The activity of 3-hydroxy-3-methylglutaryl-CoA reductase and acyl-CoA: cholesterol acyltransferase in hepatic microsomes from male, female and pregnant rats. The effect of cholestyramine treatment and the relationship of enzyme activity to microsomal lipid composition

The relationship of microsomal cholesterol and phospholipid fatty acid composition to the activities of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase and acyl-CoA: cholesterol acyltransferase was investigated in male, female virgin and pregnant rats when hepatic cholesterogenesis was stimulated by cholestyramine. Cholestyramine increased HMG-CoA reductase activity in both sexes but had no effect on microsomal free cholesterol level or acyl-CoA: cholesterol acyltransferase activity. The data suggest that during cholestyramine treatment high rates of bile acid synthesis are supported by preferential channelling of cholesterol into this pathway, whilst the substrate pool and activity of acyl-CoA:cholesterol acyltransferase are maintained unaltered. The lack of a consistent relationship among enzyme activities and microsomal lipid composition infers that HMG-CoA reductase and acyl-CoA:cholesterol acyltransferase are regulated in vivo by independent mechanisms which are unlikely to involve modulation by the physical properties of the microsomal lipid.     

Phospholipid fatty acid modification of rat liver microsomes affects acylcoenzyme A:cholesterol acyltransferase activity

The effect of phospholipid fatty acyl composition on the activity of acylcoenzyme A:cholesterol acyltransferase was investigated in rat liver microsomes. Specific phosphatidylcholine replacements were produced by incubating the microsomes with liposomes and bovine liver phospholipid-exchange protein. Although the fatty acid composition of the microsomes was modified appreciably, there was no change in the microsomal phospholipid or cholesterol content. As compared to microsomes enriched for 2 h with dioleoylphosphatidylcholine, those enriched with dipalmitoylphosphatidylcholine exhibited 30-45% less acyl-CoA:cholesterol acyltransferase activity. Enrichment with 1-palmitoyl-2-linoleoylphosphatidylcholine increased acyl-CoA:cholesterol acyltransferase activity by 20%. By contrast, dilinoleoylphosphatidylcholine abolished microsomal acyl-CoA:cholesterol acyltransferase activity almost completely. Addition of cofactors that stimulated microsomal lipid peroxidation inhibited acyl-CoA:cholesterol acyltransferase activity by only 10%, however, and did not increase the inhibition produced by submaximal amounts of dilinoleoylphosphatidylcholine. Certain of the phosphatidylcholine replacements produced changes in palmitoyl-CoA hydrolase, NADPH-dependent lipid peroxidase, glucose-6-phosphatase and UDPglucuronyl transferase activities, but they did not closely correlate with the alterations in acyl-CoA:cholesterol acyltransferase activity. Electron spin resonance measurements with the 5-nitroxystearate probe indicated that microsomal lipid ordering was reduced to a roughly similar extent by dioleoyl- or by dilinoleoylphosphatidylcholine enrichment. Since these enrichments produce widely different effects on acyl-CoA:cholesterol acyltransferase activity, changes in bulk membrane lipid fluidity cannot be the only factor responsible for phospholipid fatty acid compositional effect on acyl-CoA:cholesterol acyltransferase. The present results are more consistent with a modulation resulting from either changes in the lipid microenvironment of acyl-CoA:cholesterol acyltransferase or a direct interaction between specific phosphatidylcholine fatty acyl groups and acyl-CoA:cholesterol acyltransferase.     

Influence of diets on acyl-CoA:cholesterol acyltransferase and on acyl-CoA:retinol acyltransferase in villous and crypt cells from rat small intestinal mucosa and in the liver

Cholesterol and retinol are both esterified with long-chain fatty acid within the mucosal cells of the small intestine. The reactions are catalyzed by microsomal acyl-CoA:cholesterol and acyl-CoA:retinol acyltransferases (EC 2.3.1.26, and EC 2.3.1.-, respectively). To gain more insight into the physiological importance of these acyltransferases, they were studied in villous and crypt cells from rats either fasting or on diets which varied in fat and cholesterol content. Both enzymes had a higher activity in villous than in crypt cells. The activities in villous cells varied with feeding and fasting and the composition of diet when the animals were killed postprandially. Acyl-CoA:cholesterol acyltransferase activity went up upon cholesterol feeding whereas retinol acyltransferase in the mucosa was reduced by high-fat diets. The liver cholesterol acyltransferase activity varied with diet, it increased with both cholesterol and fat feeding, whereas retinol acyltransferase activity remained relatively constant. The results obtained suggest that different diets are of importance for cholesterol and retinol acyltransferase activities both in the intestinal mucosa and in the liver. The variation in activities of the two acyltransferases suggests that they may be different enzymes.     

Gender-related effects of chronic ethanol ingestion on rat hepatic acyl-CoA:cholesterol acyltransferase

The influence of chronic ethanol ingestion on hepatic acyl-CoA: cholesterol acyltransferase activity was investigated to determine the relationship between alcohol intake and cholesterol ester accumulation. Rats were given nutritionally complete liquid diets supplemented with 6.3% ethanol or an isocaloric equivalent of dextrin-maltose for 5 weeks. During this period, the hepatic acyl-CoA: cholesterol acyltransferase activity of ethanol-fed male rats remained constant, whereas the same activity in pair-fed controls as well as chow-fed rats exhibited a 30% decrease in activity. Unlike alcohol-fed male rats, the hepatic acyl-CoA: cholesterol acyltransferase activity of female rats decreased by approximately 30% by the fifth week of ethanol ingestion. Despite the fact that the gender of the animals led to disparate levels of acyl-CoA: cholesterol acyltransferase activity in response to ethanol ingestion, similar levels of cholesteryl ester accumulation were observed. The altered levels of acyl-CoA: cholesterol acyltransferase activity caused no significant change in the cholesterol concentration, cholesterol/phospholipid ratio, phospholipid fatty acid composition, or the membrane fluidity of the hepatic microsomes. We conclude that the altered hepatic acyl-CoA: cholesterol acyltransferase activity of ethanol-fed female rats cannot be directly responsible for ethanol-induced accumulation of cholesteryl esters.     

Identification and characterization of an acyl-CoA:triterpene acyltransferase activity in rabbit and human tissues.

Rabbit and human tissues contain substantial amounts of an unusual lipid, a fatty acid ester of a pentacyclic triterpene, that is a potent in vitro inhibitor of acyl-CoA:cholesterol acyltransferase (ACAT). A possible origin of the triterpene ester is via dietary absorption of plant triterpenes (which have a similar structure to the triterpene moiety of the animal triterpene ester), followed by fatty acid esterification of the triterpene in animal tissues. To support this idea, homogenates of rabbit and human enterocytes and liver are now shown to contain an acyl-CoA:triterpene acyltransferase activity (ATAT) which esterifies triterpene to a fatty acid. The enzyme activity was stimulated by exogenous triterpene and required ATP and coenzyme A when fatty acid was used as substrate; ATP and coenzyme A were not required when fatty acyl-CoA was used. ATAT was not inhibited by two structurally different ACAT inhibitors, which may indicate that ACAT and ATAT are different enzymes. Rat enterocytes and liver contained very little ATAT activity, consistent with the finding that rat liver contained very little triterpene ester. To establish that triterpene esterification occurs in vivo, [3H]triterpene was shown to be incorporated into triterpene ester in several organs and tissues from a rabbit given a gastric bolus of the labeled triterpene. These data provide support for the hypothesis that triterpene esters in animal tissues arise from the dietary absorption of triterpenes followed by the esterification of the triterpenes by an enzymatic activity in the animal tissues.     

Triacylglycerol synthesis in isolated fat cells. Evidence that the sn-glycerol-3-phosphate and dihydroxyacetone phosphate acyltransferase activities are dual catalytic functions of a single microsomal enzyme.

The acyl-CoA:sn-glycerol-3-phosphate acyltransferase (EC 2.3.1.15) (glycerol-P acyltransferase) and acyl-CoA:dihydroxyacetone phosphate acyltransferase (EC 2.3.1.42) (DHAP acyltransferase) activities were investigated in vitro in order to evaluate the quantitative contribution of the glycerol-P and DHAP pathways for the synthesis of triacylglycerols in isolated fat cells and to test the hypothesis that these two activities may be dual catalytic functions of a single enzyme. More than 85% of both acyltransferase activities was associated with the microsomal subcellular fraction. The microsomal glycerol-P acyltransferase activity showed an apparent Km of 8 muM for glycerol-P with a Vmax of 15.6 nmol/min/mg, while the DHAP acyltransferase activity showed an apparent Km of 40 muM for DHAP with a Vmax of 9.7 nmol/min/mg. Glycerol-P was a competitive inhibitor (Ki = 7.2 muM) of the DHAP acyltransferase, and DHAP was a competitive inhibitor (Ki = 92 muM) of the glycerol-P acyltransferase. The two acyltransferase activities showed virtual identity in their pH dependence, acyl-CoA chain length dependence, thermolability, and inactivation by N-ethylmaleimide. Trypsin, detergents, collagenase, phospholipases, and various salts and organic solvents also had similar effects on both activities. Taken as a whole, the data strongly suggest that the microsomal glycerol-P and DHAP acyltransferase activities actually represent dual functions of a single enzyme. Calculations based on the above kinetic constants and previously reported glycerol-P and DHAP pools in adipocytes suggest that the in vivo ratio of glycerol-P to DHAP acylation should be greater than 24:1.     

A novel cardiolipin-remodeling pathway revealed by a gene encoding an endoplasmic reticulum-associated acyl-CoA:lysocardiolipin acyltransferase (ALCAT1) in mouse

Cardiolipin is a major membrane polyglycerophospholipid that is required for the reconstituted activity of a number of key mitochondrial enzymes involved in energy metabolism. Cardiolipin is subjected to remodeling subsequent to its de novo biosynthesis to attain appropriate acyl composition for its biological functions. Yet, the enzyme(s) involved in the remodeling process have not been identified. We report here the identification and characterization of a murine gene that encodes an acyl-CoA:lysocardiolipin acyltransferase 1 (ALCAT1). Expression of the ALCAT1 cDNA in either insect or mammalian cells led to a significant increase in acyl-CoA:monolysocardiolipin acyltransferase and acyl-CoA: dilysocardiolipin acyltransferase activities that exhibited a dependence upon ALCAT1 enzyme levels. The recombinant ALCAT1 enzyme recognizes both monolysocardiolipin and dilysocardiolipin as substrates with a preference for linoleoyl-CoA and oleoyl-CoA as acyl donors. In contrast, no significant increases in acyltransferase activities by the recombinant ALCAT1 were detected against either glycerol-3-phosphate or a variety of other lysophospholipids as substrates, including lysophosphatidylcholine, lysophosphatidylethanolamine, and lysophosphatidylserine. Immunocytohistochemical analysis showed that the ALCAT1 enzyme is localized in the endoplasmic reticulum, which is supported by a significant ALCAT activity in isolated liver and heart microsomes. Northern blot analysis indicates that the mouse ALCAT1 is widely distributed, with the highest expression in heart and liver. In support of a role for ALCAT1 in maintaining heart function, the ALCAT1 gene is conserved among different species of vertebrates, but not in non-atrium organisms. ALCAT1 represents the first identified cardiolipin-remodeling enzyme from any living organism; its identification implies a novel role for the endoplasmic reticulum in cardiolipin metabolism.     

Effect of a high cholesterol diet on lipid metabolizing enzymes in spontaneously hypertensive rats

A high cholesterol diet induced a fatty liver and an increase in cholesterol oleate in spontaneously hypertensive rats. The activity of microsomal glycerophosphate acyltransferase in liver increased 2-3-fold to meet the increased supply of oleate, the synthesis of which was stimulated by a 10-fold increase in microsomal delta 9-desaturase activity. Hepatic fatty acid synthetase and diacylglycerol acyltransferase activities were decreased somewhat. These results, together with the fact that the large increases in hepatic cholesterol ester and triacylglycerol were not correspondingly reflected in plasma, indicated that the fatty liver resulted from decreased secretion of lipoprotein rather than increased lipogenesis. Endogenous cholesterol in liver microsomes increased 2-fold and hepatic acyl-CoA:cholesterol acyltransferase activity increased 3-fold, whereas plasma lecithin:cholesterol acyltransferase activity was unchanged. Thus, the increase in cholesterol oleate seen in spontaneously hypertensive rats fed a high cholesterol diet is due mainly to increases in acyl-CoA:cholesterol acyltransferase and delta 9-desaturase activities.     

Acylation of 2-acyl-glycerophosphocholine in guinea-pig heart microsomal fractions.

Acyl-CoA:2-acyl-sn-glycero-3-phosphocholine (GPC) acyltransferase is required for the maintenance of the asymmetric distribution of saturated fatty acids at the C-1 position of phosphatidylcholine; however, this activity has been reported to be absent in cardiac tissue. In the present study a very active acyl-CoA:2-acyl-GPC activity was detected and characterized in guinea-pig heart microsomes (microsomal fractions); the mitochondria did not appear to possess this activity. The acyl-CoA specificity of the microsomal acyl-CoA:2-acyl-GPC acyltransferase was distinct from the corresponding acyl-CoA:1-acyl-GPC acyltransferase. These differences were due to the position of the fatty acid on the lysophospholipid rather than the composition of the fatty acids. The enzyme did not exhibit a distinct preference for saturated fatty acids, as might be expected. Our results suggest that, in the heart, control of the intracellular composition and concentration of acyl-CoAs by acyl-CoA hydrolase and acyl-CoA synthetase may play an important role in maintaining the asymmetric distribution of fatty acids in phosphatidylcholine.     

1,2,3-Trithiane-5-carboxylic Acid in Raw Asparagus Shoots

New acyl coenzyme A: alcohol acyltransferase activity was found in the cell-free extract of Neurospora sp. ATCC 46892 which produces ethyl hexanoate abundantly in its culture broth. This enzyme catalyzed the esterification between ethanol and «-hexanoyl coenzyme A. It also acted on /i-butyryl coenzyme A, but not on acetyl coenzyme A. It was detected mostly in the cytoplasm. The activity was accelerated by high concentrations of sodium chloride, and unsaturated fatty acid did not inhibit it. This enzyme played a major role in biosynthesis of ethyl esters which were formed with ethanol and higher acyl coenzyme As. This is the first report of an alcohol acyltransferase which does not have alcohol acetyltransferase activity.     

Comparison of the activities of some peroxisomal and extraperoxisomal lipid-metabolizing enzymes in liver and extrahepatic tissues of the rat.

Peroxisomal (acyl-CoA oxidase and peroxisomal dihydroxyacetone-phosphate acyltransferase) and extraperoxisomal (mitochondrial fatty acid oxidation, extraperoxisomal dihydroxyacetone-phosphate acyltransferase, mitochondrial and microsomal glycerophosphate acyltransferases) lipid-metabolizing enzymes were measured in homogenates from rat liver and from seven extrahepatic tissues. Except for jejunal mucosa and kidney, extrahepatic tissues contained very little acyl-CoA oxidase activity. Peroxisomal dihydroxyacetone-phosphate acyltransferase, taken as the activity that was not inhibited by 5 mM-glycerol 3-phosphate, was present in all tissues examined, and its specific activity in liver and extrahepatic tissues was roughly of the same order of magnitude. Clofibrate treatment increased the activity of acyl-CoA oxidase in liver, and to a smaller extent also in kidney, but did not influence the activity of peroxisomal dihydroxyacetone-phosphate acyltransferase. Comparison of the activities of peroxisomal and extraperoxisomal lipid-metabolizing enzymes in extrahepatic tissues and in liver, an organ in which the contribution of peroxisomes to fatty acid oxidation and to glycerolipid synthesis has been estimated previously, suggests that, as in liver, peroxisomal long-chain fatty acid oxidation is of minor quantitative importance in extrahepatic tissues, but that in these tissues (micro)-peroxisomes are responsible for most of the dihydroxyacetone phosphate acylation and, consequently, for initiating ether glycerolipid synthesis.     

Mutagenesis and characterization of specific residues in fatty acid ethyl ester synthase: A gene for alcohol-induced cardiomyopathy

Fatty acid ethyl ester synthase-III metabolizes both ethanol and carcinogens. Structure-function studies of the enzyme have not been performed in relation to site specific mutagenesis. In this study, three residues (Gly 32, Cys 39 and His 72) have been mutated to observe their role in enzyme activity. Gly to Gln, Cys to Trp and His to Ser mutations did not affect fatty acid ethyl ester synthase activity, but His to Ser mutant had less than 9% of control glutathione S-transferase activity. The apparent loss of transferase activity reflected a 28 fold weaker binding constant for glutathione. Thus, this study indicates that Gly and Cys may not be important for synthase or transferase activities however, histidine may play a role in glutathione binding, but it is not an essential catalytic residue of glutathione S-transferase or for fatty acid ethyl ester synthase activity.     

Acyl-CoA:dihydroxyacetone phosphate acyltransferase in human skin fibroblasts: study of its properties using a new assay method

In relation to the finding that human skin fibroblasts are capable of de novo either phospholipid biosynthesis, we have studied the properties of acyl-CoA:dihydroxyacetone phosphate acyltransferase in fibroblast homogenates using a new assay method. The results indicate that the acylation of dihydroxyacetone phosphate shows an optimum at pH 5.5 with a broad shoulder of activity up to pH 6.4 and a decline in activity up to pH 8.2. At pH 5.5 the acyltransferase accepts dihydroxyacetone phosphate, but not glycerol 3-phosphate as a substrate. Furthermore, the transferase activity was found to be membrane-bound and inactivated by Triton X-100 at concentrations above 0.025% (w/v). Similar properties have been described for the enzyme as present in rat-liver and guinea-pig liver peroxisomes. These data, together with the finding that acyl-CoA:dihydroxyacetone phosphate acyltransferase is deficient in cultured skin fibroblasts from patients without peroxisomes (Zellweger syndrome), suggest that in cultured skin fibroblasts the enzyme is primarily located in peroxisomes.     

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.     

1-Acyl-sn-glycerol-3-phosphate acyltransferase in maturing safflower seeds and its contribution to the non-random fatty acid distribution of triacylglycerol

A 20,000 X g particulate preparation isolated from maturing safflower seeds catalyzed the acylation of 1-acyl-sn-glycerol 3-phosphate with acyl-CoA to form phosphatidate. The specific activity of the reaction exceeded 200 nmol min-1 mg protein-1. Although this preparation was also capable of catalyzing the acylation of sn-glycerol 3-phosphate with acyl-CoA, the hydrolysis of phosphatidate, and the acylation of 1,2-diacylglycerol, phosphatidate was the only major product when the preparation was incubated with 1-acyl-glycerol-3-P and acyl-CoA. The enzyme responsible for this phosphatidate synthesis, 1-acyl-glycerol-3-P acyltransferase, showed a strict acyl-CoA specificity. The relative order of specificity for acyl-CoA was linoleoyl = oleoyl greater than palmitoleoyl greater than elaidoyl greater than cis-vaccenoyl greater than stearoyl = palmitoyl. This observation strongly suggests that the fatty acid composition of position 2 in phosphatidate synthesized in vivo primarily depends on both the acyl-CoA specificity of the 1-acyl-glycerol-3-P acyltransferase and the fatty acid composition of the acyl-CoA pool in the cell. Thus, the absence of saturated fatty acids at position 2 of safflower triacylglycerol may be explained in terms of the acyl-CoA specificity of the 1-acyl-glycerol-3-P acyltransferase. The fatty acid moiety esterified at position 1 of glycerol-3-P also affected the effectiveness of the reaction. The 1-acyl-glycerol-3-P acyltransferase utilized 1-acyl-glycerol-3-P molecular species in the following order of effectiveness: linoleoyl = oleoyl greater than palmitoyl. With a rise in incubation temperature, the initial rates of acylation with unsaturated acyl-CoA species increased more rapidly than those for saturated acyl-CoA species. A similar tendency was observed for saturated and unsaturated acyl acceptors. These data suggest that affinity of the acyltransferase for substrates may vary in response to changes in temperature, and that 1-acyl-glycerol-3-P acyltransferase may be involved in the alteration of the individual fatty acid compositions at positions 1 and 2 of glycerolipids in tissues grown at different temperatures. Based on these findings, further metabolism of 1-acyl-glycerol-3-P acyltransferase products could be the major factor determining the non-random distribution of fatty acids in safflower triacylglycerol.     

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.     

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.     

The presence of 1-radyl-glycerophosphoethanolamine acyltransferase activity in guinea pig heart mitochondria

Ethanolamine glycerophospholipids of mammalian heart mitochondria have a high content of arachidonic acid. Since the presence of acyltransferases that acylate 1-radyl glycerophosphoethanolamine had not been reported in the organelle, it was not known whether this high arachidonate content could be attained by the deacylation-reacylation pathway. In this study we have detected the presence of acyl-CoA:1-acyl-glycerophosphoethanolamine acyltransferase and acyl-CoA:1-alkenyl-glycerophosphoethanolamine acyltransferase activities in the guinea pig heart mitochondria. Both acyltransferases were active with palmitoyl-, stearoyl-, oleoyl-, linoleoyl-, and arachidonoyl-CoAs, but the highest activities were obtained with arachidonoyl-CoA. The acyl-CoA specificities of the enzyme(s) did not reflect the fatty acid composition of the ethanolamine glycerophospholipids. The utilization of arachidonoyl-CoA by these acyltransferases in the guinea pig heart mitochondria suggests that these enzymes may play a significant role in contributing to the high arachidonate content of the ethanolamine glycerophospholipids. However, mechanisms beyond the acyl specificity of the reacylation reactions are also involved in the maintenance of the overall acyl composition of the ethanolamine glycerophospholipid in the cardiac mitochondria.     

Isolation and characterization of Chinese hamster ovary cell mutants deficient in acyl-coenzyme A:cholesterol acyltransferase activity

A protocol has been developed for isolating cholesterol ester-deficient cells from the Chinese hamster ovary cell clone 25-RA. This cell line previously was shown to be partially resistant to suppression of cholesterogenic enzyme activities by 25-hydroxycholesterol and to accumulate a large amount of intracellular cholesterol ester when grown in medium containing 10% fetal calf serum (Chang, T. Y., and Limanek, J. S. (1980) J. Biol. Chem. 255, 7787-7795). The higher cholesterol ester content of 25-RA is due to an increase in the rate of cholesterol biosynthesis and low density lipoprotein receptor activity compared to wild-type Chinese hamster ovary cells, and not due to an abnormal acyl-CoA:cholesterol acyltransferase enzyme. The procedure to isolate cholesterol ester-deficient mutants utilizes amphotericin B, a polyene antibiotic known to bind to cholesterol and to form pore complexes in membranes. After incubation in cholesterol-free medium plus an inhibitor of endogenous cholesterol biosynthesis, 25-RA cells were found to be 50-500 times more sensitive to amphotericin B killing than were mutant cells containing reduced amounts of cholesterol ester. Twelve amphotericin B-resistant mutants were isolated which retained the 25-hydroxycholesterol-resistant phenotype. These mutants did not exhibit the perinuclear lipid droplets characteristic of 25-RA cells, and lipid analysis revealed a large (up to 40-fold) reduction in cellular cholesterol ester. The acyl-CoA:cholesterol acyltransferase activities of these cholesterol ester-deficient mutants were markedly lower than 25-RA when assayed in intact cells or in an in vitro reconstitution assay. The tightest mutant characterized, AC29, was found to have less than 1% of the parental acyl-CoA:cholesterol acyltransferase activity. These mutants all have reduced rates of sterol synthesis and lower low density lipoprotein receptor activity compared to 25-RA, probably as a consequence of their reduced enzyme activities. Cell fusion experiments revealed that the phenotypes of all the mutants examined are not dominant and that the mutants all belong to the same complementation group. We conclude that these mutants contain a lesion in the gene encoding acyl-CoA:cholesterol acyltransferase or in a gene encoding a factor needed for enzyme production.     

Phosphatidylethanolamine:dolichol acyltransferase. Characterization and partial purification of a novel rat liver enzyme.

Incubation of rat or human post-heparin plasma with [3H]dolichol incorporated in liposomes consisting of dioleoyl phosphatidylcholine:dioleoyl phosphatidylethanolamine (3:1) resulted in the formation of radioactive dolichyl oleate. Non-heparinized plasma did not esterify dolichol, and, hence, the enzyme involved is probably associated with the cell surface and released into the blood by heparin. The major location of this activity was the liver, and, therefore, a partial purification of the enzyme from heparinized rat liver perfusates was performed using DEAE-Sephacel and heparin-Sepharose chromatography. The dolichol acyltransferase activity copurified with hepatic lipase activity in a lipid-protein complex of 350 kDa. Optimal acylation is achieved at pH 7.5 in the presence of 5% plasma and 20 mM Ca2+. Esterification can only be obtained when dolichol is present in a phospholipid bilayer, and the reaction is strongly stimulated by unsaturated phosphatidylethanolamine or phosphatidylserine. Radiolabeling experiments demonstrated that the primary acyl donor is phosphatidylethanolamine from which the fatty acid is transferred exclusively from position 1. Neither cholesterol nor retinol are esterified by the enzyme, and the reaction is not stimulated by acyl-CoA. Both the extracellular localization and the mechanism of transacylation clearly distinguish this new enzyme from the acyl-CoA:dolichol acyltransferase described earlier in microsomes.