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.     

Acyltransferase systems involved in phospholipid metabolism in Saccharomyces cerevisiae.

Membrane preparations from Saccharomyces cerevisiae OC-2 catalyzed the acylation of glycerophosphate, 1-acyl and 2-acyl isomers of monoacylglycerophosphate, and 1-acyl and 2-acyl isomers of monoacylglycerylphosphorylcholine. The acyl-CoA:glycerophosphate acyltransferase system (EC 2.3.1.15) showed a broad specificity for acyl-CoAs when the maximal velocities were compared under optimized conditions. The acyl-CoA:2-acylglycerophosphate acyltransferase activity was much lower than the 1-acyl-glycerophosphate acyltransferase activity. Although the 1-acylglycerophosphate acyltransferase system utilized saturated and unsaturated acyl-CoAs at comparable rates, the acylations at the 1- and 2-positions were relatively more selective for palmitate and oleate, respectively, when assayed in the presence of palmitoyl-CoA, oleoyl-CoA, 1-acylglycerophosphate, and 2-acylglycerophosphate. The acyl-CoA:1-acylglyceryl-phosphorylcholine acyltransferase system (EC 2.3.1.23) was relatively more specific for unsaturated acyl-CoAs, while the acyl-CoA:2-acylglycerylphosphorylcholine acyltransferase system (EC 2.3.1.23) utilized both palmitoyl-CoA and oleoyl-CoA at a comparable rate. Although various acyltransferase systems showed a different degree of specificity for acyl-CoAs, the positional distribution of fatty acids in the phospholipid molecules could not be explained simply by the observed specificities. Zymolyase, β-1,3-glucanase from Arthrobacter luteus, was used successfully for the protoplast formation. Subcellular fractionation of the protoplast revealed that these acyltransferase activities were localized mainly in the microsomal fraction. However, the glycerophosphate and 1-acylglycerophosphate acyltranferase activities in the mitochondrial fraction could not be explained by the contamination of microsomes in this fraction. These observations are apparently inconsistent with a current concept that the mitochondrial fraction is the major site of phospholipid synthesis in yeast.     

Characterization of sn-glycerol 3-phosphate acyltransferase from guinea pig harderian gland microsomes

Microsomal sn-glycerol 3-phosphate acyltransferase from the guinea pig Harderian gland was studied. Its specific activity (1.0 nmol/min X mg, with palmitoyl-CoA as a substrate) was almost the same as that of the rat liver microsomal enzyme. The enzyme acted on various types of acyl-CoA, the relative reaction rates being as follows: palmitoyl-CoA, 100(%); stearoyl-CoA, 30; oleoyl-CoA, 50; linoleoyl-CoA, 40; and arachidonoyl-CoA, 20. When assayed in the presence of 1 mM 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB), the activity on palmitoyl-CoA was inhibited by only 20-30%, whereas those for other acyl-CoAs were completely abolished. The DTNB-resistant activity was inhibited by 0.1 mM dihydroxyacetonephosphate and 0.5 mM dithiothreitol, whereas the DTNB-sensitive activity was not affected. Furthermore, heat treatment at 50 degrees C for 15 min abolished most of the DTNB-sensitive activity, but not the DTNB-resistant activity. These results, taken together, suggested that the microsomal fraction of the guinea pig Harderian gland contained at least two types of sn-glycerol 3-phosphate acyltransferase, and that, in contrast to in the case of rat liver microsomes, a DTNB-resistant enzyme that utilized exclusively palmitoyl-CoA was predominant.     

Intestinal Lipid Esterification and Aging in Mice and Rats

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

Microsomal fatty acyl-CoA transacylation and hydrolysis: fatty acyl-CoA species dependent modulation by liver fatty acyl-CoA binding proteins1

Liver and intestinal cytosol contain abundant levels of long chain fatty acyl-CoA binding proteins such as liver fatty acid binding protein (L-FABP) and acyl-CoA binding protein (ACBP). However, the relative function and specificity of these proteins in microsomal utilization of long chain fatty acyl-CoAs (LCFA-CoAs) for sequential transacylation of glycerol-3-phosphate to form phosphatidic acid is not known. The results showed for the first time that L-FABP and ACBP both stimulated microsomal incorporation of the monounsaturated oleoyl-CoA and polyunsaturated arachidonoyl-CoA 8–10-fold and 2–3-fold, respectively. In contrast, these proteins inhibited microsomal utilization of the saturated palmitoyl-CoA by 69% and 62%, respectively. These similar effects of L-FABP and ACBP on microsomal phosphatidic acid biosynthesis were mediated primarily through the activity of glycerol-3-phosphate acyltransferase (GPAT), the rate limiting step, rather than by protecting the long chain acyl-CoAs from microsomal hydrolase activity. In fact, ACBP but not L-FABP protected long chain fatty acyl-CoAs from microsomal acyl-CoA hydrolase activity in the order: palmitoyl-CoA>oleoyl-CoA>arachidonoyl-CoA. In summary, the data established for the first time a role for both L-FABP and ACBP in microsomal phosphatidic acid biosynthesis. By preferentially stimulating microsomal transacylation of unsaturated long chain fatty acyl-CoAs while concomitantly exerting their differential protection from microsomal acyl-CoA hydrolase, L-FABP and ACBP can uniquely function in modulating the pattern of fatty acids esterified to phosphatidic acid, the de novo precursor of phospholipids and triacylglycerols. This may explain in part the simultaneous presence of these proteins in cell types involved in fatty acid absorption and lipoprotein secretion.     

Arachidonoyl-Coenzyme A Synthetase and Nonspecific AcylCoenzyme A Synthetase Activities in Purified Rat Brain Microvessels

Purified rat brain microvessels were prepared to demonstrate the occurrence of acyl-CoA (EC 6.2.1.3) synthesis activity in the microvasculature of rat brain. Both arachidonoyl-CoA and palmitoyl-CoA synthesis activities showed an absolute requirement for ATP and CoA. This activity was strongly enhanced by magnesium chloride and inhibited by EDTA. The apparent Km values for acyl-CoA synthesis by purified rat brain microvessels were 4.0 microM and 5.8 microM for palmitic acid and arachidonic acid, respectively. The apparent Vmax values were 1.0 and 1.5 nmol X min-1 X mg protein-1 for palmitic acid and arachidonic acid, respectively. Cross-competition experiments showed inhibition of radiolabelled arachidonoyl-CoA formation by 15 microM unlabelled arachidonic acid, with a Ki of 7.1 microM, as well as by unlabelled docosahexaenoic acid, with a Ki of 8.0 microM. Unlabelled palmitic acid and arachidic acid had no inhibitory effect on arachidonoyl-CoA synthesis. In comparison, radiolabelled palmitoyl-CoA formation was inhibited competitively by 15 microM unlabelled palmitic acid, with a Ki of 5.0 microM and to a much lesser extent by arachidonic acid (Ki, 23 microM). The Vmax of palmitoyl-CoA formation obtained on incubation in the presence of the latter fatty acids was not changed. Unlabelled arachidic acid and docosahexaenoic acid had no inhibitory effect on palmitoyl-CoA synthesis. Both arachidonoyl-CoA and palmitoyl-CoA synthesis activities were thermolabile. Arachidonoyl-CoA formation was inhibited by 75% after 7 min at 40 degrees C whereas a 3-min heating treatment was sufficient to produce the same relative inhibition of palmitoyl-CoA synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification and properties of acyl-CoA:1-acyl-sn-glycero-3-phosphocholine-O-acyltransferase from bovine brain microsomes

Acyl-CoA:1-acyl-sn-glycero-3-phosphocholine-O-acyltransferase has been purified approximately 3000-fold from bovine brain microsomes by detergent solubilization followed by ion-exchange and affinity chromatography. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate revealed a single protein of molecular weight 43,000. The specificity of the purified enzyme was studied by measuring the catalytic activity with various lysophospholipids and acyl-CoA derivatives. Of the lysophospholipids tested, only lysophosphatidylcholine was a substrate. Less specificity was exhibited toward the acyl-CoA derivatives, although the enzyme showed a clear preference for arachidonoyl-CoA and little or no activity with palmitoyl-CoA or stearoyl-CoA. High concentrations of arachidonoyl-CoA inhibited the enzyme. The velocity was a sigmoidal function of the concentration of lysophosphatidylcholine (LPC) with little activity obtained below 20 microM LPC. The specificity and kinetic properties of the enzyme were altered, however, by incorporation of the enzyme into liposomes composed of a mixture of phospholipids. Decanoyl-CoA and myristoyl-CoA, which were effective substrates for the soluble enzyme, did not serve as acyl donors for the liposome-bound acyltransferase. Furthermore, the liposome-bound enzyme, in contrast to the soluble form of the enzyme, was active at concentrations of LPC below the critical micelle concentration. The liposome-bound enzyme was also substantially less susceptible to thermal denaturation and proteolytic digestion. This modulation of the acyltransferase activity by interaction with phospholipids may relate to the kinetic properties and the regulation of the enzyme in vivo.     

Peroxisomal beta-oxidation of long-chain fatty acids possessing different extents of unsaturation.

Rates of peroxisomal beta-oxidation were measured as fatty acyl-CoA-dependent NAD+ reduction, by using solubilized peroxisomal fractions isolated from livers of rats treated with clofibrate. Medium- to long-chain saturated fatty acyl-CoA esters as well as long-chain polyunsaturated fatty acyl-CoA esters were used. Peroxisomal beta-oxidation shows optimal specificity towards long-chain polyunsaturated acyl-CoA esters. Eicosa-8,11,14-trienoyl-CoA, eicosa-11,14,17-trienoyl-CoA and docosa-7,10,13,16-tetraenoyl-CoA all gave Vmax. values of about 150% of that obtained with palmitoyl-CoA. The Km values obtained with these fatty acyl-CoA esters were 17 +/- 6, 13 +/- 4 and 22 +/- 3 microM respectively, which are in the same range as the value for palmitoyl-CoA (13.8 +/- 1 microM). Myristoyl-CoA gave the higher Vmax. (110% of the palmitoyl-CoA value) of the saturated fatty acyl-CoAs tested. Substrate inhibition was mostly observed with acyl-CoA esters giving Vmax. values higher than 50% of that given by palmitoyl-CoA.     

Enzymatic basis for the formation of pulmonary surfactant lipids by acyltransferase systems

An enzymatic basis for the formation of pulmonary surfactant lipids in rat has been presented. The free fatty acid pools in lung and liver consisted mainly of palmitic, stearic, oleic, and arachidonic acids with relatively less polyunsaturated fatty acids in lung than in liver. The acyl chain specificities of the acyl-CoA synthetase systems in lung and liver microsomes were similar in that most of fatty acids found in the free fatty acid pools were effectively activated by both systems. The acyl-CoA pools had compositions significantly different from those of the free fatty acid pools in lung and liver with relatively more stearate and less polyunsaturated fatty acids. The lung acyl-CoA pool contained mainly palmitate (29%), stearate (31%), and oleate (22%) with very little polyunsaturated acyl-CoAs to compete for esterification. The use of an equimolar mixture of palmitoyl-CoA and arachidonoyl-CoA to acylate the endogenous monoacyl-glycerophosphocholine isomers in the lung microsomes yielded both the 2-palmitate and 2-arachidonate diacyl forms, whereas the major products formed by liver microsomes were the 2-arachidonate and 1-palmitate forms. These results indicate that the 1-acyl isomer is the major monoacyl-glycerophosphocholine species serving as substrate in lung microsomes, whereas both 1-acyl and 2-acyl isomers are present in liver microsomes. Thus, the enrichment of saturated and oligoenoic acids in the acyl-CoA pool combined with the predominance of the 1-acyl isomer in the acyl acceptor pool and the relatively higher selectivity for palmitoyl-CoA by the 1-acyl-GPC acyltransferase activity of lung constitute an important basis for attributing some of the formation of pulmonary surfactant lipids in rats to acyltransferase action.     

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.     

Studies on lipid metabolism in Tetrahymena pyriformis: properties of acyltransferase systems.

Membrane preparations from Tetrahymena pyriformis catalyzed the acylations of glycerophosphate, isomeric monoacylglycerophosphate, and 1-acylglycerylphosphoryl-choline. Under the optimal conditions, glycerophosphate acyltransferase and 1-acylgly-cerophosphate acyltransferase used saturated and unsaturated acyl-CoA at comparable rates. The specificities of these acyltransferase systems for various acyl-CoAs as compared with the respective maximal velocities do not directly explain the fatty acid distribution in glycerophospholipids. However, the acylation of 2-acylglycerophosphate was highly selective for palmitate when the incubations were carried out in the presence of palmitoyl-CoA, oleoyl-CoA, 1-acylglycerophosphate, and 2-acylglycerophosphate. The 1-acylglycerylphosphorylcholine acyltransferase system showed relatively higher specificity for unsaturated acyl-CoA, which is consistent with the fatty acid pattern of phospholipids. Significant amounts of diglyceride and triglyceride were formed together with phosphatidic acid from acyl-CoA and glycerophosphate, indicating that the enzymes involved in triglyceride synthesis are closely associated with acyltransferase systems involved in phosphatidate synthesis in microsomes. These acyltransferase activities were found mainly in microsomes, and to a lesser extent, in pellicles, too. No significant difference was observed in the properties of acyltransferase systems in microsomes and pellicles.     

Aging and acyl-CoA binding protein alter mitochondrial glycerol-3-phosphate acyltransferase activity

It is well known that cellular function declines with age. Since phosphatidic acid (PtdOH) biosynthesis is central to the generation of membrane phospholipids, the hypothesis that aging decreases PtdOH biosynthesis was tested. Glycerol-3-phosphate acyltransferase (GPAT) and lysophosphatidic acid acyltransferase (LAT) activities were examined in isolated mitochondria and microsomes from young and old rat liver. The results show that mitochondrial GPAT preference for palmitoyl-CoA over oleoyl-CoA was only observed if albumin or acyl-CoA binding protein (ACBP) were present in the assay in the young rats. Furthermore, mitochondrial GPAT activity was significantly reduced in the presence of albumin and ACBP in aged mitochondria using palmitoyl-CoA as the substrate. These data show, for the first time, that mitochondrial GPAT acyl-CoA preference is due to the presence of a protein that binds acyl-CoAs, not the enzyme itself, and that aging significantly reduces mitochondrial GPAT activity.     

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

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

Age-related variations in acyl-CoA:retinol acyltransferase activity and vitamin A concentration in the liver and epidermis of hairless mice

Since the factors regulating retinol esterification by acyl-CoA:retinol acyltransferase are poorly understood, we studied the age-related variations in acyl-CoA:retinol acyltransferase activity in hairless mice. Epidermis and liver were collected at intervals from birth to adolescence (0-6 weeks). Vitamin A was analyzed by high-performance liquid chromatography and acyl-CoA:retinol acyltransferase by an in vitro radioincubation assay of microsomes. Epidermal vitamin A (retinol plus retinyl esters) increased 8-10 times after birth and by the age of 3 weeks adult values were attained. This increase was accompanied by a 2-fold increase in acyl-CoA:retinol acyltransferase activity in the epidermis between 3 days and 6 weeks of age. In young animals the dependence of acyl-CoA:retinol acyltransferase on exogenous co-substrate (palmitoyl-CoA) was also lower than in adult animals. Although a pronounced age-related accumulation of retinol was recorded in the liver, the activity of acyl-CoA:retinol acyltransferase did not increase with age and there was no change in the dependence of acyl-CoA:retinol acyltransferase on exogenous palmitoyl-CoA.     

Highly selective hydrolysis of fatty acyl-CoAs by calcium-independent phospholipase A2beta. Enzyme autoacylation and acyl-CoA-mediated reversal of calmodulin inhibition of phospholipase A2 activity

Calcium-independent phospholipase A2beta (iPLA2beta) participates in numerous diverse cellular processes, such as arachidonic acid release, insulin secretion, calcium signaling, and apoptosis. Herein, we demonstrate the highly selective iPLA2beta-catalyzed hydrolysis of saturated long-chain fatty acyl-CoAs (palmitoyl-CoA approximately myristoyl-CoA > stearoyl-CoA > oleoyl-CoA approximately = arachidonoyl-CoA) present either as monomers in solution or guests in host membrane bilayers. Site-directed mutagenesis of the iPLA2beta catalytic serine (S465A) completely abolished acyl-CoA thioesterase activity, demonstrating that Ser-465 catalyzes both phospholipid and acyl-CoA hydrolysis. Remarkably, incubation of iPLA2beta with oleoyl-CoA, but not other long-chain acyl-CoAs, resulted in robust stoichiometric covalent acylation of the enzyme. Moreover, S465A mutagenesis or pretreatment of wild-type iPLA2beta with (E)-6-(bromomethylene)-3-(1-naphthalenyl)-2H-tetrahydropyran-2-one unexpectedly increased acylation of the enzyme, indicating the presence of a second reactive nucleophilic residue that participates in the formation of the fatty acyl-iPLA2beta adduct. Radiolabeling of intact Sf9 cells expressing iPLA2beta with [3H]oleic acid demonstrated oleoylation of the membrane-associated enzyme. Partial trypsinolysis of oleoylated iPLA2beta and matrix-assisted laser desorption ionization mass spectrometry analysis localized the acylation site to a hydrophobic 25-kDa fragment (residues approximately 400-600) spanning the active site to the calmodulin binding domain. Intriguingly, calmodulin-Ca2+ blocked acylation of iPLA2beta by oleoyl-CoA. Remarkably, the addition of low micromolar concentrations (5 microM) of oleoyl-CoA resulted in reversal of calmodulin-mediated inhibition of iPLA2 beta phospholipase A2 activity. These results collectively identify the molecular species-specific acyl-CoA thioesterase activity of iPLA2beta, demonstrate the presence of a second active site that mediates iPLA2beta autoacylation, and identify long-chain acyl-CoAs as potential candidates mediating calcium influx factor activity.     

Purification, molecular cloning, and genomic organization of human brain long-chain acyl-CoA hydrolase

An acyl-CoA hydrolase, referred to as hBACH, was purified from human brain cytosol. The enzyme had a molecular mass of 100 kDa and 43-kDa subunits, and was highly active with long-chain acyl-CoAs, e.g. a maximal velocity of 295 micromol/min/mg and K(m) of 6.4 microM for palmitoyl-CoA. Acyl-CoAs with carbon chain lengths of C(8-18) were also good substrates. In human brain cytosol, 85% of palmitoyl-CoA hydrolase activity was titrated by an anti-BACH antibody, which accounted for over 75% of the enzyme activity found in the brain tissue. The cDNA isolated for hBACH, when expressed in Escherichia coli, directed the expression of palmitoyl-CoA hydrolase activity and a 44-kDa protein immunoreactive to the anti-BACH antibody, which in turn neutralized the hydrolase activity. The hBACH cDNA encoded a 338-amino acid sequence which was 95% identical to that of a rat homolog. The hBACH gene spanned about 130 kb and comprised 9 exons, and was mapped to 1p36.2 on the cytogenetic ideogram. These findings indicate that the long-chain acyl-CoA hydrolase present in the brain is well conserved between man and the rat, suggesting a conserved role for this enzyme in the mammalian brain, and enabling genetic studies on the functional analysis of acyl-CoA hydrolase.     

Long-chain acyl-CoA oxidases of Arabidopsis

Full-length cDNAs coding for two distinct acyl-CoA oxidases were isolated by screening an Arabidopsis cDNA library. The genes for the two acyl-CoA oxidases have been termed AtACX1 and AtACX2. AtACX1 encodes a peptide of 664 amino acids possessing a molecular mass of 74.3 kDa. AtACX2 encodes a peptide of 691 amino acids in length with a molecular mass of 77.5 kDa. Peroxisomal targeting signals were identified in the primary sequences. AtACX1 has a putative PTS1, whereas AtACX2 has a characteristic PTS2. Expression of AtACX1 and AtACX2 in Escherichia coli gave active enzymes for enzymatic and biochemical analysis. AtACX1 was active with both medium-and long-chain saturated fatty acyl-CoAs and showed maximal activity with C14-CoA. Activity with mono-unsaturated acyl-CoAs was slightly higher than with the corresponding saturated acyl-CoA. AtACX2 was active with long-chain acyl-CoAs and showed maximal activity with C18-CoA. AtACX2 activity with mono-unsaturated acyl-CoAs was approximately twice as high as with the corresponding saturated acyl-CoA. Both enzymes have an apparent Km of approximately 5 microM with the preferred substrate. Northern analysis was conducted to determine the expression patterns of AtACX1 and AtACX2 during germination and in various tissues of a mature plant. The two genes showed generally similar expression profiles and steady-state mRNA levels in seedlings and mature tissues, but subtle differences were observed. Enzymatic analyses of plant extracts revealed that AtACX1 and AtACX2 are members of a family that includes acyl-CoA oxidases specific for shorter-chain acyl-CoAs. Through expression of antisense constructs of the individual genes, we were able to decrease long-chain oxidase activity only in antisense AtACX1 plants. Seedlings with long-chain oxidase activity reduced down to 30% of wild-type levels germinated and established normally; however, reduced root growth appeared to be a general feature of antisense AtACX1 plants.     

Carnitine-acyltransferase activity of mitochondria from mung-bean hypocotyls

Carnitine-acyltransferase activity assayed with acetyl-CoA, octanoyl-CoA, or palmitoyl-CoA is associated with the mitochondrial but not with the peroxisomes of mung-bean hypocotyls. Using mitochondria as an enzyme source, a half-maximal reaction rate is obtained with a palmitoyl-CoA concentration approximately twice that required with acetyl-CoA. In the presence of a saturating acetyl-CoA concentration the carnitine-acyltransferase activity is not enhanced by palmitoyl-CoA as additional substrate. However, palmitoylcarnitine is formed in addition to acetylcarnitine, and the formation of acetylcarnitine is competitively inhibited by palmitoyl-CoA. It is concluded that the mitochondria of mung-bean hypocotyls possess a carnitine acyltransferase of broad substrate specificity with respect to the chainlength of the acyl-CoA and that the demonstration of a carnitine-palmitoyltransferase activity in plant mitochondria does not indicate the presence of a specific carnitine long-chain acyltransferase.     

Selective increase in acylation of 1-acylglycerophosphorylcholine in livers of rats and mice by peroxisome proliferators

Rats were fed a diet containing p-chlorophenoxyisobutyric acid (clofibric acid). Activity of microsomal 1-acylglycerophosphorylcholine (1-acyl-GPC) acyltransferase in liver was increased approx. 3-fold by the treatment with clofibric acid. The treatment of rats with clofibric acid did not increase activity of microsomal 2-acyl-GPC acyltransferase. Feeding a diet containing 2,2'-(decamethylenedithio)diethanol (tiadenol), di(2-ethylhexyl)phthalate or acetylsalicylic acid also resulted in a selective increase in the activity of 1-acyl-GPC acyltransferase in rat liver. Treatment with clofibric acid increased the activity of 1-acyl-GPC acyltransferase in liver of mouse as well as rat, but did not change the activity in liver of guinea-pig. The relative rate of acylation of 1-acyl-GPC with various acyl-CoAs by hepatic microsomes was not changed by the treatment of rats with clofibric acid.