Stimulation of acyl-CoA:cholesterol acyltransferase activity in rat liver microsomes by phosphatidylcholine

Acyl-CoA:cholesterol acyltransferase (ACCAT) activity of rat liver microsomes was stimulated by phosphatidylcholine. The stimulatory effect varied with the composition of the phosphatide: dimyristyl-, dipalmityl-, distearyl- and dioleylphosphatidylcholine were stimulatory, whereas dicaproyl- and dilinoleylphosphatidylcholine were not. The results suggest that increased fluidity of the membrane induced by phosphatide is probably not involved in the stimulation of cholesterol esterification. Phosphatide exerted its effect directly on the microsomes and did not extract cholesterol or ACCAT from the microsomes to an appreciable extent.Hydrolysis of microsomal phosphatide suppressed ACCAT activity. Enztme activity was restored with the addition of phosphatidylcholine. The results suggest that phosphatide may be required for cholesterol esterification.     

Purification and spectral study of a microbial fatty acyltransferase: activation by limited proteolysis

A fatty acyltransferase with a reaction mechanism similar to that of mammalian lecithin: cholesterol acyltransferase has been purified from culture supernatants of a mutant Aeromonas salmonicida containing the cloned Aeromonas hydrophila structural gene. Typically, more than 35 mg of protein were isolated from 2 L of culture supernatant. The amino-terminal sequence, amino acid composition, and molecular weight of the purified protein corresponded to predictions based on the sequence of the gene, indicating that the signal sequence had been correctly removed during export but that no further processing had occurred. Analysis of the far-UV circular dichroic (CD) spectrum of the enzyme showed that it consists of 31% alpha-helix, 21% beta-sheet, and 16% beta-turn, with 12% of aperiodic form. Treatment of the purified protein with a variety of proteases resulted in nicking near the C-terminus. This led to an increase in enzyme activity against lipids in erythrocyte membranes and increased rate of hydrolysis of p-nitrophenyl butyrate. Activation was accompanied by a change in the CD spectrum and a change in its aggregation state. The trypsin cut site was located between the two cysteines in the enzyme. Evidence is presented that the cysteines are joined by a disulfide bond and therefore cannot participate in acyl transfer. This may distinguish the microbial enzyme from lecithin:cholesterol acyltransferase. This is the second extracellular A. hydrophila protein that we have shown can be activated by proteolysis after it is released.     

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.     

Activity of acyl-CoA: cholesterol acyltransferase and 3-hydroxy-3-methylglutaryl-CoA reductase in subfractions of hepatic microsomes enriched with cholesterol

The influence of membrane cholesterol on the activities of acyl-CoA: cholesterol acyltransferase and 3-hydroxy-3-methylglutaryl-CoA reductase was examined in three microsomal subfractions (RNA-rich, RNA-poor, and smooth) that had been enriched with cholesterol by incubation with mixed lipoproteins from hypercholesterolemic rabbit serum. Acyl-CoA: cholesterol acyltransferase activity was significantly stimulated in the three subfractions, particularly in the RNA-rich microsomal component. 3-Hydroxy-3-methylglutaryl-CoA reductase, on the other hand, was suppressed (30%) in only one (RNA-poor) of the three microsomal subfractions, despite a 1.4-fold increase in the concentration of membrane cholesterol. An attempt was made to distinguish between an effect based exclusively on an increase in available cholesterol substrate and an activation of acyl-CoA: cholesterol acyltransferase in RNA-rich microsomes enriched with cholesterol. An experimental design was devised so that substrate cholesterol was provided in the form of heated smooth microsomes and acyl-CoA: cholesterol acyltransferase was provided as a separate preparation in the form of RNA-rich microsomes. Appropriate controls were carried out to test for transfer of cholesteryl ester between the two sets of particles. The results suggested that cholesterol enhanced acyl-CoA: cholesterol acyltransferase activity by serving both as a substrate and as a non-substrate modulator.     

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.     

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.     

Membrane filter procedure for enumeration of Aeromonas hydrophila in fresh waters.

A membrane filter method (mA) for the enumeration of Aeromonas hydrophila in natural water samples was developed. The complex, primary medium employs trehalose as a fermentable carbohydrate and ampicillin and ethanol as selective inhibitors. After 20 h of incubation at 37 degrees C, an in situ mannitol fermentation test followed by an in situ oxidase test is used to further differentiate A. hydrophila from other aquatic and terrestrial microorganisms present in freshwaters. The primary medium decreases background microbial growth by about two orders of magnitude. The recoveries on mA medium from suspensions of A. hydrophila prepared from pure cultures and held for 24 h at 15 degrees C exceeded 95% of the recoveries on brain-heart infusion agar spread plates. The confirmation rate for colonies designated A. hydrophila was 98%, whereas 11% of the presumptively negative colonies were, in fact, A. hydrophila. Recoveries of A. hydrophila from fresh, surface water samples exceeded recoveries by the other methods examined.     

Correlation between changes in membrane lipid composition induced by dietary lipid and membrane-bound enzyme activity in chick liver

The activity of acyl-CoA: cholesterol acyltransferase in the liver-microsomal fraction was considerably reduced in chicks fed on diet containing unsaturated fat, whereas the activity of HMG-CoA reductase and NADPH cytochrome c reductase was not affected. The fatty acid composition of the microsomes was modified appreciably by this dietary condition and there was no change in the phospholipid or cholesterol levels. The addition of cholesterol to the fat supplemented diet resulted in a considerable increase in the microsomal cholesterol content. A decrease in HMG-CoA reductase and an increase ACAT activity was observed compared with the corresponding values from both the groups fed on a standard diet and a fat supplemented diet with no cholesterol. These results suggest that acyl-CoA: cholesterol acyltransferase is modulated by alteration in the fatty acid composition of the microsomal membrane, while the cholesterol content of the microsomes shows a close relationship with the HMG-CoA reductase activity.     

Membrane filter procedure for enumeration of Aeromonas hydrophila in fresh waters.

A membrane filter method (mA) for the enumeration of Aeromonas hydrophila in natural water samples was developed. The complex, primary medium employs trehalose as a fermentable carbohydrate and ampicillin and ethanol as selective inhibitors. After 20 h of incubation at 37 degrees C, an in situ mannitol fermentation test followed by an in situ oxidase test is used to further differentiate A. hydrophila from other aquatic and terrestrial microorganisms present in freshwaters. The primary medium decreases background microbial growth by about two orders of magnitude. The recoveries on mA medium from suspensions of A. hydrophila prepared from pure cultures and held for 24 h at 15 degrees C exceeded 95% of the recoveries on brain-heart infusion agar spread plates. The confirmation rate for colonies designated A. hydrophila was 98%, whereas 11% of the presumptively negative colonies were, in fact, A. hydrophila. Recoveries of A. hydrophila from fresh, surface water samples exceeded recoveries by the other methods examined.     

Levels of plasma and aortic lipids in young exercised rats with a diminished weight gain

Young exercised rats with a diminished weight gain had a decrease in high density lipoprotein (HDL) cholesterol and phospholipid levels in the plasma and augmented free cholesterol and phosphatide in the aorta. When the weight gain in the trained rats paralleled the gain in non-exercised animals, the values of these lipids were not altered. The levels of aortic free cholesterol in the non-exercised and exercised groups were inversely associated with concentrations of HDL-cholesterol, but were not related to the activities of lecithin cholesterol acyltransferase. In addition, the total cholesterol and phospholipid contents in the aorta negatively correlated with HDL-cholesterol concentrations. We propose that in young exercised rats with a diminished weight gain, the removal of aortic lipids is hampered due to a reduction in HDL-cholesterol.     

Lecithin:cholesterol acyltransferase regulation. Effect of fluidity of dimyristoylphosphatidylcholine vesicles

The regulation of lecithin:cholesterol acyltransferase by changes in phospholipid bilayer fluidity was investigated using pyrene excimer fluorescence to measure fluidity. Fluidity of dimyristoylphosphatidylcholine (DMPC) unilamellar vesicles was decreased by the addition of up to 20% (mol/mol) cholesterol and increased by the addition of up to 10% (mol/mol) lysoDMPC. When both cholesterol and lysoDMPC are present in the bilayer, their individual effects on fluidity are altered. These changes can be explained by complex formation between cholesterol and phospholipid as in the model of Presti et al. (Presti, F.C., Pace, R.J. and Chan, S.I. (1982) Biochemistry 21, 3831-3335). Lecithin:cholesterol acyltransferase activity with these vesicles as substrates was measured to determine whether activity can be modulated by the fluidity changes of the bilayer on which the enzyme acts. When 10% lysoDMPC, a known lecithin:cholesterol acyltransferase inhibitor, is added to the vesicles, inhibition of activity is observed. When 7.5% lysoDMPC is added to vesicles which contain either 5 or 10% cholesterol, lecithin:cholesterol acyltransferase activity increases. This increase in lecithin:cholesterol acyltransferase activity due to vesicle-fluidity increase is sufficient to overcome the decrease in activity due to lecithin:cholesterol acyltransferase inhibition. This is the first report of the ability of lysoDMPC to increase lecithin:cholesterol acyltransferase activity.     

The effects of low-density lipoprotein and cholesterol on acyl-coenzyme A: cholesterol acyltransferase activity in membranes from cultured human fibroblasts.

Membranes prepared from cultured fibroblasts were assayed for acyl-coenzyme A: cholesterol acyltransferase (ACAT) by a method that relied exclusively on the cholesterol already present on the membranes as the sterol substrate. Changes in membrane ACAT activity during incubation of fibroblasts under a variety of conditions were similar to the changes in the rate of incorporation of oleic acid into cholesteryl esters by the intact cells. The addition of low-density lipoprotein (LDL) to fibroblasts pre-incubated with lipoprotein-deficient serum led to a transient increase in membrane ACAT activity, which reached its peak after 7h and was related to the receptor-mediated uptake and degradation of the lipoprotein by the cells. However, after incubation of the membranes with a cholesterol-rich donor lipoprotein, which resulted in an equilibration of cholesterol between membranes and donor, each preparation exhibited the same activity. In contrast with these effects of LDL, incubation of the cells with non-esterified cholesterol produced a prolonged increase in ACAT activity and an increase in the activity observed after equilibration. Furthermore, ACAT activity in cells grown with linoleic acid was higher, both before and after the addition of LDL, than that of cells grown in normal medium or with palmitate. The increase in activity produced by LDL was also greater, reflecting the greater rate of degradation of LDL by the cells, and was associated with an increase in the activity observed after equilibration with donor. The results suggest that although fibroblasts can increase the amount of active enzyme on their membranes to accommodate an exceptionally high or prolonged supply of cholesterol, under normal circumstances the increase in membrane ACAT activity produced by LDL can be explained entirely by an increase in the amount of cholesterol in the substrate pool.     

Spin label studies of erythrocytes with abnormal lipid composition: comparison of red cells in a hereditary hemolytic syndrome and lecithin: Cholesterol acyltransferase deficiency

Erythrocytes from patients with familial lecithin:cholesterol acyltransferase (LCAT) deficiency have been shown to exhibit an increase in membrane fluidity which is surprisingly small in view of the extensive alterations both in membrane lipicl composition (namely, an elevation in cholesterol and phosphatidylcholine contents as well as a decrease in phosphatidylethanolamine) and in the functional properties of these cells. In the hope of deriving some information concerning the interrelationship between the structural and functional abnormalities, we have used the spin probe 5-doxyl stearic acid to investigate the temperature-dependent fluidity properties of red cells from two patients with a hereditary hemolytic syndrome (HHS) whose red cells are also characterized by qualitatively similar alterations in phosphatidylcholine and phosphatidylethanolamine but, unlike those in LCAT deficiency, have relatively normal levels of membrane cholesterol. A small increase in membrane fluidity of HHS erythrocytes equivalent to that previously observed in LCAT deficiency was found, indicating that membrane cholesterol level does not exert an important modulatory influence on membrane fluidity in these cells. It is concluded that while the distinct patterns of structural and functional erythrocyte alterations in these two disorders cannot be explained on the basis of differences in bulk membrane fluidity, the marginally increased fluidity may underlie the abnormalities in osmotic fragility and membrane p-nitrophenylphosphatase activity which are shared in common by both types of modified red cells.     

Subcellular localization of the enzymes of cholesterol biosynthesis and metabolism in rat liver

We have used isopycnic density gradient centrifugation to study the distribution of several rat liver microsomal enzymes of cholesterol synthesis and metabolism. All of the enzymes assayed in the pathway from lanosterol to cholesterol (lanosterol 14-demethylase, steroid 14-reductase, steroid 8-isomerase, cytochrome P-450, and cytochrome b5) are distributed in both smooth (SER) and rough endoplasmic reticulum (RER). The major regulatory enzyme in the pathway, hydroxymethylglutaryl-CoA reductase, also was found in both smooth and rough fractions, but we did not observe any associated with either plasma membrane or golgi. Since cholesterol can only be synthesized in the presence of these requisite enzymes, we conclude that the intracellular site of cholesterol biosynthesis is the endoplasmic reticulum. This is consistent with the long-held hypothesis. When the overall pathway was assayed by the conversion of mevalonic acid to non-saponifiable lipids (including cholesterol), the pattern of distribution obtained in density gradients verified its general endoplasmic reticulum localization. The enzyme acyl-CoA-cholesterol acyltransferase which removes free cholesterol from the membrane by esterification, was found only in the rough fraction of endoplasmic reticulum. In addition, when the RER was degranulated by the addition of EDTA, the activity of acyl-CoA-cholesterol acyltransferase not only shifted to the density of SER but was stimulated approximately 3-fold. The localization of these enzymes coupled with the stimulatory effect of degranulation on acyl-CoA-cholesterol acyltransferase activity has led us to speculate that the accumulation of free cholesterol in the RER membrane might be a driving factor in the conversion of RER to SER.     

High density lipoprotein subpopulations from galactosamine-treated rats and their transformation by lecithin:cholesterol acyltransferase

It is known that an acute hepatotoxicity is produced in rats by intraperitoneal administration of galactosamine; a consequence of this treatment is a marked deficiency of lecithin:cholesterol acyltransferase (LCAT) activity in the plasma compartment. In this study high density lipoprotein (HDL) from galactosamine-treated rats was isolated, resolved into subpopulations, and characterized. In contrast to HDL from control rats, which elutes from gel filtration columns as a single peak and has a diameter of 13.1 nm, HDL from the galactosamine-treated animals was found to elute in five major zones with diameters of 7.8-35 nm. Characterization of these subpopulations has revealed that the larger fractions are enriched in apolipoprotein E, phospholipid, and cholesterol, but contain little cholesteryl ester, while the smallest two fractions contain mainly apolipoprotein A-I, are enriched in phospholipid, and have 50-60% of their cholesterol in the ester form. Incubation of HDL from treated rats with a source of LCAT activity plus low and very low density lipoproteins caused transformation of these subpopulations into a species which, by size and composition, was essentially identical to control rat HDL. In addition, when the subpopulations were individually incubated with purified human lecithin:cholesterol acyltransferase and bovine serum albumin, there was a similar convergence toward a moderate particle size approximating control rat HDL. Cross-linking studies showed that incubation with LCAT activity reduced the heterogeneity of the treated rat HDL. We conclude that the galactosamine treatment induces a complex mixture of HDL that bears strong similarities to the small, apoA-I rich and large, apoE-rich particles seen in LCAT deficiency or secreted by hepatic cells in culture. Furthermore, these species appear to coalesce in the presence of the d greater than 1.21 g/ml fraction of control serum to yield a fairly homogeneous population that resembles control rat HDL in size, composition, and apoprotein content.     

Self-adaptive modification of red-cell membrane lipids in lecithin: Cholesterol acyltransferase deficiency. Lipid analysis and spin labeling

In a patient with lecithin: cholesterol acyltransferase deficiency, free cholesterol was markedly increased, and esterified cholesterol was diminished. In the patient's plasma, an increase in phosphatidylcholine (PC) and a decrease in sphingomyelin were observed. Concomitantly, an increase in a shorter acyl chain 16:0 was noted in PC, sphingomyelin and phosphatidylethanolamine (PE). In contrast to these results, longer chains such as 22:0 and 24:0 were decreased, especially in sphingomyelin. Unsaturated double bonds such as 18:1 was also increased in PC and PE. In the red-cell membrane lipids, the increase in free cholesterol was counteracted by an increase in PC and by a decrease in sphingomyelin and PE, reflecting changes in the patient's plasma lipids. Increased 16:0 (in PC) and decreased 18:0 and 24:0 were observed. The increased plasma free cholesterol due to metabolic defect (lecithin:cholesterol acyltransferase deficiency) led to decreased red-cell membrane fluidity. This effect appeared to be counteracted by changing phospholipid composition (increased PC and decreased sphingomyelin and PE), by increasing shorter chains (16:0), by decreasing longer chains (18:0 and 24:0) and by increasing unsaturated double bonds (18:2). These results can be interpreted as a self-adaptive modification of lecithin:cholesterol acyltransferase deficiency-induced red-cell membrane abnormalities, to maintain normal membrane fluidity. This speculation was supported by the ESR spin-label studies on the patient's membrane lipids. The normal order parameters in intact red cells and in total lipid liposomes were decreased if cholesterol-depleted membrane liposomes were prepared. Thus, the hardening effect of cholesterol appeared to be counteracted by the softening effects described above. Overall membrane fluidity in intact red cells of the lecithin:cholesterol acyltransferase-deficient patient was maintained normally, judged by order parameters in ESR spin-label studies.     

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.     

Acyl coenzyme A:cholesterol acyltransferase in neonatal chick brain

An acyl coenzyme A:cholesterol acyltransferase activity which directly incorporates palmitoyl coenzyme A into cholesterol esters using endogenous cholesterol as substrate was demonstrated in microsomal preparations from neonatal chick brain. The enzyme showed, at pH 7.4, about 2-fold greater activity than that observed at pH 5.6. Nearly 10-times higher esterifying activity was found in brain microsomes using palmitoyl coenzyme A than that with palmitic acid. The acyltransferase activity was clearly different from the other cholesterol-esterifying enzymes previously found in brain, which incorporated free fatty acids into cholesterol esters and did not require ATP or coenzyme A as cofactors. Chick brain microsomes also incorporated palmitoyl coenzyme A into phospholipids and triacylglycerols. However, most of the radioactivity from this substrate was found in the fatty acid fraction, due to the presence of an acyl coenzyme A hydrolase activity in the enzyme preparations. Therefore, the formation of palmitate was tested during all the experiments. The brain acyltransferase assay conditions were optimized with respect to protein concentration, incubation time and palmitoyl coenzyme A concentration. Microsomal activity was independent of the presence of dithiothreitol in the incubation medium and microsomes can be stored at -40 degrees C for several weeks without losing activity. Addition of fatty acid-free bovine serum albumin to brain microsomal preparations produced a considerable increase in the acyltransferase activity, while acyl coenzyme A hydrolase was clearly inhibited. Results obtained show the existence in neonatal chick brain of an acyl coenzyme A:cholesterol acyltransferase activity similar to that found in a variety of tissues from different species but not previously reported in brain.     

Acyl coenzyme A: cholesterol acyltransferase in neonatal chick brain

An acyl coenzyme A:cholesterol acyltransferase activity which directly incorporates palmitoyl coenzyme A into cholesterol esters using endogenous cholesterol as substrate was demonstrated in microsomal preparations from neonatal chick brain. The enzyme showed, at pH 7.4, about 2-fold greater activity than that observed at pH 5.6. Nearly 10-times higher esterifying activity was found in brain microsomes using palmitoyl coenzyme A than that with palmitic acid. The acyltransferase activity was clearly different from the other cholesterol-esterifying enzymes previously found in brain, which incorporated free fatty acids into cholesterol esters and did not require ATP or coenzyme A as cofactors. Chick brain microsomes also incorporated palmitoyl coenzyme A into phospholipids and triacylglycerols. However, most of the radioactivity from this substrate was found in the fatty acid fraction, due to the presence of an acyl coenzyme A hydrolase activity in the enzyme preparations. Therefore, the formation of palmitate was tested during all the experiments. The brain acyltransferase assay conditions were optimized with respect to protein concentration, incubation time and palmitoyl coenzyme A concentration. Microsomal activity was independent of the presence of dithiothreitol in the incubation medium and microsomes can be stored at −40°C for several weeks without losing activity. Addition of fatty acid-free bovine serum albumin to brain microsomal preparations produced a considerable increase in the acyltransferase activity, while acyl coenzyme A hydrolase was clearly inhibited. Results obtained show the existence in neonatal chick brain of an acyl coenzyme A:cholesterol acyltransferase activity similar to that found in a variety of tissues from different species but not previously reported in brain.     

Acyl-coenzyme A: cholesterol acyltransferase. Transfer of cholesterol to its substrate pool and modulation of activity

The preincubation at 37 degrees C of rat liver microsomal fraction, followed by re-isolation of the treated vesicles, results in a time-dependent increase in the activity of acyl-CoA: cholesterol acyltransferase. The presence of cholesterol-phospholipid (1:1, mol/mol) liposomes results in higher rate of increase in activity and under these conditions the rate of increase is liposomal cholesterol concentration-dependent. The preincubation of the microsomal fraction in the presence of [3H]cholesterol-phospholipid liposomes results in transfer of [3H]cholesterol to the re-isolated microsomal vesicles and this transfer follows first-order kinetics in respect to the donor concentration. These preincubations result also in a time-dependent and liposomal cholesterol concentration-dependent increase in the incorporation of [3H]cholesterol into the cholesteryl oleate produced on assay of cholesterol acyltransferase activity. From specific radioactivity data of the cholesteryl esters synthesised on assay of cholesterol acyltransferase in treated microsomal preparations, the rate of liposomal [3H]cholesterol equilibration with the cholesterol acyltransferase substrate pool can be calculated. The half-time of this transfer decreased with the concentration of liposomal cholesterol present during the preincubation. The activation energy for the transfer of liposomal cholesterol to the cholesterol acyltransferase substrate pool was 87.9 kJ/mol and was independent of the concentration of liposomal cholesterol. The activation energy for the rate of increase of total cholesteryl oleate was similar to this value for low concentrations of liposomal cholesterol and progressively decreased with increasing concentrations of liposomal cholesterol. The data suggest that under the present conditions, the time-dependent and temperature-dependent increase in cholesterol acyltransferase activity is due to the transfer of non-esterified cholesterol from other microsomal and/or liposomal vesicles to the vesicles that contain the enzyme and therefore to increased availability of substrate.