared from rat fed tetradecylthioacetic acid showed that tetradecylthioacetyl-CoA ester accumulated in the liver 4 h after feeding and had disappeared after 24 h. In liver extracts of the tetradecylthiopropionic acid-treated rat tetradecylthiopropionyl-CoA was not observed, but the appearance of a new long-chain acyl-CoA ester, probably a metabolite of tetradecylthiopropionic acid, was detected. This new peak reached a maximum 4h after feeding. In rat fed tetradecylthioacetic acid and 3-thiadicarboxylic acid the hepatic level of fatty acyl-CoA oxidase mRNA increased 8 h after feeding, while the acyl-CoA oxidase activity had increased after 12 h. 3. The early accumulation of specific tetradecylthioacetyl-CoA suggests that this ester may be a possible mediator of the induction of fatty acyl-CoA oxidase. The level of hepatic acyl-CoA binding protein, long-chain acyl-CoA hydrolase activity and long-chain acyl-CoA synthetase activity did not change after a single dose of all four fatty acids. Prolonged administration of 3-thia fatty acids resulted, however, in a dose- and time-dependent increase in hepatic ACBP content and ACBP mRNA level. The amount of ACBP increased in parallel to the long-chain acyl-CoA hydrolase activity. The correlated induction of fatty acyl-CoA binding protein and long-chain acyl-CoA hydrolase seems to be dependent on a sustained accumulation of total long-chain acyl-CoA esters.

Acyl Coenzyme A↗
PubMed1993

Less polar glycolipids in Alaskan pollack brain: isolation and characterization of acyl galactosyl diacylglycerol, acyl galactosyl ceramide, and acyl glucosyl ceramide.

We recently reported that glycolipid compositions of gadoid fish nerve tissues are unique in the abundance of 1) galactosyl diacylglycerol and its sulfate ester; 2) glucosyl ceramide; and 3) fatty acid ester of cerebroside (J. Lipid Res. 1992. 33: 1351-1359). The present paper reports the characterization of less polar glycolipids isolated from Alaskan pollack brain. Of twelve glycolipids purified by column chromatography, four were of the galactosyl diacylglycerol type. Chemical analysis, infrared spectrometry, and a permethylation study followed by gas chromatography-mass spectrometry revealed that they were 1,2-di-O-acyl-3-beta (6-O-acyl-D-galactopyranosyl)-sn-glycerol and 1,2-di-O-acyl-3-beta(2'-O-acyl-D-galactopyranosyl)-sn-glycerol, and mixtures of these two isomers, with slightly different fatty acid compositions, respectively. The other eight less polar glycolipids were pure forms or mixtures of isomeric forms of cerebroside fatty acid esters in which the substituted position of the acyl group on the hexose moiety varied. The permethylation study revealed that they were 6-O-acyl-beta-D-galactopyranosyl, 6-O-acyl-beta-D-glucopyranosyl, 2-O-acyl-galactosyl, 2-O-acyl-glucosyl, 3- or 4-O-acyl-galactosyl, and 3-O-acyl-glucosyl ceramides, the 6-O- and 2-O-acyl isomers being predominant. This is the first report of the natural occurrence of 1) acyl galactosyl diacylglycerol in animal tissues and 2) acyl glucosyl ceramide in nerve tissues.

Animals↗
PubMed1990

Intraorganelle localization and substrate specificities of the mitochondrial acyl-CoA: sn-glycerol-3-phosphate O-acyltransferase and acyl-CoA: 1-acyl-sn-glycerol-3-phosphate O-acyltransferase from potato tubers and pea leaves.

The mitochondrial sn-glycerol-3-phosphate and 1-acyl-sn-glycerol-3-phosphate O-acyltransferases from potato tubers and pea leaves were investigated with respect to their intraorganelle localization, their positional and substrate specificities, and their fatty acid selectivities. In mitochondria from potato tubers both enzymes were found to be located in the outer membrane. The 1-acyl-sn-glycerol-3-phosphate O-acyltransferase of pea mitochondria showed the same intraorganelle localization whereas the sn-glycerol-3-phosphate O-acyltransferase behaved like a soluble protein of the intermembrane space. The sn-glycerol-3-phosphate O-acyltransferase of both potato and pea mitochondria used sn-glycerol-3-phosphate but not dihydroxyacetone phosphate as acyl acceptor and exclusively catalyzed the formation of 1-acyl-sn-glycerol-3-phosphate which subsequently served as substrate for the second acylation reaction at its C-2 position. Both acyltransferases of potato as well as pea mitochondria showed higher activities with acyl-CoA than with the corresponding acyl-(acyl carrier protein) thioesters. When different acyl-CoA thioesters were offered separately, the sn-glycerol-3-phosphate O-acyltransferase of potato mitochondria displayed no fatty acid specificity whereas the enzyme of pea mitochondria revealed one for saturated acyl groups. On the other hand, the mitochondrial 1-acyl-sn-glycerol-3-phosphate O-acyltransferases from both potato tubers and pea leaves were more active on unsaturated than on saturated acyl-CoA thioesters. Furthermore, these enzymes preferentially used oleoyl- and linoleoyl-CoA when they were offered in a mixture with saturated ones, although the fatty acid selectivity of the pea enzyme was less pronounced than that of the potato enzyme. The sn-glycerol-3-phosphate O-acyltransferase of potato mitochondria displayed a slight preference for saturated acyl groups.

1-Acylglycerol-3-Phosphate O-Acyltransferase↗
PubMed2001

HPLC/1H NMR spectroscopic studies of the reactive alpha-1-O-acyl isomer formed during acyl migration of S-naproxen beta-1-O-acyl glucuronide.

A widely held view in drug metabolism and pharmacokinetic studies is that the initial 1-isomer to 2-isomer step in the intramolecular acyl migration of drug ester glucuronides is irreversible, and that alpha-1-O-acyl isomers do not occur under physiological conditions. We investigated this hypothesis using high-performance liquid chromatography directly coupled to proton nuclear magnetic resonance spectroscopy (HPLC/1H NMR) and mass spectrometry (LC/MS) to probe the migration reactions of S-naproxen beta-1-O-acyl glucuronide, in phosphate buffer at pH 7.4, 37 degrees C. We report the first direct observation of the alpha-1-O-acyl isomer of a drug ester glucuronide (S-naproxen) formed in a biosystem via the facile acyl migration of the corresponding pure beta-1-O-acyl glucuronide. The unequivocal identification of the reactive product was achieved using stopped-flow one-dimensional HPLC/1H NMR and two-dimensional 1H-1H total correlation spectroscopy (1H-1H TOCSY). Parallel LC/ion-trap mass spectrometry yielded the confirmatory glucuronide masses. Moreover, "dynamic" stopped-flow HPLC/1H NMR experiments revealed transacylation of the isolated alpha-1-O-acyl isomer to a mixture of alpha/beta-2-O-acyl isomers; the reverse reaction from the isolated alpha/beta-2-O-acyl isomers to the alpha-1-O-acyl isomer was also clearly demonstrated. This application of "dynamic" stopped-flow HPLC/1H NMR allows key kinetic data to be obtained on a reactive metabolite that would otherwise be difficult to follow by conventional HPLC and NMR methods where sample preparation and off-line separations are necessary. These data challenge the widely held view that the alpha-1-O-acyl isomers of drug ester glucuronides do not occur under physiological conditions. Furthermore, the similar formation of alpha-1-O-acyl isomers from zomepirac and diflunisal beta-1-O-acyl glucuronides has recently been confirmed (Corcoran et al., unpublished results). Such reactions are also likely to be widespread for other drugs that form ester glucuronides in biological systems. Ultimately, the presence of significant quantities of the kinetically labile alpha-1-O-acyl glucuronide isomer may also have toxicological implications in terms of reactivity toward cellular proteins.

Acylation↗
PubMed1993

Purification of an acyl-CoA hydrolase from rat intestinal microsomes. A candidate acyl-enzyme intermediate in glycerolipid acylation.

We have purified to apparent homogeneity an acyl-CoA hydrolase activity from rat intestinal villus cell microsomes by heparin and anion exchange and affinity chromatography. The purified 54-kDa acyl-CoA hydrolase along with several microsomal proteins form a covalent acyl-protein bond upon incubation with an activated fatty acid (acyl-CoA). The acyl moiety of the acylated acyl-CoA hydrolase is stable to denaturation and extraction with organic solvents, but is displaced by neutral hydroxylamine or mercaptoethanol, indicating a labile high energy (thio)ester linkage. The enzyme activity is inhibited by thiol-directed reagents and activated by the presence of dithiothreitol suggesting the presence of a cysteine residue(s) at or near the active site. Common serine-esterase inhibitors (NaF, phenylmethylsulfonyl fluoride) and activators (Mg2+, Ca2+) had no effect on the hydrolase activity. The enzyme hydrolyzed (transferred to water) 14-20 carbon acyl-CoA with similar efficiencies and did not utilize glycerophospholipids or mono- and diacylglycerols as potential acyl donors/acceptors. Phospholipids and mono- and diradylglycerols at concentrations below 100 microM or polyclonal antibodies raised against the purified hydrolase did not inhibit the enzyme activity. However, the acyl-CoA hydrolase activity could be immunoprecipitated from solubilized microsomes or purified enzyme preparations with corresponding decrease of the hydrolase activity in the supernatant of the immunoprecipitate. Immunoblotting studies show cross-reactivity with a protein of an identical molecular mass in other rat or human tissues. It is concluded that the microsomal acyl-CoA hydrolase deserves consideration as a candidate acyl-enzyme intermediate in glycerolipid synthesis when associated with appropriate acyltransferases.

Acylation↗
PubMed1996

Inhibition of beta-ketoacyl-acyl carrier protein synthase III (FabH) by acyl-acyl carrier protein in Escherichia coli.

beta-Ketoacyl-acyl carrier protein (ACP) synthase III (the fabH gene product) condenses acetyl-CoA with malonyl-ACP to initiate fatty acid biosynthesis in the dissociated, type II fatty acid synthase systems typified by Escherichia coli. The accumulation of malonyl-acyl carrier protein (ACP) following the inhibition of a reconstituted fatty acid synthase system by acyl-ACP implicated synthase III (FabH) as a target for acyl-ACP regulation (Heath, R. J., and Rock, C. O. (1996) J. Biol. Chem. 271, 1833-1836); therefore, the FabH protein was purified and its biochemical and regulatory properties examined. FabH exhibited a Km of 40 microM for acetyl-CoA and 5 microM for malonyl-ACP. FabH also accepted other acyl-CoAs as primers with the rank order of activity being acetyl-CoA approximately propionyl-CoA >> butyryl-CoA. FabH utilized neither hexanoyl-CoA nor octanoyl-CoA. Acyl-ACPs suppressed Fabh activity, and their potency increased with increasing acyl chain length between 12 and 20 carbon atoms. Nonesterified ACP was not an inhibitor. Acyl-ACP inhibition kinetics were mixed with respect to acetyl-CoA, but were competitive with malonyl-ACP, indicating that acyl-ACPs decrease FabH activity by binding to either the free enzyme or the acyl-enzyme intermediate. These data support the concept that the inhibition of chain initiation at the beta-ketoacyl-ACP synthase III step contributes to the attenuation of fatty acid biosynthesis by acyl-ACP.

3-Oxoacyl-(Acyl-Carrier-Protein) Synthase↗
PubMed1988

Acylation in vitro of the myelin proteolipid protein and comparison with acylation in vivo: acylation of a cysteine occurs nonenzymatically.

Characteristics of fatty acylation of myelin proteolipid protein (PLP) in vitro were compared with the corresponding process in vivo. Rapid and efficient separation of labelled PLP from other proteins and lipids was effected by extraction into chloroform/methanol/0.1 N HCl (10/10/1) and chromatography on Sephadex LH-60 in the same solvent. Covalent linkage of [3H]-palmitate to PLP was demonstrated by repetitive chromatography on LH-60, thin layer chromatography, and polyacrylamide gel electrophoresis. Reductive cleavage with sodium borohydride of PLP acylated in vitro or in vivo yielded [3H]-hexadecanol, identifying at least one of the acyl linkages as a thiolester bond. When PLP was acylated with acyl-CoA as the fatty acid donor, the reaction occurred non-enzymatically as supported by the following observations: 1) acylation activity increased with increasing pH above pH 7.5, 2) acylation activity was heat stable, 3) acylation activity was not removed from PLP during purification in organic solvents or in Triton X-100-containing buffers, and 4) acylation of tryptic fragments occurred in the absence of an exogenously added enzyme source. The relevance of in vitro fatty acylation of PLP to that in vivo was confirmed by comparison of proteolytically derived peptide maps that showed that likely the same domain of PLP was acylated in vitro and in vivo.

Acylation↗
PubMed2006

The soluble acyl-acyl carrier protein synthetase of Vibrio harveyi B392 is a member of the medium chain acyl-CoA synthetase family.

The gene encoding the unique soluble acyl-acyl carrier protein synthetase (AasS) of the bioluminescent Vibrio harveyi strain B392 has been isolated by expression cloning in Escherichia coli. This enzyme catalyzes the ATP-dependent acylation of the thiol of acyl carrier protein (ACP) with fatty acids with chain lengths from C4 to C18. The gene (called aasS) encodes a protein of 60 kDa, a hexahistidine-tagged version of which was readily expressed in E. coli and purified in large quantities. Surprisingly, the sequence of the encoded protein was significantly more similar to that of an acyl-CoA synthetase of the distantly related bacterium, Thermus thermophilus, than to that of the membrane-bound acyl-acyl carrier protein synthetase of E. coli, an enzyme that catalyzes the same reaction from a more closely related organism. Indeed, the AasS sequence can readily be modeled on the known crystal structures of the T. thermophilus acyl-CoA synthetase with remarkably high levels of conservation of the catalytic site residues. To test the possible role of AasS in the fatty aldehyde-dependent bioluminescence pathway of V. harveyi, the chromosomal aasS gene of the organism was disrupted by insertion of a kanamycin cassette by homologous recombination. The resulting aasS::kan strains retained low levels of acyl-acyl carrier protein synthetase consistent with prior indications of a second such activity in this bacterium. The mutant strains grew normally and had normal levels of bioluminescence but were deficient in the incorporation of exogenous octanoic acid into the cellular phospholipids of V. harveyi, particularly at low octanoate concentrations. These data indicate that AasS is responsible for a high-affinity and high-capacity uptake system that efficiently converts exogenous fatty acids into acyl-ACP species competent to enter the fatty acid biosynthetic cycle.

Aldehydes↗
PubMed1981

The suitability of different acyl acceptors as substrates for the acyl-Coa : 2-acyl-sn-glycero-3-phosphorylcholine acyltransferase in rat liver microsomes.

The fatty acid selectivity of the acyl-CoA : 2-acyl-sn-glycero-3-phosphorylcholine acyltransferase towards different acyl acceptors was studied in rat liver microsomes. The individual molecular species of 2-acylglycerylphosphorylcholine tested as enzyme substrates contained either palmitate, stearate, oleate, linoleate, linolenate or arachidonate with an equimolar mixture of [14C]palmitoyl-CoA plus [3H]stearoyl-CoA serving as the acyl donor. At 2-acyl-sn-glycero-3-phosphorylcholine concentrations of 16 or 64 microM, the various acyl acceptors gave generally similar reactivities, although reaction velocities with the linoleoyl, linolenoyl or arachidonoyl species were moderately greater than those with 2-stearoyl-sn-glycero-3-phosphorylcholine. Regardless of the acyl acceptor tested, little preference towards either palmitoyl-CoA or stearoyl-CoA was indicated at 64 microM 2-acyl-sn-glycero-3-phosphorylcholine whereas a distinct preference for stearate over palmitate (by 1.9--2.6-fold) was exhibited when a lower concentration (16 microM) of the acceptor was employed. The results support the potential importance of the acyl-CoA : 2-acyl-sn-glycero-3-phosphorylcholine acyltransferase for the synthesis of 1-stearoyl 2-unsaturated species of phosphatidylcholine. However, it cannot independently account for the varying palmitate : stearate ratios in the 1-position of this phospholipid when different unsaturated fatty acids reside in the 2-position.

Acyl Coenzyme A↗
PubMed1994

The activities of acyl-CoA hydrolase in lysate and subcellular fractions of human blood platelets in relation to activities of acyl-CoA:1-acyl-lysophospholipid acyltransferase.

The activities of acyl-CoA hydrolase (EC 3.1.2.2.) and acyl-CoA:1-acyl- lysophospholipid acyltransferase (EC 2.3.1.23) have been studied in subcellular fractions of human platelets. The acyl-CoA:1-acyl-lysophospholipid acyltransferase activity was higher in the 'dense-tubular-system-enriched' fraction than in the 'light-mitochondrial' fraction, using endogenously acyl-CoAs formed from labelled fatty acids, ATP, CoA and various lysophospholipids. No activity was found in the 'particle-free' fraction. No difference in specificities was observed between the incorporation of various fatty acids into different lysoPLs in the subcellular fractions compared with the platelet lysates. Generally, arachidonic, linoleic and eicosapentaenoic acids were better substrates for the acyl-CoA:1-acyl-lysophospholipid acyltransferases than oleic, docosahexaenoic and palmitic acids. The opposite was observed with the acyl-CoA hydrolase activity, palmitoyl-CoA was the substrate giving the highest activity, and eicosapentaenoyl-CoA and arachidonoyl-CoA the lowest. About 85% of the hydrolase activity was detected in the 'particle-free' fraction, with each of the six acyl-CoA derivatives tested.

1-Acylglycerophosphocholine O-Acyltransferase↗