Purification and Characterization of Rat Liver Microsomal Fatty Acid

Medical Branch, Galveston, Texas 77555. Received May 7, 1996X. Previous studies have shown that fatty acid ethyl ester synthase (FAEES) which catalyze...
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Chem. Res. Toxicol. 1997, 10, 211-218

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Purification and Characterization of Rat Liver Microsomal Fatty Acid Ethyl and 2-Chloroethyl Ester Synthase and Their Relationship with Carboxylesterase (pI 6.1) Bhupendra S. Kaphalia,† Richard R. Fritz,‡ and G. A. S. Ansari*,†,‡ Departments of Pathology and Human Biological Chemistry & Genetics, University of Texas Medical Branch, Galveston, Texas 77555 Received May 7, 1996X

Previous studies have shown that fatty acid ethyl ester synthase (FAEES) which catalyzes the formation of ethyl or 2-chloroethyl esters of long-chain fatty acids is localized in the microsomal fraction of rat liver. A recent study suggests that rat adipose tissue FAEES is similar to rat liver microsomal carboxylesterase (CE) [Tsujita and Okuda (1992) J. Biol. Chem. 267, 23489-23494]. Since the interrelationships among FAEES, 2-chloroethyl ester synthase (FACEES), and cholesterol esterase (ChE) are also not clear at present, we purified and characterized FAEES and FACEES from rat hepatic microsomes and studied their functional and structural relationships with CE and ChE. The results of these studies showed that CE, FAEES, and FACEES activities copurified during each step of purification. Although gelfiltration column chromatography of DEAE-Sephacel purified microsomal protein resolved into two peaks with an estimated molecular weight of 180 (major) and 60 kDa (minor), this paper describes characterization of only the 180 kDa protein. CE, FAEES, and FACEES activities associated with homogeneous 180 kDa protein could be inhibited by a β-esterase inhibitor (diisopropyl fluorophosphate) in an identical manner. This protein, however, showed only the hydrolytic activity, but not the synthetic activity for cholesterol oleate, indicating that it is distinct from ChE. The purified protein could be immunoprecipitated with the antibodies raised against rat adipose tissue FAEES, but not with antibodies against rat pancreatic ChE, demonstrating again that the purified protein is distinct from ChE. A single band corresponding to 60 kDa upon SDS-PAGE, under reduced denaturing conditions, indicates that the purified protein is a trimer. N-terminal amino acid sequence of the first 27 residues were identical to that of rat hepatic microsomal CE [Robbi et al. (1990) Biochem. J., 451-458] which suggests structural similarity of the purified protein with rat hepatic microsomal CE. Therefore, the functional and structural properties of the purified protein demonstrate that FAEES, FACEES, as well as CE activities are expressed by the same protein, purified in this study, which exists as a trimer (180 kDa) and is involved in biosynthesis of long-chain fatty acid esters of xenobiotic alcohols. Further studies on purification and characterization of the enzymes responsible for the esterification of xenobiotic alcohols with endogenous fatty acids from various target organs need to be conducted to determine their functional and structural interrelationships. Inhibition and induction studies of these enzyme(s) and the extent of observed toxicity could be important in understanding their role in etiology of chronic diseases induced by alcohol abuse.

Introduction As early as 1962, in vitro formation and breakdown of ethyl palmitate was shown to be catalyzed by rat adipose tissue microsomes and the liver homogenate fractions, respectively (1,2). Nonoxidative metabolism of xenobiotic alcohols via esterification with long chain fatty acids is one of the pathways for their disposition and has been suggested to play an important role in the pathophysiology of alcohol-induced diseases in humans (3-5). Myocardial infarction, neurotoxicity and pancreatitis are known to be associated with alcohol abuse. Changes in lipid composition of heart and neuronal membranes (6,7) †

Department of Pathology. Department of Human Biological Chemistry & Genetics. * Address for correspondence: G. A. S. Ansari, Ph.D., Professor Department of Pathology, The University of Texas Medical Branch, Galveston, TX 77555-0609. Phone: 409-772-3655. FAX: 409-747-1763. X Abstract published in Advance ACS Abstracts, January 1, 1997. ‡

S0893-228x(96)00079-3 CCC: $14.00

and accumulation of fatty acid ethyl esters (FAEEs)1 in the pancreas, liver, and intestine are reported in experimental animals following chronic ethanol exposure (811). In fact, nonoxidative metabolism of a large number of xenobiotic alcohols has been studied in vivo and/or in vitro (3,4,12). 2-Chloroethyl and 2-bromoethyl esters of endogenous fatty acids have been isolated and identified from the hepatic microsomal lipids of rats following the oral administration of 2-chloroethanol and 2-bromoethanol, respectively, from this laboratory (13,14) and, in vitro, using rat liver microsomes and cholesterol ester hydrolase (15). Toxicity of several fatty acid esters of xenobiotic alcohols is also known (3,4,12). 1 Abbreviations:FAEES, fatty acid ethyl ester synthase; FACEES, fatty acid 2-chloroethyl ester synthase; CE, carboxylesterase; ChE, chloesterol esterase; FAEEs, fatty acid ethyl esters; HMW, high molecular weight; LMW, low molecular weight; SDS-PAGE, sodium dodecyl sulfate-polyacryliamide gel electrophoresis; PNPA, p-nitrophenyl acetate; DFP, diisopropyl fluorophosphate.

© 1997 American Chemical Society

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Extrahepatic organs such as pancreas, heart, and brain, which are frequently damaged in alcohol abuse, are shown to have relatively high concentrations and rates of synthesis of FAEEs with minimal or no enzyme activities required for the oxidative metabolism of ethanol (5). Human subjects acutely intoxicated with ethanol at the time of death exhibited a positive correlation between the levels of FAEEs in parenchymal organs and blood alcohol (5). These esters were also considered as potential markers of alcohol abuse (16). The formation of FAEEs and their bioaccumulation in extrahepatic organs such as pancreas and heart has been implicated in the pathogenesis of pancreatitis and congestive cardiomyopathy, respectively, during alcohol abuse (9,17,18). Formation of fatty acid esters of xenobiotic alcohols was initially thought to be catalyzed by esterases through a reaction similar to the esterification of cholesterol by long-chain fatty acids (19-21). However, subsequent studies have suggested that the hepatic microsomal carboxylesterases (CEs) which belong to the multigene family of serine esterases may also play an important role in nonoxidative metabolism of xenobiotic alcohols via fatty acid conjugation (3,22). These enzymes also catalyze the hydrolysis of a wide range of xenobiotic carboxylesters and aromatic amides resulting in detoxication or activation of such compounds (22-24). In the last 12 years, several studies have demonstrated the presence of fatty acid ethyl ester synthase (FAEES), an enzyme catalyzing the formation of FAEEs, in various tissues of humans and laboratory animals (3,5). FAEES purified from rat adipose tissue (25), and rabbit and human myocardium (26,27), triacylglycerol lipase purified from human pancreas (28), cholesterol esterase (ChE) from porcine and bovine pancreas (15,29,30), and lipoprotein lipase from rat postheparin plasma (31) have been shown to catalyze the formation of FAEEs. A close interrelationship has been suggested between FAEES and CE since the first 27 amino acids of the N-terminal region of FAEES purified from rat adipose tissue is reported to be similar to that of rat liver microsomal CE (25,32). However, structural and functional interrelationships among various esterases involved in the nonoxidative metabolism of xenobiotic alcohols are poorly understood. In the present studies, FAEES and fatty acid 2-chloroethyl ester synthase (FACEES) were purified from rat liver microsomes and their relationship with CE and ChE is elucidated. These studies on the purification and elucidation of the structural, kinetic, and immunological properties of rat hepatic microsomal enzyme(s) involved in the esterification of xenobiotic alcohols with endogenous fatty acids could provide a better understanding of the mechanism of ethanol-induced cardiomyopathy and several other environmentally induced diseases.

Experimental Procedures Chemicals, Substrates, and Reagents: [1-14C]Oleic acid (56 mCi/mmol) and cholesterol [1-14C]oleate (53 mCi/mmol) were obtained from Dupont NEN, (Wilmington, DE). 2-Chloroethanol, cholesterol, oleic acid, diisopropyl fluorophosphate (DFP, inhibitor of β-esterase activity), p-nitrophenyl acetate (PNPA), Sephadex G-150-120 (particle size 40-120 µm), ampholines (pH 3.5-10 and 5-7), and porcine liver carboxylesterase (CE; EC 3.1.1.1) were from Sigma Chemical Co. (St. Louis, MO). Porcine pancreatic cholesterol esterase (ChE; EC 3.1.1.13) from Calbiochem (San Diego, CA) was used. DEAE-Sephacel was procured from Pharmacia LKB (Uppsala, Sweden). Ready gels (12%) for

Kaphalia et al. SDS-PAGE, molecular weight marker proteins, polyvinyl difluoride (PVDF) membranes (0.2 micron), and peroxidase antirabbit IgG were purchased from Bio-Rad Laboratories (Hercules, CA). Unless specified, other biochemicals and reagents used in the present study were from Sigma. 2-Chloroethyl oleate was synthesized by an acid-catalyzed reaction of 2-chloroethanol and oleic acid and characterized as described previously (13-15). Purification of FAEES and FACEES. Microsomes were prepared from the pooled livers (250 g) of male Sprague-Dawley rats, procured from Pelfreez Biologicals, AR, and FAEES and FACEES were purified at 4 °C essentially according to the method for purification of CEs described by Mentlein et al. (33). The microsomal fraction was solubilized in 225 mL of 0.1 mM Tris-HCl buffer (pH 8.5) containing 2.25 g of digitonin followed by centrifugation at 105000g for 1 h. The resultant supernatant was fractionated by ammonium sulfate precipitation. The FAEES and hydrolytic activity toward PNPA were almost completely precipitated in fractions from 40-70% ammonium sulfate saturation. The sediment following 40-70% ammonium sulfate saturation was redissolved in 15 mL 10 mM Tris-HCl buffer, pH 8.0 (buffer A), extensively dialyzed against the same buffer and subjected to ion-exchange chromatography over a DEAE-Sephacel column (25 × 2.5 cm i.d.) equilibrated with buffer A. After the protein was loaded, the column was thoroughly washed with buffer A to remove unbound proteins. The bound proteins were eluted from the column with a 1600 mL linear gradient of 0.03-0.50 M sodium chloride in buffer A. The FAEES activity as well as PNPA-hydrolyzing activity coeluted from the DEAE-Sephacel column. The active fractions were pooled, dialyzed against buffer A, and concentrated to 10 mL by Centriprep-10 membrane (Amicon). The concentrated enzyme thus obtained was subjected to gel filtration over a Sephadex G-150-120 column (90 × 2.5 cm i.d.) equilibrated with buffer A. Two peaks showing coelution of PNPA-hydrolyzing as well as FAEES activities were obtained. A major protein peak containing high molecular weight (HMW) protein with apparent molecular mass of 180 kDa eluted first in the fractions 50-75 of the column followed by a relatively smaller peak containing low molecular weight (LMW) protein(s) corresponding to an estimated molecular mass of 60 kDa in fractions 87115. Fractions corresponding to the HMW and LMW proteins were pooled separately and concentrated to 10 mL volume by Centriprep-10 membrane. Purification and characterization of only HMW fraction were further performed in this study. The HMW fraction was subjected to isoelectric focusing (IEF) on a LKB Model No. 8100 column using a mixture of 0.07 mL pH 3.5-10 and 0.63 mL pH 5-7 ampholines and 0-50% sucrose density gradient. After 24 h of focusing at 4 °C using constant voltage (1600 V) and 40 mA current, 0.8 mL fractions were collected from the column and monitored for pH, PNPAhydrolyzing and FAEES activities, and protein. Both PNPAhydrolyzing and FAEES activities were coeluted from the column and fractions containing these activities were pooled, dialyzed against 10 mM Tris-HCl buffer (pH 8.0), and further subjected to gel filtration HPLC on a 30 cm × 7.8 mm i.d., G3000SWXL column; 5 µM particle size with SWxL guard column (Tosohaas, Montgomeryville, PA) using 0.1 M sodium phosphate buffer containing 0.1 M sodium sulfate and 0.05% sodium azide (pH 6.8) as the mobile phase at a flow rate of 0.5 mL/min and monitored at 280 nm. Fractions corresponding to a peak at ∼180 kDa molecular mass, expressing FAEES as well as PNPAhydrolyzing activities, were pooled, dialyzed against 5 mM TrisHCl buffer (pH 8.0), and lyophilized. The activity of FACEES was monitored only in the pooled fractions at each step of the purification. Enzyme Assays. p-Nitrophenyl acetate (PNPA) is a common substrate for the determination of esterase activity (33-35). Therefore, esterase activity was determined by monitoring the rate of p-nitrophenol formation [(nmol/min)/mg of protein] at room temperature according to the procedure of Erlanson (34). For determining FAEES and FACEES activities, aliquots of enzyme were incubated at 37 °C for 2 h with 750 µmol ethanol

Rat Liver Microsomal FAEES and FACEES or 2-chloroethanol and 2 µmol [1-14C] oleic acid (400 DPM/nmol), respectively, in a final 2 mL volume of 50 mM sodium phosphate buffer (pH 7.4). The incubation mixture was extracted with 2 mL of chloroform twice and the esters obtained in the chloroform layer were separated by preparative thin-layer chromatography (TLC) on 500 µm thick silica gel-coated glass plates as described previously (15). The radioactivity associated with the ester fractions was measured using a Packard 1900CA Tri-Carb Liquid Scintillation Analyzer. The enzyme activities are expressed as (nmol of ester formed/h)/mg of protein. The hydrolytic activity for cholesterol oleate was determined according to the method of Labow et al. (35). A 200 µL micellar substrate containing 4 µmol of cholesterol [1-14C]oleate (150 DPM/nmol), 0.1 M sodium taurocholate, 1 M sodium chloride, and a known aliquot of enzyme preparation was incubated at 37 °C for 30 min. The incubation mixture was extracted with chloroform and subjected to TLC separation using hexane/ diethyl ether/methanol/acetic acid (90:20:5:2, v/v). Silica gel corresponding to the relative flow (Rf) of oleic acid was scraped, eluted with chloroform and radioactivity measured. For the synthetic activity toward cholesterol oleate, an emulsified solution of 2.3 µmol cholesterol, 6.2 µmol [1-14C]oleic acid (400 DPM/nmol), 4.1 µmol sodium taurocholate, 13.3 µmol ammonium chloride in 1 mL of 50 mM phosphate buffer (pH 6.0) along with 25 µL of enzyme were incubated at 37 °C for 2 h (36). The cholesterol ester formed was extracted and separated by TLC as described above. The silica gel corresponding to the Rf of cholesterol oleate was eluted with chloroform and the radioactivity determined. The synthetic and hydrolytic activities of the purified enzyme for cholesterol oleate are expressed as (nmol formed or hydrolyzed/h)/mg of protein, respectively. Kinetics and Inhibition Studies. The Km and Vmax of ethyl and 2-chloroethyl ester formation were determined using double reciprocal plots. The inhibition of PNPA-hydrolyzing, FAEES, and FACEES activities by DFP was studied by incubating 11 µg of the purified enzyme with varying concentrations (10-410-9M) of DFP at 37 °C (25). After 10 min of incubation with the inhibitor, PNPA hydrolyzing, FAEES and FACEES activities were determined as described above. Production and Purification of Antibodies. Antibodies against the HPLC purified HMW protein were raised in New Zealand male white rabbits (Alpha Diagnostic International, San Antonio, TX). The IgG fractions were purified from antiserum using ImmunePure IgG purification kit from Pierce, Rockford, IL. Western blot analysis of the digitonized rat liver microsomal proteins indicate high specificity of the antibodies. These antibodies recognize primarily a 60 kDa protein although a couple of other minor bands were also detected (data not shown). Antibodies against the FAEES purified from rat adipose tissue were provided by Dr. Tsujita, Department of Medical Biochemistry, Ehime University, Shigenobu, Onsengun, Ehime, Japan (25) and antiserum against the rat pancreatic ChE was provided by Dr. Gallo, Department of Biochemistry, The George Washington University, Washington, DC (37). Electrophoresis. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on slab gel (12%) containing 0.1% SDS from Bio-Rad under reduced denaturing conditions was performed according to the method of Laemmli (38). The gels were stained with 0.1% Coommassie Brilliant Blue R-250. Protein was determined by the method of Bradford (39) using bovine serum albumin as a standard. N-Terminal Amino Acid Sequence Analysis. The purified HMW protein transblotted onto PVDF membrane, following SDS-PAGE, was stained with 0.1% commassie blue and a single band (∼60 kDa) was cut out with a razor and subjected to sequence analysis on Applied Biosystems Model 475A protein/ peptide microsequencer (40) with an on-line model 120 microbore (phenylthio)hydantoin amino acid analyzer and a Model 900 data processor. Immunological Studies. For immunoprecipitation studies, 11 µg of purified enzyme was incubated with 0-50 µg of IgG fractions of the antibodies against FAEES purified from rat adipose tissue or preimmune IgG in a total volume of 0.2 mL of

Chem. Res. Toxicol., Vol. 10, No. 2, 1997 213

Figure 1. Gel filtration of the DEAE-Sephacel column purified protein using 10 mM Tris-HCl (pH 8.0) eluting buffer. Fractions of 4.0 mL were collected. 50 mM phosphate buffer (pH 7.4) in 2.5 mL of eppendorf tubes. After slow shaking and incubation (overnight at 4 °C), 50 µL of goat anti-rabbit IgG was added to the reaction mixtures, incubated again for 1 h, and centrifuged at 17 000 rpm for 1 h in a microcentrifuge. The PNPA-hydrolyzing, FAEES, and FACEES activities were determined in the supernatant. Western blot analysis of the purified protein was performed according to the procedure of Towbin et al. (41). Marker proteins and the purified protein along with porcine hepatic CE and pancreatic ChE resolved on SDS-PAGE gel were transblotted on PVDF transfer membrane at constant voltage (100 V) and current (250 mA) using BIO-RAD Mini Trans-Blot Electrophoretic Transfer Cell. A dilution of 1:1500 (v/v) of the antibodies against FAEES purified from rat adipose tissue, purified HMW protein or rat pancreatic ChE, and 1:500 of goat antirabbit IgG (secondary antibody) conjugated to peroxidase were used. The blots were developed with freshly prepared peroxidase substrates, hydrogen peroxide, and 4-chloro-1-naphthol.

Results Purification. PNPA-hydrolyzing as well as FAEES and FACEES activities were coprecipitated by ammonium sulfate saturation (Table 1) and copurified in successive purification steps. Ammonium sulfate (4070% saturation) precipitated approximately 66, 61, and 54% of the PNPA-hydrolyzing, FAEES, and FACEES activities, respectively, present in the digitonin-solubilized microsomes. Almost all the PNPA-hydrolyzing, and FAEES activities were adsorbed on the DEAE-Sephacel column and coeluted in a single broad peak by sodium chloride gradient. With the use of gel filtration, the DEAE-Sephacel purified enzyme was resolved into two peaks with an estimated molecular mass values of 180 kDa (high molecular weight or HMW) and 60 kDa (low molecular weight or LMW). PNPA-hydrolyzing as well as FAEES and FACEES activities, determined in both the pooled fractions of HMW and LMW proteins, were coeluted by gel filtration column chromatography (Figure 1). Further purification and characterization of HMW protein fraction, reported in this paper, showed that all three activities of the HMW fraction were focused at pI 6.1 (Figure 2). During the final step of purification by gel filtration HPLC, both PNPA-hydrolyzing and FAEES activities coeluted in a single peak corresponding to the molecular mass of ∼180 kDa (Figure 3). FACEES activity, which was determined only in the pooled fraction at each step of purification, also copurified with the PNPA-hydrolyzing and FAEES activities, indicating that these activities could not be segregated during the various steps of purification. Specific activities for the PNPA-hydrolyzing, FAEES, and FACEES of HMW protein, purified by HPLC, were found to be 30 434, 2 009, and 563 (nmol/h)/mg of protein,

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Figure 2. Isoelectric focussing of the HMW protein fraction, obtained from gel filtration, for 24 h at 1600 constant voltage and 40 mA. Fractions of 0.8 mL were collected.

Kaphalia et al.

N-terminal amino acid sequence of the first 27 residues of the purified protein was found to be Tyr-Pro-Ser-SerPro-Pro-Val-Val-Asn-Thr-Val-Lys-Gly-Lys-Val-Leu-GlyLys-Tyr-Val-Asn-Leu-Glu-Gly-Phe-Ala-Gln-. This sequence is identical to that reported for FAEES purified from rat adipose tissue (25) and CE-purified from rat liver (32). Immunological Studies. Antibodies against FAEES purified from rat adipose tissue precipitated the purified HMW protein, and the PNPA-hydrolyzing as well as FAEES and FACEES activities were associated with precipitate (Figure 6). As shown in the Western blot analysis (Figure 7), the antiserum raised against rat pancreatic ChE did not cross react with the purified protein, demonstrating its immunological dissimilarity from ChE.

Discussion

Figure 3. Purification of the focused HMW protein by HPLC using 0.1 mM phosphate buffer (pH 6.8) at a flow rate 0.5 mL/ min.

respectively. Yield and fold purification at each step of chromatographies are summarized in Table 1. The purified HMW protein also catalyzed the hydrolysis of cholesterol oleate (Table 2). However, this enzyme completely lacked synthetic activity for cholesterol oleate (data not shown). Kinetic Properties and Inhibition Studies. At a fixed ethanol concentration of 1.6 M and varying concentration of oleic acid (0.05-2.0 mM), the purified enzyme gave calculated Km of 0.8 mM, Vmax of 5714 (nmol/ h)/mg of protein and Kcat of 1028 h-1. Similarly, at a fixed 2-chloroethanol concentration of 0.8 M and varying concentration of oleic acid (0.05-1.6 mM), calculated Km of 0.33 mM and Vmax of 1935 (nmol/h)/mg of protein and Kcat of 348 h-1 were obtained. DFP, a specific inhibitor for β-esterases (CEs), inhibited all three activities (PNPA-hydrolyzing, FAEES, and FACEES) in a dose-dependent manner (Figure 4), suggesting all three activities are associated with the purified protein. Structural Properties. An estimated molecular mass of HMW protein purified by HPLC was found to be 180 kDa. SDS-PAGE of HPLC purified protein under reduced denaturing conditions showed a single band corresponding to an estimated molecular mass of 60 kDa (Figure 5), demonstrating the protein to be a trimer. The

Presence of fatty acid ethyl esters (FAEEs) in human serum (42) and tissues (5, 43) and the toxicity of these esters in isolated organs and cell lines (44-46) have been reported. In earlier studies, ethyl palmitate has been shown to cause splenic destruction and decrease phagocytic activity and immune response in several experimental animals (47,48). FAEEs exert a fluidizing effect on isolated membranes (10), increase pancreatic lysosomal fragility (45), and uncouple mitochondrial oxidative phosphorylation in vitro (44), and have been implicated in fetal alcohol syndrome (43). A large amount of the FAEEs formed intracellularly binds to mitochondrial protein which may be associated with mitochondrial dysfunctions (44). A decrease in ATP levels of rabbit pancreas (49) and an inverse relationship of FAEEs levels with amylase activity in mice (9) strongly suggest the role of FAEEs in alcohol-induced pancreatitis. Recently, we have also found that repeated oral administration of 2-chloroethyl ester of linoleic acid causes hepatic toxicity in rats (50). Acetaldehyde, the metabolic product of ethanol formed via oxidative pathway is known to induce liver diseases in alcoholism. However, the nonoxidative metabolism of alcohols via fatty acid ester formation appears to be rapid and most probably an important pathway for their disposition in extrahepatic organs frequently damaged in alcohol abuse (3-5). Esterification of fatty acid esters with ethanol is catalyzed by FAEES (5). This enzyme is distributed almost in all human and animal tissues (5,9) and has been purified from human and rabbit myocardium, and human pancreas (26-28). Its highest activity is reported in the pancreas followed by liver (5,9). Our previous studies also indicate a similar trend of fatty acid 2-chloroethyl ester synthase (FACEES) activity in rat tissues (51). In view of the toxic potential of the fatty acid esters of xenobiotic alcohols, in general, and ethanol, in particular (3,4,12), purification and characterization of the enzyme(s) involved in catalyzing the formation of FAEEs and other fatty acid esters of xenobiotic alcohols could lead to a better understanding of the mechanism(s) of alcohol and environmentally induced chronic diseases. A number of enzymes have been reported to be involved in nonoxidative metabolism of xenobiotic alcohols (3,22,51). Therefore, FAEES and FACEES as well as PNPA-hydrolyzing activities were determined throughout the purification. Copurification of PNPA-hydrolyzing, FAEES, and FACEES activities by ammonium

Rat Liver Microsomal FAEES and FACEES

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Table 1. Purification of FAEES and FACEES from Rat Liver Microsomesa

fractions digitonized microsomes 40-70% ammonium sulfate precipitate DEAE gel filtration IEF gel filtration HPLC a

total protein (mg)

PNPA-hydrolyzing activity total, specific, nmol/min (nmol/min)/mg

FAEES activity total, specific, nmol/h (nmol/h)/mg

FACEES activity total, specific, nmol/h (nmol/h)/mg

2490 1300

1.52 × 106 1.0 × 106

160 605 97 500

52 148 27 950

20 21 (1.1)

13 923 6 027 1 655 1 296

71 (3.6) 73 (3.6) 394 (19.7) 563 (28.2)

195 83 4.2 2.3

0.76 × 106 0.42 × 106 0.08 × 106 0.07 × 106

610 769 (1.3) 3 897 (6.4) 5 060 (8.3) 19 928 (32.7) 30 434 (49.9)

66 800 38 780 6 177 4 622

65 75 (1.2) 342 (5.3) 467 (7.2) 1 470 (22.6) 2 009 (30.9)

Folds purification is given in parenthesis.

Table 2. Hydrolytic Activity of Purified HMW Protein toward Cholesterol Oleatea fractions

activity, (nmol/h)/mg

digitonized microsomes 40-70% ammonium sulfate precipitate DEAE gel filtration IEF gel filtration HPLC

0.35 ( 0.09 1.42 ( 0.32 1.31 ( 0.16 1.12 ( 0.11 9.54 ( 0.32 21.70 ( 4.20

aSynthetic

activity toward cholesterol oleate was not detected.

Figure 6. Immunoprecipitation of PNPA-hydrolyzing (b), FAEES (∇), and FACEES (9) activities by antibodies against FAEES purified from rat adipose tissue.

Figure 4. Inhibition profiles of PNPA-hydrolyzing (b), FAEES (∇), and FACEES (9) activities by DFP.

Figure 7. Western blot analysis of the purified HMW protein using its antibody against (A) rat adipose tissue FAEES or purified HMW protein and (B) rat pancreatic ChE. Lanes: 1, prestained molecular weight (kDa) markers; 2 and 5, 4 µg purified HMW protein; 3, 15 µg CE from porcine liver; 4 and 6, 20 µg ChE from porcine pancreas.

Figure 5. SDS-PAGE of the HPLC purified HMW protein: Lane 1, molecular weight (kDa) markers; lane 2, 4 µg purified HMW protein.

sulfate (40-70% saturation) and their identical elution profile during various chromatographic purification steps found in this study suggest that all three activities are associated with the purified protein and do not segregate independently. This observation was supported by the results of SDS-PAGE of the purified HMW protein under reduced denaturing conditions, which showed a

single band at an approximate molecular mass value of 60 kDa, following the final step of purification by HPLC indicating that the purified HMW protein is homogeneous. Apparent molecular mass of 180 kDa by HPLC and 60 kDa upon SDS-PAGE, under reduced conditions, suggest that the purified protein is a trimer. Although the Km values determined in this study appear to be high in relation to the physiological concentration of oleic acid, nevertheless this should not undermine the physiological significance of the enzyme. As with most enzymes, the Km value determined in vitro in our studies could be different from what it would be

216 Chem. Res. Toxicol., Vol. 10, No. 2, 1997

Kaphalia et al.

Figure 8. Alignment of N-terminal amino acid sequence of purified HMW protein with the sequences of FAEES from rat adipose tissue and CE purified from various tissues of rats and other species: a, hepatic microsomal; b, rat adipose tissue; c, alveolar microphage; x, uncertain residue.

in vivo. For example Kmglucose for the enzyme aldose reductase is reported to be about 20-fold greater than the physiological concentration of glucose in the tissues (52). Furthermore, the enzyme-substrate reactions with lipophilic substrate would show this trend even more and total concentration determined in the tissue may not be the effective concentration at the site of reaction. The substrates (oleic acid, ethanol, and 2-chloroethanol), used in the present study, are lipophilic molecules, and therefore, their concentrations are expected to be far greater at the site of lipid rich endoplasmic reticulum (ER) membrane where the reaction takes place, as compared to cytosol. However, it is difficult to generate in situ conditions in aqueous medium in vitro. Immunoprecipitation and identical profiles of inhibition of PNPA-hydrolyzing, FAEES, and FACEES activities by the antibodies and DFP, respectively, further indicated that all three activities are associated with the purified HMW protein. However, inhibition of all three activities with DFP, which is a specific inhibitor for β-esterases, suggested that the protein purified in this study could be CE as the N-terminal sequence of the first 27 amino acids of the purified HMW protein is also identical to that of CE purified from rat liver endoplasmic reticulum (32). PNPA-hydrolyzing activity is known to be the best measure of all esterase activities including ChE. However, synthetic activity for fatty acid esters of xenobiotic alcohols and cholesterol oleate, molecular mass, immunoreactivity, and amino acid sequence were also used in the characterization of the purified protein. Cross reactivity of the purified protein with antibodies raised against FAEES purified from rat adipose tissue (25) and the protein purified in this study, but not with antibodies raised against ChE purified from rat pancreas by Western blot analysis, suggested that the purified HMW protein most probably is CE. The purified protein also lacked synthetic activity toward cholesterol oleate and, therefore, does not belong to the cholesterol esterase/ lipase family of enzymes. On the basis of estimated molecular mass by HPLC, estimated subunit molecular mass by SDS-PAGE, inhibition studies by DFP, Nterminal amino acid sequence, and immunological characterization, the purified HMW protein appears similar

to the CE purified and characterized earlier from rat liver microsomes ES-10 by Mentlein et al. (53), E-1 by Harano et al. (54) and FAEES purified from rat adipose tissue (25). This conclusion was further supported by an identical N-terminal amino acid sequence of the purified protein to that of rat liver microsomal CE (ES-10) reported by Robbi et al. (32). The role of CEs in the biotransformation of xenobiotic esters as well as in catalyzing the esterification of several xenobiotic compounds and/or their metabolites, alcohols, and drugs with endogenous fatty acids has long been known (3,22). CEs are glycoproteins of high mannose type and migrate as polypeptides of approximately 60 kDa molecular mass on SDS-PAGE. On the basis of charge, these esterases can be easily resolved (33) and it has been shown that CE (pI 6.1) is a trimer of approximately 180 kDa molecular mass (53). Lipases including lipoprotein lipase lack cholesterol esterase activity (55). By comparing the N-terminal sequences of the first 27 amino acids of the purified protein with FAEES purified from rat adipose tissue (25), CE from rat hepatic microsomal fraction (32), and several other CEs (Figure 8), the protein purified in the present study most probably is a HMW CE which has a subunit molecular mass of 60 kDa. The LMW protein fraction, resolved into five isoforms by IEF, also expresses PNPAhydrolyzing, and FAEES and FACEES activities (51). Although different isoforms of hepatic CEs are known to be induced and inhibited by a variety of xenobiotics (56,57), very little is known regarding the role of CEs in the potentiation and metabolic activation of xenobiotic compounds. Therefore, the role of FAEES and/or FACEES in pathogenesis of diseases of extrahepatic organs in alcohol abuse need to be investigated by using the known inducers and inhibitors of CE. These studies will also establish interrelationships between CE and FAEES and/or FACEES and a correlation of the concentration of FAEEs with the extent of damage in the target organ(s). Once formed, such esters could be easily partitioned/ retained into lipid moieties of the ER membrane due to there lipophilic nature as shown in the Fu5AH and HepG2 cells pulse-labeled with ethyl[3H]oleate (58) and accumulate in target organs at concentrations which could be toxic and cause chronic diseases. Esterification

Rat Liver Microsomal FAEES and FACEES

of membrane fatty acids can also alter the lipid-lipid and lipid-protein interactions regulating the normal membrane functions and disrupt structural properties. Further studies on purification and characterization of LMW isoforms of CE from rat hepatic microsomes and their functional and structural similarities with purified HMW protein and ChE are also needed for a better understanding of the mechanism(s) of pathogenesis of the diseases associated with nonoxidative metabolism of xenobiotic alcohols in humans.

Acknowledgment. This work was supported by National Institutes of Health grant ES 04815.

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