Effects of sphingosine, albumin and unsaturated fatty acids on the activation and translocation of phosphatidate phosphohydrolases in rat hepatocytes.

The activities of two phosphatidate phosphohydrolases were measured in cultured rat hepatocytes incubated with 0.1 mM albumin. The activity, which is inhibited by N-ethylmaleimide (PAP-1) is located in the cytosolic and membrane fractions. PAP-1 activity is stimulated by Mg2+ and it can be translocated from the cytosol to the membranes by relatively low (0.5-1 mM) concentrations of fatty acids. In addition, higher concentrations (1-3 mM) of fatty acids cause an increase in the total PAP-1 activity. Translocation of PAP-1 activity in the hepatocytes is preferentially promoted by unsaturated fatty acids (C18:1, C18:2, C18:3, C20:4 and C20:5), rather than by saturated acids (C14:0, C16:0, C18:0). Increasing the extracellular concentration of albumin from 30 microM to 1 mM displaces PAP-1 activity from the membrane fraction. Sphingosine, but not staurosporine, can inhibit the redistribution of PAP-1 activity induced by oleate. The amphiphilic amines, sphingosine, chlorpromazine and propranolol, also decrease membrane-bound PAP-1 activity in the absence of fatty acids, but they do not alter, significantly, the activity of the cytosolic PAP-1. In the presence of 1 mM oleate, sphingosine, chlorpromazine and propranolol decrease the translocation of PAP-1 from the cytosol to the membranes. The phosphohydrolase activity, which is insensitive to N-ethylmaleimide (PAP-2), is specifically located in the plasma membrane (Jamal, Z., Martin, A., Gomez-Mu?oz, A. and Brindley, D.N. (1991) J. Biol. Chem. 266, 2988-2996) and it is not stimulated by Mg2+. Saturated fatty acids, albumin, sphingosine and propranolol have no significant effects on PAP-2 activity. However, chlorpromazine decreases PAP-2 activity by about 14%. Linolenate, arachidonate and eicosapentaenoate at 1 mM also produced small (7-10%) decreases in PAP-2 activity. It is proposed that both PAP-1 and PAP-2 activities may be involved in signal transduction, although the main function of PAP-1 seems to be involved in the synthesis of glycerolipids.     

EFFECT OF FATTY-ACID DOUBLE-BOND POSITION ON THE SELECTIVITY OF RAT-BRAIN ENZYMES: THE INCORPORATION OF OLEIC AND CIS-VACCENIC ACIDS INTO LYSOLECITHIN IN VITRO

Abstract— —Selectivity in the esterification of fatty acids to lysolecithin by rat-brain enzymes in vitro was investigated using free fatty acids (activation plus esterification) and CoA esters (esterification) of two naturally-occurring monoenoic fatty-acid isomers, oleic acid [18:1 (n - 9)] and cis-vaccenic acid [18:1 (n - 7)]. Esterification of free acids to l-acyl-sn-glycero-3-phosphorylcholine (1-acyl GPC) was dependent on CoA and ATP, and was stimulated by MgCl₂ and NaF. Under comparable conditions, fatty-acid activation (acyl-CoA synthetase [acid: CoA ligase (AMP)] EC 6.2.1.3.) appeared to be rate-limiting to 1-acyl GPC acyltransferase (acyl-CoA:l-acylglycero-3-phosphocholine O-acyltrans-ferase, EC 2.3.1.23.), since rates were always less with free fatty acids than with the CoA esters. A comparison of substrate curves obtained with free fatty acids and CoA esters suggests a preference for oleic acid during activation. Acyltransferase activity with 2-acyl GPC was similar with both acyl-CoA isomers, whereas with 1-acyl GPC, activity with oleoyl-CoA consistently exceeded that with cis-vaccenoyl-CoA. This difference between patterns of selectivity in esterification of positions 1 and 2 of lecithin suggests that separate enzymes catalyze the two reactions. The transfer of the isomers to the 2 position was affected in a similar manner by changes in pH and temperature, as well as in protein, fatty acid (or acyl-CoA), and 1-acyl GPC concentrations. Patterns of incorporation with simultaneous incubation of both isomers suggests one enzyme. Differences in acyltransferase activity with the two isomerie acyl-CoA's were observed in subcellular distribution, activity changes with brain maturation, and loss of activity on preincubation of microsomes at 45C. From these results it is not certain whether oleic and cis-vaccenic acids are esterified to the 2 position by separate enzymes, or by one enzyme with different affinities for the isomers. However, the investigation clearly indicates that acyltransferases, and possibly acyl-CoA synthetases in brain possess selectivity related to subtle differences in double-bond position. These selectivities probably are important in determining the specific fatty-acid composition of the complex lipids of brain.     

Acyl-CoA:cholesterol acyltransferase in human small intestine: its activity and some properties of the enzymic reaction

Esterification of endogenous cholesterol in human small intestinal mucosa by acyl-CoA:cholesterol acyltransferase (ACAT, EC 2.3.1.26) was studied using [1-14C]oleoyl-CoA as substrate. The reaction was linear for 2 min only. The esterification of cholesterol was stimulated by albumin, but this effect was dependent on the oleoyl-CoA concentration. When the albumin concentration was 5 g/liter, maximal esterification was obtained with 35 microM oleoyl-CoA. The pH optimum was 7.2-7.8. The ACAT specific activity was highest in microsomal preparations from jejunum (0.21 +/- 0.19 (n = 18) nmol cholesteryl oleate . mg microsomal protein-1 . min-1), and lower in proximal duodenum and distal ileum. Whole homogenates of biopsies had about 1/4 of the activity of the corresponding microsomal preparation. Microsomal preparations from jejunum contained acyl-CoA hydrolase (EC 3.1.2.2) which under the prevailing conditions had a maximal activity of 4.4 nmol oleate formed . microsomal protein-1 . min-1. The high activity of intestinal ACAT in man renders it possible that this enzyme plays a role in cholesterol absorption.     

Acyl-CoA: retinol acyltransferase (ARAT) and lecithin:retinol acyltransferase (LRAT) activation during the lipocyte phenotype induction in hepatic stellate cells

We have examined retinol esterification in the established GRX cell line, representative of hepatic stellate cells, and in primary cultures of ex vivo purified murine hepatic stellate cells. The metabolism of [3H]retinol was compared in cells expressing the myofibroblast or the lipocyte phenotype, under the physiological retinol concentrations. Retinyl esters were the major metabolites, whose production was dependent upon both acyl-CoA:retinol acyltransferase (ARAT) and lecithin:retinol acyltransferase (LRAT). Lipocytes had a significantly higher esterification capacity than myofibroblasts. In order to distinguish the intrinsic enzyme activity from modulation of retinol uptake and CRBP-retinol content of the cytosol in the studied cells, we monitored enzyme kinetics in the purified microsomal fraction. We found that both LRAT and ARAT activities were induced during the conversion of myofibroblasts to lipocytes. LRAT induction was dependent upon retinoic acid, while that of ARAT was dependent upon the overall induction of the fat storing phenotype. The fatty acid composition of retinyl-esters suggested a preferential inclusion of exogenous fatty acids into retinyl esters. We conclude that both LRAT and ARAT participate in retinol esterification in hepatic stellate cells: LRAT's activity correlates with the vitamin A status, while ARAT depends upon the availability of fatty acyl-CoA and the overall lipid metabolism in hepatic stellate cells.     

Monounsaturated fatty acyl-coenzyme A is predictive of atherosclerosis in human apoB-100 transgenic, LDLr-/- mice

ACAT2, the enzyme responsible for the formation of cholesteryl esters incorporated into apolipoprotein B-containing lipoproteins by the small intestine and liver, forms predominantly cholesteryl oleate from acyl-CoA and free cholesterol. The accumulation of cholesteryl oleate in plasma lipoproteins has been found to be predictive of atherosclerosis. Accordingly, a method was developed in which fatty acyl-CoA subspecies could be extracted from mouse liver and quantified. Analyses were performed on liver tissue from mice fed one of four diets enriched with one particular type of dietary fatty acid: saturated, monounsaturated, n-3 polyunsaturated, or n-6 polyunsaturated. We found that the hepatic fatty acyl-CoA pools reflected the fatty acid composition of the diet fed. The highest percentage of fatty acyl-CoAs across all diet groups was in monoacyl-CoAs, and values were 36% and 46% for the n-3 and n-6 polyunsaturated diet groups and 55% and 62% in the saturated and monounsaturated diet groups, respectively. The percentage of hepatic acyl-CoA as oleoyl-CoA was also highly correlated to liver cholesteryl ester, plasma cholesterol, LDL molecular weight, and atherosclerosis extent. These data suggest that replacing monounsaturated with polyunsaturated fat can benefit coronary heart disease by reducing the availability of oleoyl-CoA in the substrate pool of hepatic ACAT2, thereby reducing cholesteryl oleate secretion and accumulation in plasma lipoproteins.     

Fatty acid ethyl ester synthesis by human liver microsomes

Fatty acid ethyl esters are a family of non-oxidative metabolites of ethanol present in many tissues after ethanol consumption. In this report we demonstrate the existence in human liver of an acyl-CoA: ethanol acyltransferase activity which may be responsible in part for the synthesis of these compounds in vivo. The effects of oleoyl-CoA and ethanol concentrations, presence or absence of bovine serum albumin and detergent, pH and enzyme concentration on this activity have been determined. Acyl-CoA: ethanol acyltransferase activity is localised in the membrane-bound fraction. Using inhibitors directed against related enzyme activities, it has been shown that the activity is not related to serine-dependent carboxylesterases or acyl-CoA: cholesterol acyltransferase, but that it may be associated with acyl-CoA hydrolase activity. We have also compared acyl-CoA: ethanol acyltransferase activity with fatty acid ethyl ester synthase activity in microsomes and cytosol from the same liver. Our data indicate that these activities are comparable in vitro (on a units/g liver basis), and suggest that both may be significant in vivo.     

The biosynthesis of hepatic cholesterol esters and triglycerides is impaired in mice with a disruption of the gene for stearoyl-CoA desaturase 1

Stearoyl-CoA desaturase (SCD) is a microsomal enzyme required for the biosynthesis of oleate and palmitoleate, which are the major monounsaturated fatty acids of membrane phospholipids, triglycerides, and cholesterol esters. Two well characterized isoforms of SCD, SCD1 and SCD2, exist in the mouse. Most mouse tissues express SCD1 and 2 with the exception of the liver, which expresses mainly the SCD1 isoform. We found that asebia mice homozygous for a natural mutation of the gene for SCD1 (SCD-/-) are deficient in hepatic cholesterol esters and triglycerides despite the presence of normal activities of acyl-CoA:cholesterol acyltransferase and glycerol phosphate acyltransferase, the enzymes responsible for cholesterol ester and triglyceride synthesis, respectively, in the liver of these mice. Feeding diets supplemented with triolein or tripalmitolein to the SCD-/- mice resulted in an increase in the levels of 16:1 and 18:1 in the liver but failed to restore the 18:1 and 16:1 levels of the cholesterol ester and triglycerides to the levels found in normal mice. The SCD-/- mouse had very low levels of triglycerides in the VLDL and LDL lipoprotein fractions compared with the normal animal. Transient transfection of an SCD1 expression vector into Chinese hamster ovary cells resulted in increased SCD activity and esterification of cholesterol to cholesterol esters. Taken together, our observations demonstrate that the oleoyl-CoA and palmitoleyl-CoA produced by SCD1 are necessary to synthesize enough cholesterol esters and triglycerides in the liver and suggest that regulation of SCD1 activity plays an important role in mechanisms of cellular cholesterol homeostasis.     

Localization of acyl-coenzyme A: cholesterol acyltransferase in villus and crypt cells of chick intestine

Endogenous cholesterol esterification by acyl-CoA:cholesterol acyltransferase (EC 2.3.1.26) was studied in isolated enterocytes obtained from chick duodenal, jejunal, and ileal villi and crypts, using [14C]oleoyl-CoA as substrate. The maximal specific activity in each cell fraction was found in chick jejunum, followed by duodenum and ileum. Jejunal upper and mid villi showed higher specific activities than lower villi and crypts. Epithelial cells isolated from chick intestine also incorporated oleoyl-CoA into different lipids using the endogenous substrates. Upper and mid villus cells showed the maximal incorporation of oleoyl-CoA into triglycerides in duodenum and jejunum. Levels of oleoyl-CoA incorporation into phospholipids were higher than those found in the synthesis of triglycerides or cholesterol esters, whatever may be the cell fraction considered. Upper villus cells also showed the highest specific activity in the incorporation of oleoyl-CoA into phospholipids. The acyl-CoA hydrolase specific activity was practically similar in all the cell fractions obtained from chick duodenum, jejunum, and ileum.     

beta-Oxidation of the coenzyme A esters of elaidic, oleic, and stearic acids and their full-cycle intermediates by rat heart mitochondria.

beta-Oxidation rates for the CoA esters of elaidic, oleic and stearic acids and their full-cycle beta-oxidation intermediates and for the carnitine esters of oleic and elaidic acids were compared over a wide range of substrate and albumin concentrations in rat heart mitochondria. The esters of elaidic acid were oxidized at about half the rate of the oleic acid esters, while stearoyl-CoA was oxidized equally as rapid as oleoyl-CoA. The full-cycle beta-oxidation intermediates of elaidoyl-CoA (trans-16 : 1 delta 7, -14 : 1 delta 5, and -12 : 1 delta 3) were found to be oxidized at rates nearly equal to those for the corresponding intermediates of oleoyl-CoA. Therefore, after the first cycle of beta-oxidation, oleoyl-CoA and elaidoyl-CoA are oxidized at nearly equal rates. The activity of fatty acyl-CoA dehydrogenase was higher with elaidoyl-CoA and its full-cycle intermediates as substrates than with the corresponding cisisomers. It was concluded that the slower oxidation rate of elaidic acid is not due to slower oxidation of any of its full-cycle beta-oxidation intermediates, nor to slower activity of fatty acyl-CoA dehydrogenase, nor to outer mitochondrial carnitine acyltransferase. Possible explanations to account for the slower oxidation rate of elaidic acid are discussed.     

Study of the coinduction by fatty acids of catalase A and acyl-CoA oxidase in standard and mutant Saccharomyces cerevisiae strains

Evidence is presented that Saccharomyces cerevisiae can metabolize fatty acids via the inducible peroxisomal beta-oxidation pathway even when these acids are not the sole carbon source. The fatty acids of chain length of C10-C18 induce acyl-CoA oxidase simultaneously with catalase A but have no effect on catalase T and acyl-CoA dehydrogenase. The coinduction of both acyl-CoA oxidase and catalase A is recorded in strains with both active catalase A and T or displaying only catalase A activity. In mutants lacking catalase A, the induction of acyl-CoA oxidase is observed without a concomitant increase in catalase activity. After centrifugation in a linear Ficoll gradient of the particulate fraction from the cells grown on ethanol and oleate the activity of acyl-CoA oxidase cosediments with catalase A. The relationship of catalase A to acyl-CoA oxidase is discussed.     

Involvement of cytosol proteins in oleate activation of rabbit liver fructose-1,6-diphosphatase.

Dialyzed rabbit liver cytosol was specifically freed of endogenous fructose-1,6-diphosphatase by immunoadsorption on a column of Sepharose-immobilized anti-fructose-1,6-diphosphatase. This material increased the specific activity of homogeneous enzyme to the maximal rate observed with EDTA and shifted the pH optimum from 8.4 to 7.4. With oleate or other fatty acids as activators, the hydrolysis of fructose-1,6-diphosphatase by enzyme, at neutral pH, showed nonlinear initial rates dropping to lower linear rates. Cytosol activator acted synergistically with oleate both to increase neutral enzyme activity and to maintain the high initial catalytic rates. After sucrose density centrifugation or gel filtration, the cytosol had no effect by itself, but still potentiated oleate activation. The factor was destroyed by treatment with subtilisin or trypsin, but all attempts to identify a unique protein component in cytosol were unsuccessful. The presence of Na dodecyl-SOJ, deoxycholate, or urea did not improve the resolution of the factor, but these compounds did lower the K50 for activation by cytosol. Since fatty acids are the only unique compounds which have been isolated from cytosol which activated fructose-1,6-diphosphatase, it appears that soluble proteins can act as natural carriers for the fatty acids. This was supported by the fact that both dialyzed rabbit alpha-globulins and muscle phosphofructokinase also acted synergistically with oleate in a manner similar to cytosol. Phosphatidic acid and phosphatidylserine activated fructose-1,6-diphosphatase, and their action was synergistic with oleate. Glutathione (1 mM) activated the enzyme 5-fold at pH 7.3 and its effects were additive with oleate and cytosol or alpha-globulins.     

Incorporation of lipoxygenase products into cholesteryl esters by acyl-CoA:cholesterol acyltransferase in cholesterol-rich macrophages.

Macrophages which were incubated with acetylated low-density lipoproteins, resulting in cholesteryl ester accumulation, incorporated the monohydroxyeicosatetraenoic acids (5-, 15-, and 12-HETEs) into cholesteryl esters. The esterification of these hydroxy fatty acids to cholesterol by total membrane preparations of cholesterol-rich macrophages was dependent on the synthesis of the fatty acyl-CoA derivative, and was catalysed by acyl-CoA:cholesterol acyltransferase (ACAT). Stimulation of membrane ACAT activity by 25-hydroxycholesterol increased the synthesis of cholesteryl 12-HETE by 40%. In contrast, inhibiting ACAT activity by progesterone and compound 58-035 decreased cholesteryl 12-HETE production by 60% and 90% respectively. Although 5-, 15- and 12-HETE were esterified to cholesterol by ACAT, these monohydroxy fatty acids were less optimal as substrates compared with oleic acid or arachidonic acid. The hydrolysis and release of 12-HETE and the other monohydroxyeicosatetraenoic acids from intracellular cholesteryl esters and phospholipids occurred at a faster rate than for the more conventional fatty acids, oleate and arachidonate. Cholesteryl esters which contain hydroxy fatty acids therefore provide only a transient storage for lipoxygenase products, as these fatty acids are released into the medium as readily as hydroxy fatty acids found in phospholipids and triacylglycerols. The data provide evidence, for the first time, of an ACAT-dependent esterification of the lipoxygenase products 5-, 15- and 12-HETEs to cholesterol in the macrophage-derived foam cell. The channelling of these monohydroxy fatty acids to cholesteryl esters provides a mechanism which can alter the amount of lipoxygenase products incorporated into cellular phospholipids, thus averting deleterious changes to cell membranes. ACAT, by catalysing the esterification of monohydroxyeicosatetraenoic acids to cholesterol, could play a key role in regulating the amount of lipoxygenase products in the pericellular space of the cholesterol-enriched macrophage.     

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.     

Fatty acid biosynthesis in Erlich cells. The mechanism of short term control by exogenous free fatty acids.

We have examined the mechanism by which extracellular free fatty acids regulate fatty acid biosynthesis in Ehrlich ascites tumor cells. De novo biosynthesis in intact cells was inhibited by stearate greater than oleate greater than palmitate greater than linoleate. The amount of citrate and long chain acyl-CoA in the cells was not changed appreciably by the addition of free fatty acids to the incubation medium, indicating than free fatty acids do not regulate fatty acid biosynthesis by changing the total intracellular content of these metabolites. By measuring the incorporation of labeled free fatty acids into acyl-CoA, however, it was determined that the fatty acid composition of the acyl-CoA poolwas changed dramatically to reflect the composition of the exogenous free fatty acids. The relative inhibitory effects of different free fatty acids appear to depend on the ability of their acyl-CoA derivatives to regulate acyl-CoA carboxylase activity. The acyl-CoA concentration needed to produce 50% inhibition of purified Ehrlich cell carboxylase was found to be 0.68 mum for stearoyl-CoA, 1.6 mum for oleoyl-CoA, 2.2 mum for palmitoyl-CoA, 23 mum for myristoyl-CoA, 30 mum for lauroyl-CoA, and 37 mum for linoleoyl-CoA. In contrast to their effects on de novo synthesis, all of the free fatty acids added except stearate stimulated chain elongation in intact cells. Microsomal chain elongation, the major system for elongation in Ehrlich cells, also was regulated by the composition of the cellular acyl-CoA pool. Lauroyl-CoA, myristoyl-CoA, and palmitoyl-CoA were good substrates for elongation by isolated microsomes; oleoyl-CoA, and linoleoyl-CoA were intermediate; and stearoyl-CoA was a very poor substrate. We conclude that free fatty acids regulate fatty acid biosynthesis by changing the composition of the cellular acyl-CoA pool. These changes control the rate of malonyl-CoA production and, because of the acyl-CoA substrate specificity of the microsomal elongation system, modulate the amount of malonyl-CoA used for chain elongation.     

Translocation of CTP: phosphocholine cytidylyltransferase from cytosol to membranes in HeLa cells: stimulation by fatty acid, fatty alcohol, mono- and diacylglycerol

Addition of oleate, oleyl alcohol, or palmitate to HeLa cell medium resulted in a rapid stimulation of PC synthesis and activation of CTP: phosphocholine cytidylyltransferase. Stimulation was optimal with 0.35 mM oleate, 0.3 mM oleyl alcohol and 5 mM palmitate, or 1 mM palmitate if EGTA were added to the medium. The cytidylyltransferase was activated by translocation of the inactive cytosolic form to membranes. In untreated cells approx. 30% of the total cytidylyltransferase was membrane bound, while in treated cells, 80-90% was membrane associated. Addition of bovine serum albumin (10 mg/ml) to cells previously treated with oleate (0.35 mM) rapidly removed cellular fatty acid, and the membrane-bound cytidylyltransferase activity returned to approx. 30%. Similar results were obtained by extraction of membranes with albumin in vitro. Although 95% of the free fatty acid was extracted, 30-40% of the membrane cytidylyltransferase remained bound. Translocation of cytidylyltransferase between isolated cytosol and microsomal fractions was promoted by addition of oleate, palmitate, oleyl alcohol, and monoolein. Addition of diacylglycerol, lysophosphatidylcholine, lysophosphatidylethanolamine, calcium palmitate, and detergents such as Triton X-100, cholate or Zwittergent did not stimulate translocation of the enzyme. Addition of oleoyl-CoA promoited translocation, however, 40% of it was hydrolyzed releasing free oleic acid. Cytosolic cytidylyltransferase bound to microsomes pre-treated with phospholipase C, which had 7-fold elevated diacylglycerol content. Fatty acid-promoted translocation was blocked by Triton X-100, but not by 1 M KCl. These results suggest that a variety of compounds with differing head group size and charge, and number of hydrocarbon chains can function as translocators, and that hydrophobic rather than ionic interactions mediate the binding of cytidylyltransferase to membranes.     

Some properties and subcellular distribution of acyl-coenzyme A: retinol acyltransferase activity in rat testes

The present study was conducted to examine esterification of retinol by testicular microsomes. The microsomes were isolated from rat testes and were incubated under varying assay conditions with [3H]retinol. [3H]Retinylpalmitate was identified by reversed-phase high-performance liquid chromatography as an esterified product. The rate of esterification was increased by the addition of a fatty acyl-CoA. Coenzyme A esters of oleic, palmitic and stearic acids were equally effective substrates for retinol esterification. A 17-fold increase was observed in the presence of palmitoyl-CoA when microsomes had been pretreated with hydroxylamine, a reagent that reacts with coenzyme A thioesters to form hydroxamic acids. The esterifying activity was stimulated by the addition of dithiothreitol (4 mM) and fatty acid-free bovine serum albumin (1 mg/ml). The optimal concentrations for retinol and palmitoyl-CoA were 40 microM and 30-40 microM, respectively. The enzyme activity was inhibited by p-hydroxymercuribenzoate, sodium taurocholate and 5,5'-dithiobis-(2-nitrobenzoic acid), but not by EDTA. The enzyme activity was highest in microsomes (36%). However, some activity was present in mitochondria (29%). These results clearly show the presence of a fatty acyl-CoA: retinol acyltransferase that catalyzes the esterification of retinol in rat testes.     

Evidence that oleoyl-CoA and ATP-dependent elongations coexist in rapeseed (Brassica napus L.).

The elongation of different substrates was studied using several subcellular fractions from Brassica napus rapeseed. In the presence of malonyl-CoA, NADH and NADPH, very-long-chain fatty acid (VLCFA) synthesis was observed from either oleoyl-CoA (acyl-CoA elongation) or endogenous primers (ATP-dependent elongation). No activity was detected using oleic acid as precursor. Acyl-CoA and ATP-dependent elongation activities were mainly associated with the 15 000 g/25 min membrane fraction. Reverse-phase TLC analysis showed that the proportions of fatty acids synthesized by these activities were different. Acyl-CoA elongation increased up to 60 microM oleoyl-CoA, and ATP-dependent elongation was maximum at 1 mM ATP. Both activities increased with malonyl-CoA concentration (up to 200 microM). Under all conditions tested, acyl-CoA elongation was higher than ATP-dependent elongation, and, in the presence of both ATP and oleoyl-CoA, the elongation activity was always lower. ATP strongly inhibited acyl-CoA elongation, whereas ATP-dependent elongation was slightly stimulated by low oleoyl-CoA concentrations (up to 15 microM) and decreased in the presence of higher concentrations. CoA (up to 150 microM) had no effect on the ATP-dependent elongation, whereas it inhibited the acyl-CoA elongation. These marked differences strongly support the presence in maturing rapeseed of two different elongating activities differently modulated by ATP and oleoyl-CoA.     

Characterization of an acyl-coenzyme a thioesterase associated with the envelope of spinach chloroplasts

The enzymic hydrolysis of acyl-coenzyme A occurs in intact and purified chloroplasts. The different components of spinach chloroplasts were separated after a slight osmotic shock and the purified envelope membranes were shown to be the site of very active acyl-CoA thioesterase activity (EC 3.1.2.2.). The enzyme, which had a pH optimum of 9.0, was not affected by sulfhydryl reagents or by serine esterase inhibitors. However, the acyl-CoA thioesterase was strongly inhibited by unsaturated fatty acids, especially oleic acid, at concentrations above 100 micromolar. In marked contrast, saturated fatty acids had only a slight effect on the thioesterase activity. Substrate specificities showed that the velocity of the reaction increased with the chain length of the substrate from decanoyl-CoA to myristoyl-CoA and then decreased with the chain length from myristoyl-CoA to stearoyl-CoA. Interestingly, oleoyl-CoA was only slowly hydrolyzed. These results suggest that the envelope acyl-CoA thioesterase coupled with an envelope acyl-CoA synthetase may be involved in a switching system which indirectly allows acyl transfer from acyl carrier protein derivatives to unsaturated acyl-CoA derivatives and ensures the predominance of unsaturated 18 carbon fatty acids in plants. Furthermore, the position of both acyl-CoA thioesterase and synthetase in the envelope membranes suggest that these two enzymes may be involved in the transport of oleic acid from the stroma phase to the cytosol compartment of the leaf cell.     

Acyl specificity in triglyceride synthesis by lactating rat mammary gland.

We have studied the specificity of the acyl-CoA:diglyceride acyltransferase reaction in lactating rat mammary gland to provide a rational explanation at the enzyme level for the nonrandom distribution of fatty acids in milk fat triglycerides. Acyl-CoA:diglyceride acyltransferase activity was measured using various diglyceride and radioactive acyl-CoA substrates; products were identified as triglycerides by thin-layer and gas-liquid chromatography. Most of the enzymatic activity was located in the microsomal fraction and showed a broad specificity for the acyl donors tested C10, C12, C14, C16, C18, and C18:1 CoA esters). The acyltransferase activity was highly specific for sn-1,2-diglyceride enantiomers; rac-1,3- and sn-2,3-diglycerides were relatively inactive. The acyl-CoA specificity was not affected by the type of 1,2-diglyceride acceptor offered, although dilaurin was the best acceptor and sn-1,2-dilaurin greater than sn-1,2-dimyristin greater than sn-1,2-dipalmitin greater than sn-1,2-distearin. We have previously shown that in the microsomal fraction from lactating rat mammary gland, the acyltransferase activities concerned with the conversion of sn-glycero-3-phosphate to diacylglycerophosphate show a very marked specificity for long chain acyl-CoA's. Therefore, we conclude that the predominant localization of long chain fatty acids in the 1 and 2 positions, and of shorter chain fatty acids in the 3 position of the glycerol backbone, results at least in part from the specificities of the mammary gland acyltransferases.