Cholesterol Is Superior to 7-Ketocholesterol or 7α-Hydroxycholesterol as an Allosteric Activator for Acyl-coenzyme A:Cholesterol Acyltransferase 1*

We compared the abilities of cholesterolversus various oxysterols as substrate and/or as activator for the enzyme acyl-coenzyme A:cholesterol acyltransferase (ACAT), by monitoring the activity of purified human ACAT1 in response to sterols solubilized in mixed micelles or in reconstituted vesicles. The results showed that 5α,6α-epoxycholesterol and 7α-hydroxycholesterol are comparable with cholesterol as the favored substrates, whereas 7-ketocholesterol, 7β-hydroxycholesterol, 5β,6β-epoxycholesterol, and 24(S),25-epoxycholesterol are very poor substrates for the enzyme. We then tested the ability of 7-ketocholesterol as an activator when cholesterol was measured as the substrate, andvice versa. When cholesterol was measured as the substrate, the addition of 7-ketocholesterol could not activate the enzyme. In contrast, when 7-ketocholesterol was measured as the substrate, the addition of cholesterol significantly activated the enzyme and changed the shape of the substrate saturation curve from sigmoidal to essentially hyperbolic. Additional results show that, as an activator, cholesterol is much better than all the oxysterols tested. These results suggest that ACAT1 contains two types of sterol binding sites; the structural requirement for the ACAT activator site is more stringent than it is for the ACAT substrate site. Upon activation by cholesterol, ACAT1 becomes promiscuous toward various sterols as its substrate.

Acyl-coenzyme A:cholesterol acyltransferase (ACAT) 1 is a membrane-bound enzyme located in the endoplasmic reticulum (ER). It is present in a variety of cell types and tissues and utilizes two lipophilic substrates, cholesterol and long-chain fatty acyl-coenzyme A, to catalyze the formation of neutral lipid cholesteryl esters. In mammals, two ACAT isoforms exist (ACAT1 and ACAT2) (reviewed in Ref. 1). The tissue distribution of ACAT1 is essentially ubiquitous, whereas that of ACAT2 is more restricted. The physiological roles of these isoforms in various tissues are under active investigation. At the single cell level, ACAT participates in controlling the cel-lular membrane cholesterol level. Unlike many other enzymes involved in cholesterol metabolism, regulation of ACAT by sterol occurs at the post-translational level. In mixed micelles or in reconstituted vesicles, both ACAT1 and ACAT2 display a sigmoidal response to cholesterol as their substrates (2). These results are consistent with the concept that the ACAT activity is allosterically regulated by membrane cholesterol content in the ER (reviewed in Ref. 3).
Oxysterols are sterols containing a second oxygen atom, present as a carbonyl, hydroxyl, or epoxide group in rings A or B or in the side chain, in addition to the C3 hydroxyl group. A large number of oxysterols are found in various locations, including food products, plasma, or inside the cells (reviewed in Refs. 4 and 5). They are produced by various enzymes in vivo, and/or by chemical oxidation in vitro. 24-Hydroxycholesterol, 25-hydroxycholesterol, 27-hydroxycholesterol, and 7␣-hydroxycholesterol are the four main oxysterols enzymatically derived (5,6) and can be found in the plasma and inside certain cell types. 7-Ketocholesterol, 7␣-hydroxycholesterol, 7␤-hydroxycholesterol, and 27-hydroxycholesterol are the four major oxysterols found in human atherosclerotic lesions (5,7). In addition, 7-ketocholesterol, 7␣-hydroxycholesterol, 7␤-hydroxycholesterol, 5␣,6␣-epoxycholesterol, and 5␤,6␤-epoxycholesterol are the major oxysterols present in oxidized low density lipoprotein preparations in vitro (8,9). Oxysterols possess a wide range of biological properties and may play regulatory roles in cholesterol metabolism. For example, when added to the culture medium of intact cells, most oxysterols, including 7␣-hydroxycholesterol, 7-ketocholesterol, and 25-hydroxycholesterol, greatly suppressed the cholesterol biosynthesis rate (10,11). Several oxysterols are high affinity ligands for the nuclear receptor LXR␣1 (13,14), and one of them, 24(S),25epoxycholesterol, has been proposed as a participant in cholesterol regulation in the liver (12). On the other hand, the roles of oxysterols in controlling cholesterol homeostasis in vivo are still under debate, partly because in various mammalian systems examined, various oxysterols are present in very low concentrations, with much shorter half-lives relative to cholesterol (15).
Oxysterols have also been shown to have effects on ACAT activity. For example, when added to medium of tissue culture cells, oxysterols such as 7-ketocholesterol or 25-hydroxycholesterol, in addition to their suppressive effect on cholesterol biosynthesis rate, stimulated cholesterol esterification rate and increased ACAT activity (16). When cholesterol was added in the same manner, it failed to provide the same response. Despite numerous studies, the mechanism of oxysterol-mediated activation of cholesteryl ester biosynthesis had not been clarified. It could be due to the presence of a putative oxysterol binding site present in ACAT1 or could be due to the ability of oxysterol to mobilize cellular cholesterol to the ER; other mechanism(s) could not be ruled out. We and other investigators used various crude cell extract systems and had demonstrated the apparent activation of ACAT by 25-hydroxycholesterol in vitro (17)(18)(19). However, in these studies, the enzyme ACAT and the sterols (cholesterol and 25-hydroxycholesterol) serving as substrate and/or activator were present in different membranes. Thus, one could not rule out the possibility that the apparent activation by 25-hydroxycholesterol was due to its ability to translocate cholesterol from a cholesterol-rich membrane to a cholesterol-poor membrane where ACAT is located (discussed in Ref. 19). Studies were also performed attempting to determine the sterol substrate specificity of ACAT. When individual sterols were delivered in vesicle form to the enzyme, Cases et al. (20) have shown that ACAT seemed to utilize various oxysterols much more efficiently than cholesterol. Again, these studies involved the use of crude enzyme extracts, with ACAT and sterols present in different membranes. Among various sterols, the ability to move from the donor membrane to the ACAT-containing membrane differs greatly (21). The transfer rates for oxysterols were much faster (more than 10 times) than those for cholesterol; these differences could greatly mask the true sterol specificity for the enzyme. Thus, the intrinsic substrate specificity of ACAT could not be determined from these studies. In oxidized low density lipoprotein-loaded macrophages, large amounts of esterified oxysterols and esterified cholesterol are present in the cytosolic fraction of the cells; these cytosolic steryl esters are the products of ACAT reaction (7). How ACAT can utilize oxysterols in the presence of large amounts of cellular cholesterol is not clear, partly because the relative specificity of ACAT toward oxysterols and cholesterol as substrate or as activator has not been clarified.
In the current work, we compared the abilities of several selected oxysterols versus cholesterol as ACAT substrates or as ACAT activators. The oxysterols evaluated included, as representatives of ring A or B oxysterols, 7␣-, and 7␤-hydroxycholesterol, 7-ketocholesterol, and 5␣,6␣-and 5␤,6␤-epoxycholesterol and, as a representative of side-chain oxysterols, 24(S),25epoxycholesterol. We first placed sterols and the enzyme in mixed micelles (22), using human ACAT1 purified to homogeneity as the enzyme source and analyzed the enzyme activity in response to varying sterol concentrations. In mixed micelles, the sterol is in direct contact with the enzyme in solution form (23). This assay system also avoids the formation of sterol microdomain(s). On the other hand, the environment provided by the mixed micelles is not close to that of ACAT1 under physiological conditions, because ACAT1 is an integral membrane protein residing in the ER. We therefore tested the validity of information learned from using the mixed micelles system by using the reconstituted vesicle system (24,25). The latter system provides an environment close to that of ACAT1 under physiological conditions. In addition, the sterol and the enzyme ACAT1 reside in the same vesicles, thus eliminating the sterol transfer step between two different vesicles prior to enzyme catalysis. Our results show that ACAT1 can accommodate cholesterol, 5␣,6␣-epoxycholesterol, and 7␣-hydroxycholesterol as its three preferred substrates. In contrast, for activation of ACAT1, cholesterol is superior to all of the oxysterols tested, including 7-ketocholesterol, 7␣-hydroxycholesterol, 7␤hydroxycholesterol, 5␣,6␣-epoxycholesterol, 5␤,6␤-epoxycholesterol, or 7␤-hydroxycholesterol. Thus, the structural requirement for sterol as an ACAT activator is more stringent than it is for sterol as an ACAT substrate.

Methods
Enzyme Purification-The source of enzyme was recombinant human ACAT1 expressed in insect Hi5 cells and purified to electrophoretic homogeneity. The purification procedure was as described previously (22,28), using nickel column chromatography and ACAT1 monoclonal column chromatography. For some of the experiments reported in this work (see Fig. 4), the enzyme source used was HisACAT1 ⌬1-65 (29). The ACAT1 monoclonal antibody only recognizes the N terminus of the enzyme. The HisACAT1 ⌬1-65 enzyme lacked the N-terminal and thus could only be partially purified, using nickel column chromatography (29).
ACAT Enzyme Activity Assay-The enzyme was assayed in sterol/ PC/taurocholate mixed micelles or in reconstituted vesicles conducted as described previously (22), using the radioactive substrate [ 3 H]oleoyl coenzyme A at 4 ϫ 10 4 dpm/nmol. The mixed micelles were prepared as described previously (22), containing varying sterol concentrations, reported as mol % sterol/sterol ϩ PC as indicated in various figures, in 11.2 mM PC and 18.6 mM taurocholate. For assays that contained two different sterols, each sterol/PC/taurocholate mixed micelles sample was made separately and then mixed together and used within 6 h. The reconstituted vesicles were prepared essentially as described previously (30,31). In the current work, we used taurocholate instead of cholate as the detergent to prepare the bile salt/PC/sterol micelles, with taurocholate at 18.6 mM and the PC at 11.2 mM. We then used cholestyramine to remove bile salt from mixed micelles, which rapidly led to vesicle formation. Control experiments using radioactive taurocholate showed that after two treatments of cholestyramine, more than 99% of the taurocholate was removed from the resultant vesicles.
Examining the Sterol Solubility in Mixed Micelles-The sterol solubility in micellar solutions was monitored by subjecting the solutions to ultracentrifugation (at 50,0000 ϫ g for 40 min) followed by ultrafiltration through a Sartorius Centrisart ultrafiltration device that contains a membrane with a molecular mass cut-off of 300 kDa as described (32). After the ultracentrifugation and the ultrafiltration steps, the sterols were quantitated according to the method described previously (33). Briefly, the lipids present in the untreated samples or in the supernatants after treatment were extracted with chloroform/methanol (2:1), spotted onto the TLC plate, and separated using the solvent system hexane/acetone/acetic acid ϭ 80:20:1 (v/v/v). The plate was sprayed with a 0.05% solution of primulin dye and then scanned by using the STORM 860 imaging system to detect the laser-excited fluorescent signals. Spots of a given sterol were quantitated by integration of variable pixel intensities using the ImageQuant software. Standard curves were produced by quantitating increasing amounts (10 -200 g) of a given sterol sample spotted in parallel lanes.
Chemical Synthesis of Sterol Oleate Esters-Various nonradioactive sterol oleate esters were used as internal markers and visualized by iodine staining after TLC analysis. Other than cholesteryl oleate and 25-hydroxycholesterol (from Steraloids), the sterol esters described in the current work were not commercially available. They were chemically synthesized based on the general instruction provided by Molecular Probes, Inc. (Eugene, OR). Typically, 5 mol of a given sterol was weighed in an amber vial, and 0.166 ml of methylene chloride was added and swirled briefly. 0.075 ml of triethylamine, 3 mg of oleic anhydride, and 0.042 mg of 4-dimethylaminopyridine as catalyst and 2 l of 0.1% 2,6-di-tert-butyl-p-cresol in methylene chloride as antioxidant were then added. The vial was sealed under nitrogen and stirred with a magnetic stir bar overnight in the dark. 12 h later, the same amounts of oleic anhydride, 4-dimethylaminopyridine, and 2,6-di-tertbutyl-p-cresol described before were added, and the reaction was con-tinued for an additional 10 h. The sterol oleates synthesized were used as the internal markers for TLC without purification. When TLC plates were run in a petroleum ether/ether/acetic acid (90:10:1) solvent system, the R f values for oleates of the following sterols were as follows: cholesterol, 0.87; 5␣,6␣-epoxycholesterol, 0.51; 5␤,6␤-epoxycholesterol, 0.58; 7-ketocholesterol, 0.36; 7␣-hydroxycholesterol, 0.21; 7␤-hydroxycholesterol, 0.28; ␤-sitosterol, 0.89; 25-hydroxycholesterol, 0.30; 24(S),25-epoxycholesterol, 0.46.

RESULTS
In order to quantitatively compare the abilities of various sterols that serve as ACAT substrate and/or activator, a single phase system composed of the enzyme, phospholipid, and sterol is needed. We had previously developed a mixed micelles system for measuring ACAT activity. This system consisted of mixing egg PC, taurocholate, and cholesterol at appropriate ratios, followed by bath-sonication at cold temperature until optical clearance. The enzyme ACAT solubilized in low concentration of the detergent CHAPS was then added to the mixed micelles before the reaction began. It is possible that the micelles prepared by sonication may still contain a minor portion of sterol in the form of supersaturated microcrystals and/or multilamellar or unilamellar vesicles. To remove microcrystals and/or vesicles from the micelles, Moschetta et al. (32) developed a simple and effective procedure that involved ultracentrifugation and ultrafiltration. We used this procedure to monitor the quality of the mixed micelles that contained either cholesterol, or 7-ketocholesterol, or 5␣,6␣-epoxycholestanol. The results showed that essentially 100% of these three sterols existed in the micellar phase at concentrations from 0.25 to 2 mM (which is equivalent to 0.022-0.15 mol % sterol/sterol ϩ PC) (data not shown). Other sterols, including 5␤,6␤-epoxycholesterol, ␤-sitosterol, 7␣-hydroxycholesterol, and 7␤-hydroxycholesterol, behaved in the same manner (results not shown). In contrast, oxysterols that contain the hydroxy groups at the side chain, including 24-hydroxycholesterol, 25-hydroxycholesterol, and 27-hydroxycholesterol, could only produce clear micellar solutions at no more than 0.5 mM (0.043 mol % sterol/ sterol ϩ PC). Therefore, in our current study, we have focused our effort on comparing various sterols that readily form mixed micelles. We compared the ability of seven sterols to serve as substrates of ACAT by varying their concentrations from 0.047 to 2.0 mM, which amount to 0.004 -0.15 mol % sterol/sterol ϩ PC. In some experiments, the highest concentrations were extended to 2.3 mM (0.17 mol % sterol/sterol ϩ PC). The solubility of 24(S),25-epoxycholesterol was higher than that of oxysterols with hydroxy groups at the side chain; this property allowed us to study 24(S),25-epoxycholesterol at concentrations between 0.0047 and 0.1 mol % sterol/sterol ϩ PC. The results (Fig. 1) show that 5␣,6␣-epoxycholestanol, cholesterol, and 7␣-hydroxycholesterol are the three best substrates. All three sterols exhibit sigmoidal substrate saturation curves; the K 0.5 values (the concentration at which half-maximal velocity is achieved) for these three sterols were similar, varying between 0.7 and 0.9 mol % sterol/sterol ϩ PC. At sterol concentrations above K 0.5 , 5␣,6␣-epoxycholestanol is a slightly better substrate than cholesterol, whereas 7␣-hydroxycholesterol is ϳ70% as efficient as cholesterol as the substrate (results of three independent experiments). The other three sterols tested, 7-ketocholesterol, 7␤-hydroxycholesterol, and ␤-sitosterol (a major plant sterol) were all vastly inferior to cholesterol as the substrate. In addition, 24(S),25-epoxycholesterol tested at concentrations up to 0.1 mol % sterol/sterol ϩ PC is also a poor substrate. The inset showed the same data using a smaller scale to report the ACAT activity. It demonstrates that ACAT1 can definitely use any of the latter four sterols as substrates although in a far less efficient manner. We next tested the effect of cholesterol when 7-ketocholesterol serves as the substrate, and vice versa. The results ( Fig. 2A) show that cholesterol added at low concentrations (from 0.01 to 0.1 mol % sterol/sterol ϩ PC) significantly activated the enzyme when 7-ketocholesterol was measured as the substrate. It also changed the substrate saturation curve from being sigmoidal to essentially hyperbolic. Calculations (using the software program Prism) showed that the Hill coefficient decreased from 3.0 without cholesterol to 1.1 with cholesterol added at 0.1 mol % sterol/sterol ϩ PC. The K 0.5 value decreased from 0.13 mol % sterol/sterol ϩ PC without cholesterol to 0.04 mol % sterol/sterol ϩ PC with cholesterol. The V max value increased from 150 nmol/min without cholesterol to 200 nmol/min with cholesterol. The activation effect by cholesterol shown in Fig. 2A could not be explained, because cholesterol is a better substrate and binds to the catalytic site more efficiently. If this were the case, cholesterol should have caused severe inhibition when 7-ketocholesterol was measured as the substrate. When cholesterol was measured as the substrate, 7-ketocholesterol added at low concentrations (from 0.005 to 0.025 mol % sterol/sterol ϩ PC) caused slight inhibition of the enzyme (Fig. 2B). The inhibition was more prominent at higher cholesterol concentrations. Thus, the inhibitory effect of 7-ketocholesterol is presumably through substrate competition. 7-Ketocholesterol did not alter the sigmoidicity of the cholesterol substrate saturation curve. Calculations showed that the K 0.5 value for cholesterol stayed at 0.07 mol % sterol/sterol ϩ PC, and the Hill coefficient stayed around 2.2, with or without 7-ketocholesterol added.
Human ACAT1 is a homotetrameric enzyme (34). We had previously shown that by deleting the first 65 amino acid residues from the N-terminal, the enzyme could be converted to a dimeric form. This form was designated as hACAT1 ⌬1-65 . The dimeric enzyme is 5-10 times more active than the native ACAT1 in terms of catalytic efficiency (29). We compared the effect of cholesterol and 7-ketocholesterol using hACAT1 ⌬1-65 as the enzyme source. The results show (Fig. 3A) that choles- terol significantly activated the enzyme activity when 7-ketocholesterol was measured as the substrate, whereas 7-ketocholesterol added had minimal effect on the enzyme activity when cholesterol was measured as the substrate (Fig. 3B). Thus, the ability of cholesterol to activate the enzyme does not require the enzyme to exist at the tetrameric form.
We next tested the effect of cholesterol when 5␣,6␣-epoxycholestanol was measured as the substrate, and vice versa. The results showed that cholesterol added at low concentrations significantly activated the enzyme when 5␣,6␣-epoxycholesterol was used as the substrate, whereas 5␣,6␣-epoxycholesterol added inhibited the enzyme when cholesterol was measured as the substrate (data not shown). This result suggested that cholesterol may be a better activator than 5␣,6␣-epoxycholesterol, although the latter sterol is a slightly better substrate than cholesterol (Fig. 1). To test this interpretation, we next used 7-ketocholesterol as the substrate at two different concentrations (at 0.08 or 0.12 mol % sterol/sterol ϩ PC) and compared cholesterol versus various other sterols as indicated, including 5␣,6␣-epoxycholesterol, for their abilities to activate the enzyme. The results (Fig. 4) showed that among all the sterols tested, cholesterol is the only sterol that caused significant activation of the enzyme.
In intact cells, ACAT resides mainly in the ER and uses sterol present in the ER as the enzymatic substrate (3). To measure ACAT activity in a system that is similar to the ER, we next tested the effects of various sterols present in vesicle form. To avoid the step of sterol transfer between donor vesicles to the vesicles where ACAT resides, we had previously developed a reconstituted vesicle system by diluting the ACAT solubilized in detergent into a large excess of preformed vesicles with defined sterol and PC composition (24). We now used the reconstituted vesicle system to compare the abilities of choles-terol, 5␣,6␣-epoxycholesterol, and 7-ketocholesterol to serve as ACAT substrates. The results show (Fig. 5) that 5␣,6␣-epoxycholesterol and cholesterol are much better substrates than 7-ketocholesterol. For each of the sterols tested, the enzyme responded to the sterol content present in the vesicles in a sigmoidal-like manner. We next tested the effect of cholesterol on ACAT activity when 7-ketocholesterol was measured as the substrate, and vice versa. The results show that cholesterol added at low concentrations significantly activated the enzyme when 7-ketocholesterol (Fig. 6A) was measured as the substrate, whereas 7-ketocholesterol (Fig. 6B) added inhibited the enzyme, presumably through substrate competition, when cholesterol was measured as the substrate. In data not shown, we also tested the effect of cholesterol on ACAT activity when 5␣,6␣-epoxycholestanol was measured as the substrate, and vice versa, and have obtained essentially the same results as described in Fig. 6, A and B. To further test this finding using the reconstituted vesicle system, we used 7-ketocholesterol as the variable substrate and compared cholesterol and 5␣,6␣epoxycholesterol, added at 0.4 mol % sterol/sterol ϩ PC, for their ability to activate the enzyme. The results (Fig. 7) show that cholesterol is superior to 5␣,6␣-epoxycholesterol as the activator. Thus, the results using the reconstituted vesicles system fully corroborated the results using the mixed micelles system. DISCUSSION In the current work, we employed the mixed micelles assay system to compare the abilities of various sterols to serve as a substrate for ACAT1. The results show that cholesterol and 5␣,6␣-epoxycholesterol are the two best substrates tested. 7␣-Hydroxycholesterol is the third best substrate, being 70% as efficient, whereas other sterols such as 7-ketocholesterol were 10% or less as efficient. Modification of cholesterol by including the 7␣-hydroxy moiety in steroid ring B is moderately tolerated as the enzymatic substrate. We then developed a method to compare the abilities of various sterols serving as ACAT activators. The results show that cholesterol is far superior as an activator to all other sterols tested, including 5␣,6␣-epoxycholesterol or 7␣-hydroxycholesterol. Thus, the structural specificity of the activator site is much more stringent than its sterol substrate site. The results obtained by using the mixed micelles have essentially been confirmed by using the reconstituted vesicles system.
In bile salt-based mixed micelles, a sterol-specific microdomain(s) has not been reported. In PC-based vesicles, the physical properties of cholesterol, 5␣,6␣-epoxycholestanol, 7␣-hydroxycholesterol, 7␤-hydroxycholesterol, and 7-ketocholesterol have previously been shown to be similar to those of cholesterol (19,35,36). Therefore, we believe that the sterol specificity demonstrated in our current work is mainly due to the intrinsic specificity of the enzyme, not due to subtle difference in biophysical properties of sterols in micelles or in vesicles. We also show that the ability of cholesterol to serve as an activator is preserved when the oligomeric structure of ACAT changes from the tetrameric form to a dimeric form. To explain these results, we propose the following model: ACAT1 contains a sterol substrate site and an allosteric sterol activator site. The activator site is restricted to cholesterol only, whereas the substrate site is more promiscuous. When 7-ketocholesterol is the substrate, activation by cholesterol at low concentration decreases the K 0.5 value as well as increasing the V max value toward 7-ketocholesterol. When 5␣,6␣-epoxycholesterol is the substrate, activation by cholesterol at low concentration decreases the K 0.5 value without affecting the V max value of the enzyme toward 5␣,6␣-epoxycholesterol. In the future, using various biochemical approaches that include photoaffinity labeling and sitespecific mutagenesis, studies can test the validity of this model.
In oxidized low density lipoprotein-loaded macrophages, a large amount of esterified oxysterols including 7-ketocholesterol are found; those present in the cytosol were derived from ACAT reaction (7). These observations could be explained based on our model; ACAT1 present in macrophages is activated by cholesterol in the cholesterol-rich environment and becomes more efficient in using 7-ketocholesterol as an alternative substrate, thus causing an ample amount of 7-ketocholesterol to be esterified. In addition, as shown in our current work, ␤-sitosterol (a major plant sterol) is a poor ACAT1 substrate and a poor activator. Our model predicts that ␤-sitosterol may become a much better substrate of ACAT1 when the enzyme is under high cholesterol conditions. In the future, this prediction could be tested under various physiological conditions. Related to our current work, Brown et al. (37) have recently shown that when added as cyclodextrin complex, cholesterol, 7-ketocholesterol, 7␣-hydroxycholesterol, or other structurally related sterols added to the ER membranes in vitro causes conformational change of the sterol-sensing protein SCAP. Oxysterols with hydroxy groups at the side chain, such as 25hydroxycholesterol or 27-hydroxycholesterol, fail to initiate this change. SCAP is a key factor involved in sterol-specific transcriptional regulation of cholesterol biosynthesis. The sterol specificity demonstrated in their system strongly suggests that it is cholesterol itself that acts on the sterol regulatory machinery. The authors did not test the abilities of the sterols to cause conformational change of SCAP in a dose-dependent manner. Our results reported here show that cholesterol is superior to either 7-ketocholesterol or 7␣-cholesterol as an ACAT activator in vitro. Both ACAT1 and SCAP are integral membrane proteins that mainly reside in the ER. Our results thus reinforce the results of Brown et al. (37), supporting the concept that the content of cholesterol rather than the content of an oxysterol such as 7-ketocholesterol or 7␣-hydroxycholesterol located in the ER plays a pivotal role in the regulation of intracellular cholesterol metabolism. The fact that 7-ketocholesterol added in intact cells caused significant stimulation in esterification of cholesterol (16) could be explained by its ability to cause translocation of cholesterol from the plasma membrane to the ER, where ACAT resides (3,38).
Due to their limited solubility in mixed micelles, we could not perform extensive testing on various oxysterols with hydroxymoiety at the side chain. We did, however, find that 25-hydroxycholesterol at concentrations up to 0.05 mol % was also a poor substrate for ACAT1. When cholesterol was measured as a substrate, 25-hydroxycholesterol added at low concentration failed to increase ACAT activity, whereas cholesterol added at low concentration greatly increased the ACAT activity in utilizing 25-hydroxycholesterol as the substrate (results not shown). In lipid membranes, the oxysterols with hydroxy groups at the side chain are very different from cholesterol in terms of their biophysical properties (21,35,36). Kauffman et al. (39) proposed that 25-hydroxycholesterol and 27-hydroxycholesterol might prefer to interact with PC in "reverse orientation" (i.e. with its hydrophilic side chain lining up with the polar moiety of the PC molecules in membranes). Based on earlier studies and the current results, if oxysterols serve as important regulators for ACAT activity in intact cells, they may act through certain novel mechanism(s) rather than by incorporating into the ER membranes and being recognized by ACAT as a preferred activating molecule.