© 1996 American Heart Association, Inc.
Articles |
Acyl-Coenzyme A:Cholesterol O-Acyltransferase Is Not Identical to Liver Microsomal Carboxylesterase
From the Department of Medical Laboratory Sciences and Technology, Division of Clinical Chemistry (M.A.D., I.B., S.E.H.A.) and the Department of Internal Medicine (K.E.), Karolinska Institutet, Huddinge University Hospital, Sweden.
Correspondence to Dr Stefan E.H. Alexson, Department of Medical Laboratory Sciences and Technology, Division of Clinical Chemistry, Karolinska Institutet, Huddinge University Hospital, S-141 86 Huddinge, Sweden.
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Abstract |
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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.
Key Words: acyl-coenzyme A hydrolase • cholesterol O-acyltransferase • acyl-coenzyme A • carboxylesterase
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Introduction |
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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 2.3.1.26). 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.
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Methods |
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[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.
Animals
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 Assay
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/(molxcm).
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
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/Lxcm).
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Results |
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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.
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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.
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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.
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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).
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PD 138142-15 was found to be a slightly more potent ACAT
inhibitor than DL-MA (the calculated IC50 value
was 1.2 µmol/L) but had no effect on carboxylesterase activity (Fig 3B and Table).
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Discussion |
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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.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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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.
Received May 30, 1995; accepted December 13, 1995.
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References |
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