Acyl-Coenzyme A:Cholesterol O-Acyltransferase Is Not Identical to Liver Microsomal Carboxylesterase
Abstract Acyl-coenzyme A (CoA):cholesterol O-acyltransferase (ACAT) is responsible for esterification of cholesterol in the cell. The enzyme has never been purified, but two cDNA sequences coding for this enzyme were recently reported. One of the sequences was identical to human liver carboxylesterase. We have used inhibitors to elucidate the relation between microsomal carboxylesterase, acyl-CoA hydrolase (ACH), and ACAT activities in rat liver. Low concentrations of serine esterase inhibitors strongly inhibited carboxylesterase and acyl-CoA hydrolase activities but stimulated ACAT activity. At higher concentrations, ACAT activity was also inhibited. A sulfhydryl-modifying agent was found to be a potent inhibitor of ACAT without affecting carboxylesterase activity. Similarly, two specific ACAT inhibitors, dl-melinamide and PD 138142-15, inhibited ACAT activity but did not affect carboxylesterase or ACH activities. Our data thus exclude ACAT as a liver microsomal carboxylesterase. The complex inhibition patterns observed with serine esterase inhibitors indicate that carboxylesterases and ACHs may interfere with ACAT activity by competing for the substrate. It is obvious that final identification of ACAT requires demonstration of an active homogenous protein.
- Received May 30, 1995.
- Accepted December 13, 1995.
Esterification of free cholesterol to fatty acyl esters is a key reaction for maintaining a constant concentration of free cholesterol within the cell. This reaction is also important under pathological conditions; for example, during the development of atherosclerotic lesions, when massive accumulation of cholesterol ester occurs in the vessel wall. Most of the intracellular esterification of cholesterol is catalyzed by ACAT (EC 126.96.36.199). ACAT is a membrane-bound enzyme localized in the endoplasmic reticulum, catalyzing the esterification of cholesterol with CoA-activated fatty acids, mainly oleoyl-CoA. Despite considerable efforts during many years, only partial purifications of the enzyme from pig1 and rat liver2 have been achieved. Two groups have studied the properties of ACAT using radiation inactivation, a method that may offer an estimation of the molecular size of membrane-bound enzymes without prior purification. Billheimer et al3 found that ACAT and long-chain ACH, a member of the microsomal carboxylesterase multigene family,4 5 6 are closely associated and that both units are required for ACAT activity. In contrast, Erickson et al7 reported results suggesting that ACAT and ACH have different active sites and accordingly are not part of a common functional unit.
Recently, two cDNA sequences stated to correspond to ACAT were published by two independent groups. Chang et al8 cloned a cDNA from a human macrophage cDNA library, which after transfection into an ACAT-deficient Chinese hamster ovary cell line resulted in expression of ACAT activity. Furthermore, expression of the human ACAT cDNA in Sf9 insect cells showed that the resulting ACAT had catalytic properties similar to those of ACAT enzyme present in human and hamster cell lines.9 A second cDNA encoding ACAT, which in transfection experiments also expressed ACAT activity, was cloned from a human liver cDNA library by Becker at al.10 However, the predicted amino acid sequence showed no similarity to the sequence published by Chang et al but was identical to human liver carboxylesterase. These data appear conflicting and may reflect a complex interaction of ACAT with other protein(s), as suggested from the radiation inactivation experiments.3
The aim of the present study was to investigate the relation of ACAT to microsomal ACHs and carboxylesterases. We have used a number of different inhibitors to characterize the active sites of these enzymes. The effects of treatment of microsomes with serine esterase inhibitors, the ACAT inhibitors DL-MA and PD 138142-15, and the sulfhydryl-reactive compound p-HMB were studied. We conclude that ACAT, ACH, and carboxylesterase are different enzymes. Thus, ACAT is not identical to liver microsomal carboxylesterase, and a model implying that ACH is the acyl-binding component of ACAT is not plausible.
[1-14C]Oleoyl-CoA (60 mCi/mmol), obtained from Du Pont de Nemours GmbH, was diluted with unlabeled oleoyl-CoA (Sigma) to a final concentration of 2.5 mmol/L and stored under nitrogen at −20°C. [3H]Cholesteryl oleate was from Du Pont. BNPP, p-HMB, DTNB, p-nitrophenyl acetate, Triton WR-1339, cholesteryl oleate, palmitoyl-CoA, and human serum albumin (fraction V, essentially fatty acid free) were from Sigma. DFP was purchased from Aldrich Chemical Company, Inc. Cholesterol (crystalline pure), DMSO, and silica gel 60 plates for TLC were obtained from Merck. DL-MA was generously provided by Sumitomo Pharmaceuticals Co. PD 138142-15 was kindly supplied by Parke-Davis.
Normal male Sprague-Dawley rats weighing about 220 g were obtained from Alab (Sollentuna, Sweden). The rats were fed standard rat chow (R3, Ewos) and had access to water ad libitum. The animals were killed in the morning by CO2 anesthesia followed by decapitation. The livers were removed and immediately chilled in ice-cold sucrose medium (0.3 mol/L sucrose, 50 mmol/L Tris-HCl, 1 mmol/L EDTA, 50 mmol/L NaCl; pH 7.4).
Preparation of Liver Microsomes
Livers were homogenized in sucrose medium (described above) and centrifuged at 10 000g for 30 minutes. The supernatant was then centrifuged at 105 000g for 70 minutes. The resulting microsomal pellet was suspended in 100 mmol/L potassium phosphate buffer containing 1 mmol/L EDTA (pH 7.4), at ≈20 mg/mL microsomal protein. The protein concentration was determined by the method of Bradford,11 using bovine serum albumin as standard, and the final protein concentration was adjusted to 2.5 mg/mL with phosphate buffer. Freshly prepared microsomes were used in all experiments except when the inhibition with DFP and PD 138142-15 was studied.
ACAT activity was measured as the formation of labeled cholesteryl ester from [1-14C]oleoyl CoA and cholesterol according to the original procedure of Goodman et al.12 Standard ACAT assays included 250 μg microsomal protein, 1.0 mg human serum albumin, 25 nmol [1-14C]oleoyl-CoA, 20 μg cholesterol added as an aqueous dispersion in Triton WR-1339 (600 μg), and 100 mmol/L potassium phosphate buffer containing 1 mmol/L EDTA (pH 7.4). The total volume of each incubation was 1.0 mL. Inhibitors were dissolved in suitable solvents at different inhibitor concentrations. BNPP and DFP were dissolved in 0.1 mol/L potassium phosphate buffer; p-HMB and PD 138142-15 were dissolved in DMSO and added to the incubation mixture in a volume of 20 to 25 μL; DL-MA was dissolved in acetone, and 10 μL of the inhibitor was used in each assay. The control samples received the same solvents as used in the inhibitor preparation. All components except oleoyl-CoA were preincubated for 20 minutes at 37°C, except that when the inhibition of DL-MA was studied, the preincubation time was 7 minutes. The reaction was started by the addition of [1-14C]oleoyl-CoA. After 5 to 14 minutes, the incubations were stopped by the addition of chloroform/methanol (2:1, vol/vol) and vigorous shaking. To each sample, 10 μg of cholesteryl oleate was added as a carrier, [3H]cholesteryl oleate (20 000 cpm) was added as internal standard, and 1.0 mL of 0.9% NaCl was added to improve the separation of the two phases. The chloroform phase was removed, evaporated under nitrogen, and lipids were separated by TLC, by using hexane/ethyl acetate (95:5, vol/vol). To identify cholesteryl oleate, the TLC plates were exposed to iodine vapor. The spots corresponding to cholesteryl oleate were scraped off and placed in scintillation vials. The overall yield was calculated by measuring the recovery of [3H]cholesteryl oleate in each sample. Blank incubations were run in parallel, and the radioactivity was subtracted from the values obtained from each measurement. The incorporated radioactivity was quantitated by liquid scintillation counting and used to calculate the ACAT activity, which was expressed as nanomoles of [14C]cholesteryl oleate formed per milligram of microsomal protein per minute.
Determination of ACH Activity
ACH activity was assayed in principle according to Alexson and Nedergaard,13 using palmitoyl-CoA as substrate. In some cases, when inhibition with BNPP and DFP was studied, an aliquot of the ACAT incubation mixture was removed just before the incubation was stopped, and placed on ice. Aliquots of 100 to 125 μL of the incubation mixture (corresponding to 25 to 30 μg of microsomal protein) were then used for the measurement of ACH activity. The medium consisted of 25 mmol/L Tris-HCl, 200 mmol/L KCl, 0.05 mmol/L DTNB (pH 7.4), and microsomal protein, in a final volume of 1.0 mL. The concentration of palmitoyl-CoA was 50 μmol/L. The reaction was followed at 412 nm with a Hitachi U 3000 spectrophotometer, and hydrolase activity was calculated from the formula E412=13 600/(mol×cm).
The effect of DL-MA was studied in isolated microsomes. The inhibitor was added in 10 μL of acetone and incubated with the microsomal protein (125 μg) in 1.0 mL of hydrolase medium for 20 minutes at room temperature, and the reaction was initiated by addition of palmitoyl-CoA.
Carboxylesterase activity was measured with 1 mmol/L p-nitrophenyl acetate as substrate in PBS, in principle as described.14 For the inhibition studies with BNPP, DFP, and p-HMB, 40 μL of ACAT incubation mixture (about 10 μg of microsomal protein) was used. Inhibition of carboxylesterase with DL-MA and PD 138142-15 (measured with about 10 μg of microsomal protein) was determined after 20 minutes’ preincubation at room temperature. The liberated p-nitrophenol was measured at 420 nm, and the activity was calculated from the formula E420=3.06/(mmol/L×cm).
ACAT activity was determined in microsomes as the formation of labeled cholesteryl ester after incubation with [1-14C]oleoyl-CoA and exogenous cholesterol. The ACAT activity in untreated microsomes was about 1.5 nmol·min−1·mg protein−1, which is in good agreement with other published results.3 The incubation time varied between 5 and 14 minutes, which was within the linear range for the product formation (data not shown). To study the possible interaction of the ACAT enzyme with microsomal esterases, we investigated the effects of different inhibitors on ACAT, ACH, and carboxylesterase activities. The calculated IC50 values for the inhibitors tested are given in the Table⇓. The effects of all inhibitors except DFP were studied in the presence and absence of Triton WR-1339. The results showed that Triton WR-1339 did not exhibit any effect on the inhibition pattern. The ACAT activity was, however, ≈50% lower when cholesterol was dispersed in ethanol instead of Triton WR-1339.
Effects of Serine Esterase Inhibitors
Inhibition With BNPP
The sensitivity of ACAT, ACH, and carboxylesterase activities to the specific serine esterase inhibitor BNPP was measured in isolated microsomes. The concentrations of BNPP used ranged from 10 μmol/L to 5 mmol/L. ACAT activity increased with increasing concentrations of BNPP, up to 130% of control at 100 μmol/L BNPP. The activity decreased at higher concentrations, with 50% inhibition occurring at 0.59 mmol/L and 90% inhibition at about 2 mmol/L (Fig 1A⇓ and Table⇑). ACH and carboxylesterase activities were much more sensitive to BNPP. Both enzymes were inhibited to about 50% at 10 μmol/L BNPP, and carboxylesterase was inhibited to 90% at 100 μmol/L. However, ACH activity was never completely inhibited; the residual activity of ACH at 5 mmol/L BNPP was about 20%, which is probably owing to the presence of an ACH activity that is not sensitive to BNPP. For clarity, the effects of BNPP at low concentrations are illustrated in Fig 1B⇓.
Inhibition With DFP
The effect of DFP was tested at different concentrations. Both ACH and carboxylesterase activities were almost completely inhibited at the lowest concentration (10 μmol/L) tested. DFP was a less efficient inhibitor of ACAT activity than BNPP. The calculated concentration of DFP required for 50% inhibition was 6.1 mmol/L (Table⇑).
The effects of the serine esterase inhibitors BNPP and DFP on ACH and carboxylesterase activities were very similar, and the concentration of these inhibitors needed for 50% inhibition was in the micromolar range. These results are in accordance with previous data demonstrating that liver microsomal ACH and carboxylesterases are serine esterases.5 However, the inactivation of ACAT activity occurred at much higher concentrations (mmol/L) of these serine esterase inhibitors, strongly suggesting that ACAT does not contain a similar active site. Furthermore, preincubation of the microsomes with 1 mmol/L of BNPP (a concentration causing about 75% inhibition) followed by 10-fold dilution resulted in stimulation of ACAT activity (results not shown). Thus, BNPP does not bind irreversibly by phosphorylation to an active-site serine in the ACAT protein.
Effects of p-HMB
To investigate whether sulfhydryl groups (cysteines) are involved in the enzymatic activity of ACAT, we tested the effects of p-HMB. This substance was a relatively potent inhibitor of ACAT activity, with a calculated IC50 value of 14.7 μmol/L (Fig 2⇓ and Table⇑). However, it was a much less potent inhibitor of carboxylesterase activity, with only about 20% decrease of the activity at a concentration of 40 μmol/L of p-HMB. At the highest concentration tested (500 μmol/L) the activity was decreased only about 30%. The effect of p-HMB on ACH activity was not determined in our standard ACH assay because of the reaction of p-HMB with DTNB. However, p-HMB is a very strong inhibitor of purified ACH when measured as hydrolysis of the thioester bond at 232 nm (S. Alexson, unpublished results, 1995). Thus, the inhibitory effect of p-HMB indicates an involvement of cysteines for the activities of ACAT and ACH but not for the carboxylesterase activity.
Effects of ACAT Inhibitors
We have tested two structurally different ACAT inhibitors, DL-MA which is a fatty acid amide, and PD 138142-15, a novel water-soluble ACAT inhibitor that contains a sulfonylurea nucleus.15
DL-MA has been shown to be a potent inhibitor of ACAT activity in microsomes from rabbit intestinal mucosa, with an IC50 of about 0.5 μmol/L.16 17 In our inhibition studies, DL-MA inhibited ACAT activity, with a calculated IC50 value of 1.9 μmol/L, whereas the activities of ACH and carboxylesterase were unaffected (Fig 3A⇓ and Table⇑).
The two cDNAs reported to correspond to ACAT8 10 encode completely different proteins. Both these proteins increased ACAT activity when expressed in heterologous systems. This finding raises the question of whether there are two ACAT enzymes or whether ACAT activity may be dependent on the interaction of more than one enzyme. The radiation inactivation experiments on isolated microsomes suggested that ACAT and an ACH may form a functional unit.3 We have recently purified a long-chain ACH from rat liver microsomes, which was shown to be structurally related to carboxylesterases.6 This ACH is a 60-kD serine esterase that is inhibited by serine esterase inhibitors and sulfhydryl reagents.4 5 In the present study we conducted a number of inhibition experiments to explore the relation between microsomal ACAT, carboxylesterases, and ACH activities and to investigate the possible involvement of the amino acids serine and cysteine in the active sites of these enzymes. Incubation of microsomes with BNPP resulted in opposite effects on ACH and carboxylesterase activities on one hand and ACAT activity on the other. Whereas carboxylesterase activity was completely inhibited and ACH activity was inhibited to 75% to 80%, at low concentrations, ACAT activity was stimulated. The increased ACAT activity at low concentrations of serine esterase inhibitor could be explained by increased substrate availability owing to inhibition of ACH activity or inhibition of cholesteryl ester hydrolase activity.18 The incomplete inhibition of ACH activity with BNPP is in accordance with previous data in which we found that immunoprecipitation with a polyclonal antibody to microsomal ACHs (corresponding to esterases pI 6.2/6.4,5 ) precipitates about 75% of the total activity.6 Recently a different microsomal ACH was purified from rat intestine, which was found to be insensitive to another serine esterase inhibitor, PMSF, but stimulated by DTT.19 Thus, the residual activity in rat liver microsomes, which is insensitive to serine esterase inhibitors, may be owing to the presence of an ACH identical to the ACH expressed in the intestine. Interestingly, ACAT activity was inhibited at high concentrations of BNPP (fully inhibited at 2.5 mmol/L) and DFP (almost fully inhibited at 10 mmol/L), suggesting that, like several carboxylesterases and lipases,20 21 22 23 ACAT may contain an active-site serine involved in the catalytic function. However, the high concentrations needed and our observation that the effects of BNPP were reversible indicate that the effects of BNPP and DFP were nonspecific.
ACAT activity was strongly inhibited at low concentrations of p-HMB, suggesting the involvement of a cysteine for ACAT activity. The concentration of p-HMB needed for inactivation of ACAT is in the same range as found by Erickson et al7 but clearly lower than the concentrations reported by Kinnunen et al24 for rabbit liver. In our experiments, carboxylesterase activity was found to be almost unaffected by p-HMB.
The selective ACAT inhibitors DL-MA and PD 138142-15 were very potent inhibitors of ACAT activity without affecting ACH or carboxylesterase activities. Billheimer et al3 have suggested that ACH activity is present in microsomes in association with a larger inhibitor protein, possibly ACAT. The most plausible interpretation of our data, showing that inhibition of ACH activity results in a small (about 30%) but reproducible increase in ACAT activity, is that ACH activity may inhibit or limit the activity of ACAT. Erickson et al7 have also observed different effects of inhibitors on ACAT and ACH activities. In agreement with our data, they found that a cysteine-modifying compound (p-HMB) inhibited ACAT activity, but in contrast to our data, ACH activity was stimulated more than twofold. Taken together, these data clearly demonstrate that rat liver microsomal carboxylesterases (including pI 6.2/6.4, which express thioesterase and carboxylesterase activities) are not identical to ACAT.
Apparently, overexpression of one of the microsomal carboxylesterases in Chinese hamster ovary cells results in increased ACAT activity and cholesteryl ester accumulation.10 However, liver microsomes contain several carboxylesterase isoenzymes that are also active on different lipid substrates, one of which has been reported to exhibit significant cholesteryl ester hydrolase activity.18 As demonstrated by our inhibition experiments, changes in the relative activity of the different esterases may affect overall lipid metabolism and result in changes in ACAT activity. It is clear that the conclusive identification of ACAT requires demonstration of an active protein in pure form.
Selected Abbreviations and Acronyms
|ACAT||=||acyl-coenzyme A:cholesterol O-acyltransferase|
This work was supported by grants from the Bank of Sweden Tercentenary Foundation, the Swedish Natural Science Research Council, Magnus Bergvalls Stiftelse, Åke Wibergs Stiftelse, and the Swedish Medical Research Council.
Billheimer JT, Cromley DA, Kempner ES. The functional size of acyl-coenzyme A (CoA):cholesterol acyltransferase and acyl-CoA hydrolase as determined by radiation inactivation. J Biol Chem. 1990;265:8632-8635.
Mentlein R, Berge RK, Heymann E. Identity of purified monoacylglycerol lipase, palmitoyl-CoA hydrolase and aspirin-metabolizing carboxylesterase from rat liver microsomal fraction. Biochem J. 1985;232:479-483.
Erickson SK, Lear SR, McCreery MJ. Functional sizes of hepatic enzymes of cholesteryl ester metabolism determined by radiation inactivation. J Lipid Res. 1994;35:763-769.
Chang CC, Huh HY, Cadigan KM, Chang T-Y. Molecular cloning and functional expression of human acyl-coenzyme A:cholesterol acyltransferase cDNA in mutant Chinese hamster ovary cells. J Biol Chem. 1993;268:20747-20755.
Cheng D, Chang CCY, Qu X-m, Chang T-Y. Activation of acyl-coenzyme A:cholesterol acyltransferase by cholesterol or by oxysterol in a cell-free system. J Biol Chem. 1995;270:685-695.
Becker A, Böttcher A, Lackner KJ, Fehringer P, Notka F, Aslanidis C, Schmitz G. Purification, cloning, and expression of a human enzyme with acyl coenzyme A:cholesterol acyltransferase activity, which is identical to liver carboxylesterase. Arterioscler Thromb. 1994;14:1346-1355.
Goodman DS, Deykin D, Shiratori T. The formation of cholesteryl esters with rat liver enzymes. J Biol Chem. 1964;230:1335-1345.
Alexson SE, Nedergaard J. A novel type of short- and medium-chain acyl-CoA hydrolases in brown adipose tissue mitochondria. J Biol Chem. 1988;263:13564-13571.
Beaufay HA, Amar-Costesec A, Feytmans H, Thines-Sempoux D, Wibo M, Robbi M, Berthet J. Analytical study of microsomes and isolated subcellular membranes from rat liver, I: biochemical methods. J Cell Biol. 1974;61:188-200.
Sliskovic DR, Krause BR, Picard JA, Anderson M, Bousley RF, Hamelehle KL, Homan R, Julian TN, Rashidbaigi ZA, Stanfield RL. Inhibitors of acyl-CoA:cholesterol O-acyl transferase (ACAT) as hypocholesterolemic agents, 6: the first water-soluble ACAT inhibitor with lipid-regulating activity. J Med Chem. 1994;37:560-562.
Morinaga O. The intestinal absorption of cholesterol: the role of acyl-CoA:cholesterol acyltransferase and mixed micelle. J Kyoto Prefectural Univ Med. 1987;96:511-523.
Lehner R, Kuksis A. Purification of an acyl-CoA hydrolase from rat intestinal microsomes: a candidate acyl-enzyme intermediate in glycerolipid acylation. J Biol Chem. 1993;268:24726-24733.
Ozols J. Isolation and characterization of 60-kilodalton glycoprotein esterase from liver microsomal membranes. J Biol Chem. 1987;262:15316-15321.
Ozols J. Isolation, properties, and the complete amino acid sequence of a second form of 60-kDa glycoprotein esterase: orientation of the 60-kDa proteins in the microsomal membrane. J Biol Chem. 1989;264:12533-12545.
Robbi M, Beaufay H, Octave JN. Nucleotide sequence of cDNA coding for rat liver pI 6.1 esterase (ES-10), a carboxylesterase located in the lumen of the endoplasmic reticulum. Biochem J. 1990;269:451-458.