ACBP and cholesterol differentially alter fatty acyl CoA utilization by microsomal ACAT

Microsomal acyl CoA:cholesterol acyltransferase (ACAT) is stimulated in vitro and/or in intact cells by proteins that bind and transfer both substrates, cholesterol, and fatty acyl CoA. To resolve the role of fatty acyl CoA binding independent of cholesterol binding/transfer, a protein that exclusively binds fatty acyl CoA (acyl CoA binding protein, ACBP) was compared. ACBP contains an endoplasmic reticulum retention motif and significantly colocalized with acyl-CoA cholesteryl acyltransferase 2 (ACAT2) and endoplasmic reticulum markers in L-cell fibroblasts and hepatoma cells, respectively. In the presence of exogenous cholesterol, ACAT was stimulated in the order: ACBP > sterol carrier protein-2 (SCP-2) > liver fatty acid binding protein (L-FABP). Stimulation was in the same order as the relative affinities of the proteins for fatty acyl CoA. In contrast, in the absence of exogenous cholesterol, these proteins inhibited microsomal ACAT, but in the same order: ACBP > SCP-2 > L-FABP. The extracellular protein BSA stimulated microsomal ACAT regardless of the presence or absence of exogenous cholesterol. Thus, ACBP was the most potent intracellular fatty acyl CoA binding protein in differentially modulating the activity of microsomal ACAT to form cholesteryl esters independent of cholesterol binding/transfer ability.     

Multisite promiscuity in the processing of endogenous substrates by human carboxylesterase 1

Human carboxylesterase 1 (hCE1) is a drug and endobiotic-processing serine hydrolase that exhibits relatively broad substrate specificity. It has been implicated in a variety of endogenous cholesterol metabolism pathways including the following apparently disparate reactions: cholesterol ester hydrolysis (CEH), fatty acyl Coenzyme A hydrolysis (FACoAH), acyl-Coenzyme A:cholesterol acyltransfer (ACAT), and fatty acyl ethyl ester synthesis (FAEES). The structural basis for the ability of hCE1 to perform these catalytic actions involving large substrates and products has remained unclear. Here we present four crystal structures of the hCE1 glycoprotein in complexes with the following endogenous substrates or substrate analogues: Coenzyme A, the fatty acid palmitate, and the bile acids cholate and taurocholate. While the active site of hCE1 was known to be promiscuous and capable of interacting with a variety of chemically distinct ligands, these structures reveal that the enzyme contains two additional ligand-binding sites and that each site also exhibits relatively non-specific ligand-binding properties. Using this multisite promiscuity, hCE1 appears structurally capable of assembling several catalytic events depending, apparently, on the physiological state of the cellular environment. These results expand our understanding of enzyme promiscuity and indicate that, in the case of hCE1, multiple non-specific sites are employed to perform distinct catalytic actions.     

Triglyceride and cholesteryl ester hydrolysis in a cell culture model of smooth muscle foam cells

Cultured rabbit aortic smooth muscle cells were converted to foam cells by exposure to sonicated lipid droplets of defined composition using an inverted culture technique. Uptake of the lipid droplets by the cells was shown to be dependent on the time of exposure to the droplets and on the mass of droplets presented to the cells. A comparison of the hydrolysis of triolein and cholesteryl oleate by cells that had been exposed to isotropic lipid droplets containing equimolar amounts of the two lipids revealed that the rate of hydrolysis of triglyceride was 3 to 4 times faster than that for cholesteryl ester. The hydrolysis of cholesteryl oleate from cells loaded with the isotropic droplets was approximately 1.5 times as fast as that from cells loaded with anisotropic droplets containing only cholesteryl oleate. A comparison of the hydrolysis of cholesteryl ester in the presence and absence of Sandoz compound 58-035, an inhibitor of acyl CoA:cholesterol acyl transferase, by cells loaded with isotropic droplets showed that about 30% of the free cholesterol liberated by hydrolysis was reesterified regardless of the mass of free cholesterol. We conclude that cultured smooth muscle cells have a greater capacity to hydrolyze triglyceride than cholesteryl ester, and that the rate of hydrolysis of cholesteryl ester appears to be related to the physical state of the droplet in which the cholesteryl ester is stored. In addition, it appears that the smooth muscle cells have a cholesteryl ester cycle that is inefficient in the reesterification of excess free cholesterol.     

Effect of dietary fat saturation on acylcoenzyme A:-cholesterol acyltransferase activity of Ehrlich cell microsomes.

Ehrlich cells grown in mice fed coconut oil diets (highly saturated) contain about twice as much cholesteryl ester as those grown in mice fed sunflower oil diets (highly polyunsaturated). Acylcoenzyme A: cholesterol acyltransferase (ACAT) activity was 30-100% higher in microsomes prepared from the cells grown on coconut oil (M(c)) than in those prepared from the cells grown on sunflower oil (M(s)). Increased ACAT activity was noted in M(c) with either [1-(14)C]palmitoyl CoA or [1,2-(3)H]cholesterol as the labeled substrate. This occurred at all acyl CoA concentrations tested and, in the [1,2-(3)H]cholesterol assay, with palmitoyl, oleoyl, or linoleoyl CoA as the substrate. The pH optimum for ACAT activity was the same with M(c) and M(s), pH 7.0. ACAT activity obeyed Michaelis-Menten kinetics at palmitoyl CoA concentrations between 1 and 10 micro M. Substrate inhibition occurred at higher concentrations. Kinetic analysis with [1-(14)C]palmitoyl CoA as the substrate indicated that the apparent K(m) for M(c) was 33% smaller than for M(s). There was no difference, however, in apparent V(max) values. The cholesterol and phospholipid contents of M(c) and M(s) were similar, but their fatty acid compositions differed considerably. M(c) contained 2.7 times more monoenoic fatty acid and only half as much polyenoic fatty acid as M(s). Our results indicate that dietary modification of the microsomal fatty acid composition is associated with alterations in the activity of ACAT, an enzyme that is tightly bound to the microsomes. These changes in ACAT activity may be partly responsible for the differences in cholesteryl ester contents of Ehrlich cells grown in mice fed the coconut and sunflower oil diets.     

Modulation of endosomal cholesteryl ester metabolism by membrane cholesterol

Cells acquire cholesterol in part by endocytosis of cholesteryl ester containing lipoproteins. In endosomes and lysosomes cholesteryl ester is hydrolyzed by acidic cholesteryl ester hydrolase producing cholesterol and fatty acids. Under certain pathological conditions, however, such as in atherosclerosis, excessive levels of cholesteryl ester accumulate in lysosomes for reasons that are poorly understood. Here, we have studied endosomal and lysosomal cholesteryl ester metabolism in cultured mouse macrophages and with cell-free extracts. We show that net hydrolysis of cholesteryl ester is coupled to the transfer of cholesterol to membranes. When membrane cholesterol levels are low, absorption of cholesterol effectively drives cholesteryl ester hydrolysis. When cholesterol levels in acceptor membranes approach saturation or when cholesterol export is blocked, cholesterol is re-esterified in endosomes. These results reveal a new facet of cellular cholesterol homeostasis and provide a potential explanation for cholesteryl ester accumulation in lysosomes of atherosclerotic cells.     

cAMP stimulates cholesteryl ester clearance to high density lipoproteins in J7774 macrophages.

The regulation by cAMP of cholesteryl ester hydrolysis and net depletion of cellular cholesteryl ester (cholesteryl ester clearance) in J774 murine macrophages was explored. Using Sandoz 58035 to selectively inhibit acyl CoA:cholesterol acyltransferase, we showed that the absolute rate of cholesteryl ester hydrolysis was stimulated 2-fold in J774 cells by the cAMP analogues 8-(4-chlorophenylthio)adenosine 3':5'-cyclic monophosphate and dibutyryl-cAMP. The rate of hydrolysis was also stimulated by prostaglandin E1, by cholera toxin, and by a mixture of forskolin and isobutylmethylxanthine, but was not affected by epinephrine or dibutyryl-cGMP. These data demonstrate that cholesteryl ester hydrolysis in J774 cells can be stimulated by cAMP-dependent protein kinase. Cholesteryl ester clearance from J774 cells was achieved upon incubation with high density lipoproteins (HDL) plus CPT-cAMP but not with HDL alone. HDL-mediated cholesteryl ester clearance was dependent on the concentration of both HDL and CPT-cAMP. The data suggest that the defect responsible for the lack of HDL-mediated cholesteryl ester clearance in J774 cells involves a failure to modulate cAMP levels.     

Characterization of a second sterol‐esterifying enzyme in Toxoplasma highlights the importance of cholesterol storage pathways for the parasite

Lipid bodies are eukaryotic structures for temporary storage of neutral lipids such as acylglycerols and steryl esters. Fatty acyl‐CoA and cholesterol are two substrates for cholesteryl ester (CE) synthesis via the ACAT reaction. The intracellular parasite Toxoplasma gondii is incapable of sterol synthesis and unremittingly scavenges cholesterol from mammalian host cells. We previously demonstrated that the parasite expresses a cholesteryl ester‐synthesizing enzyme, TgACAT1. In this article, we identified and characterized a second ACAT‐like enzyme, TgACAT2, which shares 56% identity with TgACAT1. Both enzymes are endoplasmic reticulum‐associated and contribute to CE formation for storage in lipid bodies. While TgACAT1 preferentially utilizes palmitoyl‐CoA, TgACAT2 has broader fatty acid specificity and produces more CE. Genetic ablation of each individual ACAT results in parasite growth impairment whereas dual ablation of ACAT1 and ACAT2 is not tolerated by Toxoplasma. ΔACAT1 and ΔACAT2 parasites have reduced CE levels, fewer lipid bodies, and accumulate free cholesterol, which causes injurious membrane effects. Mutant parasites are particularly vulnerable to ACAT inhibitors. This study underlines the important physiological role of ACAT enzymes to store cholesterol in a sterol‐auxotrophic organism such as Toxoplasma, and furthermore opens up possibilities of exploiting TgACAT as targets for the development of antitoxoplasmosis drugs.     

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.     

The triacylglycerol synthesis enzyme DGAT1 also catalyzes the synthesis of diacylglycerols, waxes, and retinyl esters

The final step of triacylglycerol biosynthesis is catalyzed by acyl CoA:diacylglycerol acyltransferase (DGAT) enzymes. The two known DGATs, DGAT1 and DGAT2, are encoded by unrelated genes. Although both DGAT1 and DGAT2 knockout mice have reduced tissue triacylglycerol contents, they have disparate phenotypes, prompting us to investigate whether the two enzymes have unrecognized functional differences. We now report that DGAT1 exhibits additional acyltransferase activities in vitro, including those of acyl CoA:monoacylglycerol acyltransferase (MGAT), wax monoester and wax diester synthases, and acyl CoA:retinol acyltransferase (ARAT), which catalyze the synthesis of diacylglycerols, wax esters, and retinyl esters, respectively. These activities were demonstrated in in vitro assays with membranes from insect cells or homogenates from COS7 cells overexpressing DGAT1. Wax synthase and ARAT activities were also demonstrated in intact COS7 cells expressing DGAT1. Additionally, cells and tissues from DGAT1-deficient mice exhibited reduced ARAT activity, and the mice had increased levels of unesterified retinol in their livers on a high-retinol diet. Our findings indicate that DGAT1 can utilize a variety of acyl acceptors as substrates in vitro and suggest that these activities may be relevant to the in vivo functions of DGAT1.     

Effects of LDL enriched with different dietary fatty acids on cholesteryl ester accumulation and turnover in THP-1 macrophages

LDL enriched with either saturated, monounsaturated, n-6 polyunsaturated, or n-3 polyunsaturated fatty acids were used to study the effects of dietary fatty acids on macrophage cholesteryl ester (CE) accumulation, physical state, hydrolysis, and cholesterol efflux. Incubation of THP-1 macrophages with acetylated LDL (AcLDL) from each of the four diet groups resulted in both CE and triglyceride (TG) accumulation, in addition to alterations of cellular CE, TG, and phospholipid fatty acyl compositions reflective of the individual LDLs. Incubation with monounsaturated LDL resulted in significantly higher total and CE accumulation when compared with the other groups. After TG depletion, intracellular anisotropic lipid droplets were visible in all four groups, with 71% of the cells incubated with monounsaturated AcLDL containing anisotropic lipid droplets, compared with 30% of cells incubated with n-3 AcLDL. These physical state differences translated into higher rates of both CE hydrolysis and cholesterol efflux in the n-3 group. These data suggest that monounsaturated fatty acids may enhance atherosclerosis by increasing both cholesterol delivery to macrophage foam cells and the percentage of anisotropic lipid droplets, while n-3 PUFAs decrease atherosclerosis by creating more fluid cellular CE droplets that accelerate the rate of CE hydrolysis and the efflux of cholesterol from the cell.     

Technical Advance: a novel technique for the sensitive quantification of acyl CoA esters from plant tissues

We report a novel, highly sensitive and selective method for the extraction and quantification of acyl CoA esters from plant tissues. The method detects acyl CoA esters with acyl chain lengths from C4 to C20 down to concentrations as low as 6 fmol in extracts. Acyl CoA esters from standard solutions or plant extracts were derived to their fluorescent acyl etheno CoA esters in the presence of chloroacetaldehyde, separated by ion-paired reversed-phase high-performance liquid chromatography, and detected fluorometrically. This derivitization procedure circumvents the selectivity problems associated with previously published enzymatic methods, and methods that rely on acyl chain or thiol group modification for acyl CoA ester detection. The formation of acyl etheno CoA esters was verified by mass spectrometry, which was also used to identify unknown peaks from chromatograms of plant extracts. Using this method, we report the composition and concentration of the acyl CoA pool during lipid synthesis in maturing Brassica napus seeds and during storage lipid breakdown in 2-day-old Arabidopsis thaliana seedlings. The concentrations measured were in the 3--6 microM range for both tissue types. We also demonstrate the utility of acyl CoA profiling in a transgenic B. napus line that has high levels of lauric acid. To our knowledge, this is the first time that reliable estimates of acyl CoA ester concentrations have been made for higher plants, and the ability to profile these metabolites provides a valuable new tool for the investigation of gene function.     

Polyunsaturated fatty acids up-regulate hepatic scavenger receptor B1 (SR-BI) expression and HDL cholesteryl ester uptake in the hamster.

Diets rich in polyunsaturated fatty acids lower plasma HDL cholesterol concentrations when compared to diets rich in saturated fatty acids. We investigated the mechanistic basis for this effect in the hamster and sought to determine whether reduced plasma HDL cholesterol concentrations resulting from a high polyunsaturated fat diet are associated with a decrease in reverse cholesterol transport. Animals were fed semisynthetic diets enriched with polyunsaturated or saturated fatty acids for 6 weeks. We then determined the effect of these diets on the following parameters: 1) hepatic scavenger receptor B1 (SR-BI) mRNA and protein levels, 2) the rate of hepatic HDL cholesteryl ester uptake, and 3) the rate of cholesterol acquisition by the extrahepatic tissues (from de novo synthesis, LDL and HDL) as a measure of the rate of reverse cholesterol transport. Compared to saturated fatty acids, dietary polyunsaturated fatty acids up-regulated hepatic SR-BI expression by approximately 50% and increased HDL cholesteryl ester transport to the liver; as a consequence, plasma HDL cholesteryl ester concentrations were reduced. Although dietary polyunsaturated fatty acids increased hepatic HDL cholesteryl ester uptake and lowered plasma HDL cholesterol concentrations, there was no change in the cholesterol content or in the rate of cholesterol acquisition (via de novo synthesis and lipoprotein uptake) by the extrahepatic tissues.These studies indicate that substitution of polyunsaturated for saturated fatty acids in the diet increases SR-BI expression and lowers plasma HDL cholesteryl ester concentrations but does not affect reverse cholesterol transport.     

Role of acyl-coenzyme A:cholesterol acyltransferase-1 in the control of hepatic very low density lipoprotein secretion and low density lipoprotein receptor expression in the mouse and hamster

Cholesteryl esters present in nascent very low density lipoproteins are generated in a reaction catalyzed by acyl CoA:cholesterol acyltransferase (ACAT). To examine the effect of cholesteryl esters on the secretion of apoB-containing lipoproteins, we transiently overexpressed human (h) ACAT-1 in the livers of low density lipoprotein (LDL) receptor(-/-) mice using adenovirus-mediated gene transfer. Overexpression of hACAT-1 increased hepatic total and esterified cholesterol but did not reduce hepatic free cholesterol due to a compensatory increase in the rate of de novo cholesterol synthesis. Overexpression of hACAT-1 markedly increased the plasma concentration and hepatic secretion of apoB-containing lipoproteins but had no effect on the clearance of very low density lipoprotein-apoB from plasma indicating that cholesteryl esters play an important role in regulating the assembly and secretion of apoB-containing lipoproteins. ACAT activity has been implicated in the regulation of the LDL receptor pathway by dietary fatty acids. It has been hypothesized that unsaturated fatty acids, by enhancing ACAT activity, reduce the amount of free cholesterol in a putative regulatory pool that feeds back on LDL receptor expression. We directly tested this hypothesis in hamsters by transiently overexpressing hACAT-1 in the liver. Enhanced cholesterol esterification in the liver resulted in a compensatory increase in de novo cholesterol synthesis but no induction of LDL receptor expression suggesting that fatty acids regulate LDL receptor expression via a mechanism independent of ACAT.     

Effects of high-density lipoprotein2 on cholesterol transport and acyl-coenzyme A:cholesterol acyltransferase activity in P388D1 macrophages

High-density lipoproteins are the putative vehicles for cholesterol removal from monocyte-derived macrophages, which are an important cell type in all stages of atherosclerosis. The role of HDL₂, an HDL subclass that accounts for most variation in plasma HDL-cholesterol concentration, in cholesterol metabolism in monocyte-derived macrophages is not known. In this study, the dose-dependent effects of HDL₂ on cellular cholesterol mass, efflux, and esterification, and on cellular cholesteryl ester (CE) hydrolysis using the mouse macrophage P388D1 cell line was investigated. HDL₂ at low concentrations (40 μg protein/ml) decreased CE content without affecting cellular free cholesterol content (FC), CE hydrolysis, or cholesterol biosynthesis. In addition, HDL₂ at low concentrations reduced cellular acyl-coenzyme A:cholesterol acyltransferase (ACAT) activity and increased FC efflux from macrophages. Thus, HDL₂ has two potential roles in reverse cholesterol transport. In one, HDL₂ is an acceptor of macrophage FC. In the other, more novel role, HDL₂ increases the availability of macrophage FC through the inhibition of ACAT. Elucidation of the mechanism by which HDL₂ inhibits ACAT could identify new therapeutic targets that enhance the transfer of cholesterol from macrophages to the liver.     

Effects of dietary n-3 fatty acids on the composition of cholesteryl esters and triglycerides in plasma and liver perfusate of the rat

The effects of polyunsaturated fatty acids of the omega-3 family (PUFA n-3), (addition of fish oil), on the molecular composition of cholesteryl esters and triglycerides in plasma and liver perfusate of rats were studied. Rats fed a diet rich in saturated fatty acids (addition of lard) served as controls. Supplemention with PUFA n-3 not only decreases the plasma concentrations of free cholesterol, cholesteryl esters, and triglycerides, it also significantly alters the plasma composition of cholesteryl esters and triglycerides. Analyses of liver perfusate indicate a decrease in triglycerides secretion by in vitro perfused liver and reciprocal changes in relative contents of cholesteryl esters fractions with C(16) and C(20) acyl chains. This finding may be a result of chain-shortening of long-chain fatty acids probably in peroxisomal beta-oxidative system. Alterations in plasma cholesteryl esters and triglycerides composition of the fish oil group could be affected further by additional factors such as increased plasma cholesterol esterification activity and presence of triglyceride species of intestinal origin.     

Promoting export of macrophage cholesterol: the physiological role of a major acute-phase protein,serum amyloid A 2.1

We show that murine macrophages that have ingested cell membranes as a source of cholesterol exhibit a marked increase in acyl-CoA:cholesterol acyl transferase (ACAT) activity. Exposure of these macrophages to acute-phase high-density lipoprotein (HDL) results in a marked reduction of ACAT and enhancement of cholesteryl ester hydrolase (CEH) activities, phenomena not seen with native HDL. These complementary but opposite effects of acute-phase HDL on the two enzyme systems that regulate the balance between esterified (storage) cholesterol and unesterified (transportable) cholesterol are shown to reside with serum amyloid A (SAA) 2.1, an acute-phase apolipoprotein of HDL whose plasma concentration increases 500- to 1,000-fold within 24 h of acute tissue injury. Mild trypsin treatment of acute-phase HDL almost completely abolishes the apolipoprotein-mediated effects on the cholesteryl ester cycle in cholesterol-laden macrophages. The physiological effect of SAA2.1 on macrophage cholesterol is to shift it into a transportable state enhancing its rate of export, which we confirm in tissue culture and in vivo. The export process is shown to be coupled to the ATP binding cassette transport system. Our findings integrate previous isolated observations about SAA into the sphere of cholesterol transport, establish a function for a major acute-phase protein, and offer a novel approach to mobilizing macrophage cholesterol at sites of atherogenesis.     

Carnitine acyltransferases and their influence on CoA pools in health and disease

Cells contain limited and sequestered pools of Coenzyme A (CoA) that are essential for activating carboxylate metabolites. Some acyl-CoA esters have high metabolic and signalling impact, so control of CoA ester concentrations is important. This and transfer of the activated acyl moieties between cell compartments without wasting energy on futile cycles of hydrolysis and resynthesis is achieved through the carnitine system. The location, properties of and deficiencies in the carnitine acyltransferases are described in relation to their influence on the CoA pools in the cell and, hence, on metabolism. The protection of free CoA pools in disease states is achieved by excretion of acyl-carnitine so that carnitine supplementation is required where unwanted acyl groups build up, such as in some inherited disorders of fatty acid oxidation. Acetyl-carnitine improves cognition in the brain and propionyl-carnitine improves cardiac performance in heart disease and diabetes. The therapeutic effects of carnitine and its esters are discussed in relation to the integrative influence of the carnitine system across CoA pools. Recent evidence for sequestered pools of activated acetate for synthesis of malonyl-CoA, for the synthesis of polyunsaturated fatty acids and for the inhibition of carnitine palmitoyltransferase 1 to regulate fatty acid oxidation is reviewed.     

12-[(5-iodo-4-azido-2-hydroxybenzoyl)amino]dodecanoic acid: biological recognition by cholesterol esterase and acyl-CoA:cholesterol O-acyltransferase

Potential probes of protein cholesterol and fatty acid binding sites, namely, 12-[(5-iodo-4-azido-2-hydroxybenzoyl)amino]dodecanoate (IFA) and its coenzyme A (IFA:CoA) and cholesteryl (IFA:CEA) esters, were synthesized. These radioactive, photoreactive lipid analogues were recognized as substrates and inhibitors of acyl-CoA:cholesterol O-acyltransferase (ACAT) and cholesterol esterase, neutral lipid binding enzymes which are key elements in the regulation of cellular cholesterol metabolism. In the dark, IFA reversibly inhibited cholesteryl [14C]oleate hydrolysis by purified bovine pancreatic cholesterol esterase with an apparent Ki of 150 microM. Cholesterol esterase inhibition by IFA became irreversible after photolysis with UV light and oleic acid (1 mM) provided 50% protection against inactivation. Incubation of homogeneous bovine pancreatic cholesterol esterase with IFA:CEA resulted in its hydrolysis to IFA and cholesterol, indicating recognition of IFA:CEA as a substrate by cholesterol esterase. The coenzyme A ester, IFA:CoA, was a reversible inhibitor of microsomal ACAT activity under dark conditions (apparent Ki = 20 microM), and photolysis resulted in irreversible inhibition of enzyme activity with 87% efficiency. IFA:CoA was also recognized as a substrate by both liver and aortic microsomal ACATs, with resultant synthesis of 125IFA:CEA. IFA and its derivatives, IFA:CEA and IFA:CoA, are thus inhibitors and substrates for cholesterol esterase and ACAT. Biological recognition of these photoaffinity lipid analogues will facilitate the identification and structural analysis of hitherto uncharacterized protein lipid binding sites.     

Effects of substrate composition on the esterification and hydrolysis activity of lysosomal acid sterol ester hydrolase

Lipid microemulsions with various core and surface lipid compositions were prepared by co-sonication of cholesteryl esters, triolein (TO), egg phosphatidylcholine (egg PC), and cholesterol. The heterogeneous emulsion particle mixture was purified by gel filtration and particles with the size and general organization of low density lipoproteins were obtained. These lipid microemulsion particles were used for studies of the cellular metabolism of lipoprotein-derived cholesterol and cholesteryl esters as catalyzed by the enzyme acid sterol ester hydrolase (EC 3.1.1.13). The hydrolysis of cholesteryl oleate (CO) was more than twice and that of cholesteryl linoleate (CL) more than three times faster than the hydrolysis of cholesteryl stearate (CS) over the temperature range 25-39.6 degrees C. Both the synthesis and hydrolysis of cholesteryl esters were insensitive to the physical state of the microemulsion cores. The synthesis of cholesteryl esters by this enzyme was also insensitive to the ratios of cholesterol and egg PC in the microemulsion surface layers. Incorporation of triolein into the microemulsion cholesteryl ester core slightly increased the rate of cholesteryl ester synthesis. A decreasing fatty acyl chain length (C18:0 to C14:0) and an increasing degree of unsaturation (C18:0 to C18:2) enhanced the synthesis rate. It is suggested that the hydrolysis and synthesis of cholesteryl esters in microemulsions (and lipoproteins) take place only in the particle surface layer and that the rate of catalysis is directly dependent on the amount of substrate in this surface layer.