Rat liver bile acid CoA:amino acid N-acyltransferase
2003; Elsevier BV; Volume: 44; Issue: 12 Linguagem: Inglês
10.1194/jlr.m300128-jlr200
ISSN1539-7262
AutoresDongning He, Stephen Barnes, Charles N. Falany,
Tópico(s)Metabolism and Genetic Disorders
ResumoBile acid CoA:amino acid N-acyltransferase (BAT) is responsible for the amidation of bile acids with the amino acids taurine and glycine. Rat liver BAT (rBAT) cDNA was isolated from a rat liver λZAP cDNA library and expressed in Sf9 insect cells using a baculoviral vector. rBAT displayed 65% amino acid sequence homology with human BAT (hBAT) and 85% homology with mouse BAT (mBAT). Similar to hBAT, expressed rBAT was capable of forming both taurine and glycine conjugates with cholyl-CoA. mBAT, which is highly homologous to rBAT, forms only taurine conjugated bile acids (Falany, C. N., H. Fortinberry, E. H. Leiter, and S. Barnes. 1997. Cloning and expression of mouse liver bile acid CoA: Amino acid N-acyltransferase. J. Lipid Res. 38: 86–95). Immunoblot analysis of rat tissues detected rBAT only in rat liver cytosol following homogenization and ultracentrifugation. Subcellular localization of rBAT detected activity and immunoreactive protein in both cytosol and isolated peroxisomes. Rat bile acid CoA ligase (rBAL), the enzyme responsible for the formation of bile acid CoA esters, was detected only in rat liver microsomes. Treatment of rats with clofibrate, a known peroxisomal proliferator, significantly induced rBAT activity, message, and immunoreactive protein in rat liver. Peroxisomal membrane protein-70, a marker for peroxisomes, was also induced by clofibrate, whereas rBAL activity and protein amount were not affected.In summary, rBAT is capable of forming both taurine and glycine bile acid conjugates and the enzyme is localized primarily in peroxisomes in rat liver. Bile acid CoA:amino acid N-acyltransferase (BAT) is responsible for the amidation of bile acids with the amino acids taurine and glycine. Rat liver BAT (rBAT) cDNA was isolated from a rat liver λZAP cDNA library and expressed in Sf9 insect cells using a baculoviral vector. rBAT displayed 65% amino acid sequence homology with human BAT (hBAT) and 85% homology with mouse BAT (mBAT). Similar to hBAT, expressed rBAT was capable of forming both taurine and glycine conjugates with cholyl-CoA. mBAT, which is highly homologous to rBAT, forms only taurine conjugated bile acids (Falany, C. N., H. Fortinberry, E. H. Leiter, and S. Barnes. 1997. Cloning and expression of mouse liver bile acid CoA: Amino acid N-acyltransferase. J. Lipid Res. 38: 86–95). Immunoblot analysis of rat tissues detected rBAT only in rat liver cytosol following homogenization and ultracentrifugation. Subcellular localization of rBAT detected activity and immunoreactive protein in both cytosol and isolated peroxisomes. Rat bile acid CoA ligase (rBAL), the enzyme responsible for the formation of bile acid CoA esters, was detected only in rat liver microsomes. Treatment of rats with clofibrate, a known peroxisomal proliferator, significantly induced rBAT activity, message, and immunoreactive protein in rat liver. Peroxisomal membrane protein-70, a marker for peroxisomes, was also induced by clofibrate, whereas rBAL activity and protein amount were not affected. In summary, rBAT is capable of forming both taurine and glycine bile acid conjugates and the enzyme is localized primarily in peroxisomes in rat liver. Amidation of bile acids in the liver with glycine or taurine prior to their excretion into bile is an important biochemical event in bile acid metabolism in humans as well as in most other mammals (1Hofmann A.F. Enterohepatic circulation of bile acids.in: Schultz S.G. Handbook of Physiology - The Gastrointestinal System III. American Physiological Society, Bethesda, MD1989: 567-596Google Scholar). The conjugation of bile acids with glycine or taurine significantly lowers the pKa of the unconjugated bile acids, ensuring a sustained aqueous solubility for bile acids over the wide range of pH conditions found within the enterohepatic circulation. In addition, biliary excretion of bile acids is a driving force for bile acid flow (2Hofmann A.F. Mysels K.J. Bile acid solubility and precipitation in vitro and in vivo: the role of conjugation, pH, and Ca2+ ions.J. Lipid Res. 1992; 33: 617-626Abstract Full Text PDF PubMed Google Scholar). Since the ability of the liver to form bile acid amidates is very high, less than 1% of the bile acids in bile under normal circumstances are unconjugated (1Hofmann A.F. Enterohepatic circulation of bile acids.in: Schultz S.G. Handbook of Physiology - The Gastrointestinal System III. American Physiological Society, Bethesda, MD1989: 567-596Google Scholar). Amidation of bile acids, therefore, has a fundamentally important role in biliary physiology, both in health and in disease. Bile acids are the major metabolites of cholesterol and are synthesized in the liver. The conjugation of bile acids with glycine or taurine is a two-step process involving the successive action of the enzymes bile acid CoA ligase (BAL) and bile acid CoA:amino acid N-acyltransferase (BAT). Our laboratories have previously purified BAT activity from human liver (3Johnson M. Barnes S. Kwakye J. Diasio R.B. Purification of human liver cholyl CoA: amino acid N-acyltransferase.J. Biol. Chem. 1991; 262: 10227-10233Abstract Full Text PDF Google Scholar) and cloned a cDNA encoding human BAT (hBAT) from a human liver cDNA library (4Falany C.N. Johnson M. Barnes S. Diasio R.B. Molecular cloning and expression of human liver bile acid CoA:amino acid:N-acyltransferase.J. Biol. Chem. 1994; 266: 19375-19379Abstract Full Text PDF Google Scholar). Human BAT (hBAT) expressed in bacterial and COS cells utilizes both glycine and taurine as substrates (4Falany C.N. Johnson M. Barnes S. Diasio R.B. Molecular cloning and expression of human liver bile acid CoA:amino acid:N-acyltransferase.J. Biol. Chem. 1994; 266: 19375-19379Abstract Full Text PDF Google Scholar). Additionally, our laboratories have cloned and expressed mouse BAT (mBAT) and determined its chromosomal location (5Falany C.N. Fortinberry H. Leiter E.H. Barnes S. Cloning and expression of mouse liver bile acid CoA: Amino acid N-acyltransferase.J. Lipid Res. 1997; 38: 86-95Abstract Full Text PDF PubMed Google Scholar). Analysis of the enzymatic properties of mBAT showed that, in contrast to hBAT, mBAT is a taurine-specific conjugating form of BAT (5Falany C.N. Fortinberry H. Leiter E.H. Barnes S. Cloning and expression of mouse liver bile acid CoA: Amino acid N-acyltransferase.J. Lipid Res. 1997; 38: 86-95Abstract Full Text PDF PubMed Google Scholar). There is marked species variability in the relative proportions of glycine and taurine conjugates that are present in bile due to the specific properties of BAT in each species (6Kwakye J. Johnson M. Barnes S. Diasio R.B. Bile acid CoA:amino acid N-acyltransferases from dog, human, pig, rabbit and rat livers - a wide variation of kinetic and substrate properties.Comp. Biochem. Physiol. 1991; 100B: 131-136Google Scholar) and substrate availability (7Hardison W.G.M. Hepatic taurine concentration and dietary taurine as regulators of bile acid conjugation with taurine.Gastroenterology. 1978; 75: 71-75Abstract Full Text PDF PubMed Scopus (113) Google Scholar). Some species such as dogs, cats, and mice synthesize only taurine conjugates, whereas other species such as rats and humans synthesize both taurine- and glycine-conjugated bile acids (8Hagey L.R. Bile Acid Biodiversity Invertebrates and Its Chemistry and Evolutionary Implications. University of California at San Diego, San Diego1992Google Scholar). Furutani et al. (9Furutani M.S. Arii S. Higashitsuji H. Mise M. Fukumoto M. Takano S. Nakayama H. Imamura M. Fujita J. Reduced expression of kan-1 (encoding putative bile acid-CoA-amino acid N-acyltransferase) mRNA in livers of rats after partial heptectomy and during sepsis.Biochem. J. 1995; 311: 203-208Crossref PubMed Scopus (22) Google Scholar) identified a rat liver mRNA whose expression was down-regulated by systemic infection with Escherichia coli. Sequencing of the corresponding cDNA (termed Kan-1) revealed that Kan-1 had 65% homology with hBAT. This led to the assumption that Kan-1 was rat liver BAT (rBAT). Subsequent sequence analysis also revealed that Kan-1 had high homology (85–86%) to mBAT (5Falany C.N. Fortinberry H. Leiter E.H. Barnes S. Cloning and expression of mouse liver bile acid CoA: Amino acid N-acyltransferase.J. Lipid Res. 1997; 38: 86-95Abstract Full Text PDF PubMed Google Scholar). In the rat, the principal biliary bile acids are taurine conjugates with a glycine-taurine ratio approaching 1:6 (v/v) (10Subbiah M. Kuksis A. Mookerjea S. Secretion of bile salts by intact and isolated rat livers.Can. J. Biochem. 1969; 47: 847-854Crossref PubMed Scopus (41) Google Scholar). This has led many investigators to assume that either taurine is the preferred amino acid substrate or that there are multiple forms of BAT with different substrate specificities. To investigate whether Kan-1 exclusively utilizes taurine as a substrate or utilizes both taurine and glycine, the cDNA for Kan-1 was isolated from a rat liver λZAP cDNA library and the functional enzyme expressed in Sf9 insect cells. The active enzyme was capable of forming both taurine and glycine conjugates with several bile acids. Because Kan-1 utilizes both glycine and taurine as substrates, it is similar to hBAT and will be referred to as rBAT. Since our laboratory has recently cloned and expressed rat liver BAL (11Falany C.N. Xie X. Wheeler J. Wang J. Smith M. He D. Barnes S. Molecular cloning and expression of rat liver bile acid CoA ligase.J. Lipid Res. 2002; 43: 2062-2071Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar), we have the ability to undertake studies using rat BAL (rBAL) and rBAT to better understand the mechanism for the formation and physiological properties of bile acid conjugates and the cellular localization(s) of these enzymes. In previous studies, it has been suggested that rBAT is localized in rat liver peroxisomes (12Kase B. Bjorkhem I. Peroxisomal bile acid-CoA:amino-acid N-acyltransferase in rat liver.J. Biol. Chem. 1989; 264: 9220-9223Abstract Full Text PDF PubMed Google Scholar) even though hBAT had been isolated from the cytosolic fraction of human liver (3Johnson M. Barnes S. Kwakye J. Diasio R.B. Purification of human liver cholyl CoA: amino acid N-acyltransferase.J. Biol. Chem. 1991; 262: 10227-10233Abstract Full Text PDF Google Scholar). To confirm these previous results, rats were treated with clofibrate, a peroxisomal proliferative agent, and the effects on rBAT and rBAL expression in liver were monitored. Also, specific polyclonal antibodies and selective enzymatic assays were utilized to investigate the localization of rBAT and rBAL during the isolation of liver peroxisomes. Clofibrate was purchased from ICN Biomedicals Inc. (Aurora, OH). OptiPrep and tobacco etch virus (TEV) protease were purchased from Invitrogen (Carlsbad, CA). Rabbit anti-PMP70 polyclonal antibody was obtained from Zymed Laboratories Inc. (South San Francisco, CA). [α-32P]dCTP (800 μCi/mmol) and [3H]glycine (10 μCi/mmol) were from Amersham Pharmacia Biotech (Piscataway, NJ). [3H]taurine (29 μCi/mmol) was from American Radiolabeled Chemicals Inc. (St. Louis, MO). The STAT-60 RNA isolation reagent was purchased from TEL-TEST (Friendswood, TX). The expression vectors pQE-30 and pQE-31 were from Qiagen (Valencia, CA). Quickhyb was purchased from Stratagene (La Jolla, CA). The pFastBacHTb baculovirus expression system and CellFECTIN reagent were obtained from Life Technologies (Gaithersburg, MD). Cholyl CoA was synthesized from cholic acid and CoA by the method of Shah and Staple (13Shah P. Staple E. Synthesis of coenzyme A esters of some bile acids.Steroids. 1968; 12: 571-576Crossref PubMed Scopus (32) Google Scholar) with modifications as described previously (3Johnson M. Barnes S. Kwakye J. Diasio R.B. Purification of human liver cholyl CoA: amino acid N-acyltransferase.J. Biol. Chem. 1991; 262: 10227-10233Abstract Full Text PDF Google Scholar). Due to the difficulty of expressing full-length rBAT with enzymatic activity in several prokaryotic expression vectors, the pFastBacHTb Baculovirus expression system was utilized to generate enzymatically active rBAT in Sf9 insect cells. Primers were designed according to the published rBAT sequence (9Furutani M.S. Arii S. Higashitsuji H. Mise M. Fukumoto M. Takano S. Nakayama H. Imamura M. Fujita J. Reduced expression of kan-1 (encoding putative bile acid-CoA-amino acid N-acyltransferase) mRNA in livers of rats after partial heptectomy and during sepsis.Biochem. J. 1995; 311: 203-208Crossref PubMed Scopus (22) Google Scholar) to amplify full-length open reading frame of the rBAT from a rat liver λZap cDNA library by PCR. The forward primer (5′-TATAGAATTCAAATGGCCAAGCTGACAGCTG-3′) incorporates an EcoRI restriction site (underlined), and the reverse primer (5′-TATAAAGCTTCCACTCACAGCTGACTGTTG-3′) incorporates a HindIII restriction site (underlined) to facilitate subcloning of the rBAT sequence into the EcoRI-HindIII sites of the pFastBacHTb vector to generate an rBAT cDNA plasmid construct with a 6-His tag on the amino end that can be cleaved by TEV protease. The nucleotide sequence of rBAT is identical to the Kan-1 sequence reported by Furutani et al. (9Furutani M.S. Arii S. Higashitsuji H. Mise M. Fukumoto M. Takano S. Nakayama H. Imamura M. Fujita J. Reduced expression of kan-1 (encoding putative bile acid-CoA-amino acid N-acyltransferase) mRNA in livers of rats after partial heptectomy and during sepsis.Biochem. J. 1995; 311: 203-208Crossref PubMed Scopus (22) Google Scholar). DH10Bac cells were then transformed with the pFastBacHTb-rBAT plasmid for transposition of the rBAT DNA into the bacmid to form recombinant bacmids. Cells with recombinant rBAT bacmids were selected to isolate the large (>23 kb) bacmid DNA with a modified miniprep technique (Life Technologies). To generate recombinant baculovirus particles containing rBAT, 9 × 105 Sf9 cells in mid-log phase were seeded in a 6-well plate in 2 ml/well of Sf-900 II serum-free medium (SFM) containing penicillin (50 U/ml) and streptomycin (50 mg/ml), then allowed to attach at 28°C for 1.5 h. For transfection, bacmid DNA and CellFECTIN reagent were each diluted in 100 ml Sf-900 II SFM without antibiotics, then gently mixed and incubated for 45 min at room temperature. The Sf9 cells were washed once with 2 ml Sf-900 II SFM without antibiotics and overlaid with the bacmid/CellFECTIN solution diluted with 200 ml Sf-900 II SFM. The cells were incubated at 28°C for 5 h, then the diluted bacmid/CellFECTIN complexes were removed and replaced with fresh Sf-900 II SFM containing antibiotics. After incubation at 28°C for 72 h, the baculovirus remained in the supernate from each well after removal of cells by centrifugation at 500 g for 5 min. To obtain cytosolic rBAT, Sf9 cells in mid-log phase in suspension culture were infected with the recombinant baculovirus and incubated in a 135 rpm orbital shaker at 28°C for 48 h. Infected cells were harvested by centrifugation at 500 g for 5 min. Cell pellets were resuspended in 50 mM Tris-HCl (pH 8.5) containing 5 mM 2-mercaptoethanol, 100 mM KCl, 1 mM PMSF, and 1% Nonidet P-40, and lysed by brief sonication. The cytosolic fraction was obtained by centrifugation at 100,000 g for 45 min. Ni-NTA affinity chromatography was performed to purify the His-tagged rBAT protein from the cytosol. The 6-His tag was subsequently cleaved by incubation with TEV protease at 4°C for 6 h. Purified rBAT was recovered from the cleavage reaction in the flow-through fraction of a second Ni-NTA affinity column. Expressed rBAT activities were measured in Sf9 cell cytosol initially to monitor expression or with purified rBAT for enzyme kinetic applications as described below. rBAT activity was determined using the radioassay described by Johnson, Barnes, and Diasio (14Johnson M. Barnes S. Diasio R.B. A radioassay of bile acid coenzyme A:glycine/taurine: N-acyltransferase using n-butanol solvent extraction procedure.Anal. Biochem. 1989; 182: 360-365Crossref PubMed Scopus (13) Google Scholar) in which [3H]amino acids are conjugated to unlabeled cholyl-CoA to form 3H-labeled bile acid conjugates. The standard assay mixture contained 100 mM potassium phosphate (pH 8.4), 1.15 mM cholyl-CoA, and 0.025 μCi of the corresponding 3H-labeled amino acid in a total volume of 100 μl. Reactions were initiated by the addition of cholyl-CoA, incubated at 37°C for 30 min, and terminated by addition of 1 ml 100 mM sodium phosphate (pH 2.0) containing 1% SDS. Radioactive conjugates were then extracted from unreacted labeled amino acid with water-saturated n-butanol and quantified by scintillation spectroscopy. The BAL enzyme assay was modified from the procedure of Killenberg and Jordan (15Killenberg P.G. Jordan J.T. Purification and characterization of bile acid-CoA: amino acid: N-acyltransferase from rat liver.J. Biol. Chem. 1978; 253: 1005-1010Abstract Full Text PDF PubMed Google Scholar). The BAL reaction mixture contained 20 μM chenodeoxycholic acid (CDCA), 5 mM ATP, 50 μM CoA, 5 mM MgCl2, 50 mM NaF, and 2 μM [11,12-3H]CDCA (25 μCi/mmol) (Amersham Pharmacia Biotech) in 0.1 M Tris-HCl (pH 8.5) in a total reaction volume of 80 μl. Reactions were initiated by addition of the rBAL enzyme (microsomal fraction) in a 20 μl volume and incubated for 20 min at 37°C. Methanol-perchloric acid (45%:1.5%, v/v) was added to stop reactions. For control reactions, the methanol-perchloric acid solution was added prior to the enzyme fraction. Reactions were then extracted twice with 3 ml of water-saturated diethyl ether. The amount of [3H]CDCA-CoA in the aqueous phase was determined by scintillation spectroscopy. Initial rates of rBAT amino acid conjugation reaction were determined at various concentrations (0.1, 0.2, 0.4, 0.8, 1.0, 2.0, 4.0, 8.0, 10.0, and 20.0 mM) of taurine or glycine in the presence of 1.15 mM cholyl-CoA. Protein concentration and incubation time were selected so that no more than 15% of the limiting substrate was consumed in the reaction. Kinetic parameters were determined using the Enzyme Kinetics program (Trinity Software). Liver homogenates were prepared from young adult male Sprague Dawley rats as per the OptiPrep protocol (Invitrogen). Excised liver was transferred to ice-cold homogenization medium [0.25 M sucrose, 1 mM EDTA, 0.1% ethanol, 10 mM HEPES-NaOH (pH 7.4)], minced, and homogenized with a Potter-Elvehjem teflon-glass homogenizer. The homogenate was centrifuged at 500 g for 10 min. The pellet was resuspended in homogenization buffer, then rehomogenized and centrifuged. The resulting supernatant fraction was centrifuged at 4,000 g for 10 min to remove nuclei and heavy mitochondria. To recover the light mitochondrial fraction as well as the cellular cytosol, the supernatant fraction was centrifuged at 17,000 g for 15 min. This supernate was centrifuged at 100,000 g for 50 min to recover cytosol and the microsomal fraction. To purify peroxisomes, the light mitochondrial pellet was resuspended in homogenization solution with a Dounce homogenizer and mixed with an equal volume of OpitPrep (50% iodixanol). The suspension was centrifuged at 180,000 g for 3 h in a fixed-angle rotor (Beckman 50.2 Ti). Fractions were collected from a self-generated gradient by upward displacement. For analysis of subcellular rBAT and rBAL distribution, immunoblot analysis was performed with each gradient fraction as well as with the cytosolic and microsomal fractions as described below. A rabbit anti-mBAT polyclonal antibody was generated using the full-length expressed mBAT protein (5Falany C.N. Fortinberry H. Leiter E.H. Barnes S. Cloning and expression of mouse liver bile acid CoA: Amino acid N-acyltransferase.J. Lipid Res. 1997; 38: 86-95Abstract Full Text PDF PubMed Google Scholar). The full-length mBAT was expressed in E. coli M15 cells by transformation with the recombinant mBAT/pQE-30 expression vector, purified utilizing the 6-His tag, and utilized for the production of antibody. To raise the rabbit anti-rBAL polyclonal antibody, a partial-length 464 amino acid fragment of rBAL was utilized (aa 227–690) (11Falany C.N. Xie X. Wheeler J. Wang J. Smith M. He D. Barnes S. Molecular cloning and expression of rat liver bile acid CoA ligase.J. Lipid Res. 2002; 43: 2062-2071Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). This rBAL fragment was generated with an amino-terminal 6-His tag in the pQE-31 expression vector, and purified for the subsequent production of antibody. To identify the peroxisomal-containing fractions, a polyclonal rabbit antibody to peroxisomal marker protein (PMP70) was utilized (Zymed). For immunoblot analysis, 120 μg rat liver cytosolic protein, 100 μg microsomal protein, or 80 μg rat liver subcellular fraction protein was resolved by SDS-PAGE and electrotransferred to nitrocellulose membranes. Membranes were blocked with 5% nonfat milk followed by incubation with a 1:1,000 dilution of rabbit anti-mBAT antibody, a 1:2,000 dilution of rabbit anti-rBAL antibody, or a 1:2,000 dilution of rabbit anti-rat PMP70 antibody. Membranes were then incubated with a 1:60,000 dilution of goat anti-rabbit IgG conjugated with horseradish peroxidase. Immunoconjugates were visualized using the Supersignal West Pico System (Pierce). RNA STAT-60 was used to isolate total RNA from several different tissues (heart, liver, spleen, lung, kidney, brain, and testis) of an adult male Sprague Dawley rat. RNA (20 μg) was resolved by electrophoresis in a 1% agarose-formaldehyde gel and transferred to a nylon membrane. To prepare probe, rBAT cDNA was amplified by PCR and purified from an agarose gel (Qiaquick Gel Extraction kit). The rBAT cDNA was labeled with [32P]dCTP with the Prime It Oligolabeling Kit (Stratagene) to a specific activity of ∼2.5 × 106 cpm/ng. Hybridization was carried out for 1.5 h at 68°C in Quickhyb (Stratagene). The membrane was washed twice in 2× SSC/0.1% SDS for 15 min at RT and twice in 0.1× SSC/0.1% SDS for 15 min at 60°C. Autoradiography was performed at −70°C with an intensifying screen. The expression of BAT in rat intestinal tissue was analyzed by RT-PCR amplification of intestinal RNA. RNA was isolated from adult Sprague Dawley rats using STAT60. The RNA was converted to cDNA using a SuperScript II RT kit (Invitrogen). Amplification of BAT mRNA was carried out using Red-Taq (Sigma) at a range of temperatures in a Tgradient PCR machine (Biometra). The forward primer was 5′-ATGGCCAAGCTGACAGCTG and the reverse primer was 5′-CCACTCACAGCTGACTGTTG. Amplification of PCR products was analyzed by agarose gel electrophoresis and ethidium bromide staining. Young adult female Sprague-Dawley rats (178 g to 192 g) were purchased from Charles River (Wilmington, MA). Animals were allowed to acclimate in the animal care facility for 1 week and were maintained on a 12 h light/dark cycle at a temperature of 18°C to 22°C. Rats were allowed free access to food (Purina Certified Rodent Diet) and water for the duration of the study. After acclimatization, animals were randomized into experimental groups of three rats per group. For peroxisomal induction, clofibrate in corn oil was administered daily by gavage at a dose of 250 mg/kg for 7 days. Control animals received an equivalent volume of corn oil. For the preparation of cytosolic and microsomal fractions of liver, rats were weighed and then anesthetized with ether prior to surgical removal of livers, which were blotted and weighed. A portion of each liver was homogenized with a motor-driven Teflon pestle-glass homogenizer in 10 v (w/v) of ice-cold 0.07 M Tris-HCl (pH 7.4) containing 0.17 M KCl and 2 mM EDTA. The homogenate was centrifuged at 10,000 g for 20 min at 4°C, then the resulting supernate was centrifuged at 100,000 g for 50 min at 4°C to generate cytosolic and microsomal fractions. The microsomal pellet was resuspended in 100 mM potassium phosphate buffer (pH 7.4) containing 1 mM EDTA and 1 mM PMSF. The protein concentration of each microsomal and cytosolic fraction was determined with the Bio-Rad protein assay using a γ globulin standard then samples were aliquoted and frozen at −70°C until use. BAL and BAT expression, immunoreactivity, and enzyme activities were expressed as means ± SD. The effect of clofibrate on these parameters was carried out using Student's t-test. Initial attempts to express rBAT in E. coli with vectors such as pMAL-p2× and pKK233-2 were unsuccessful due to proteolytic degradation of the expressed protein. In contrast, the baculovirus expression system with Sf9 insect cells generated high levels of stable rBAT expression. To evaluate rBAT expression, 200 μg of Sf9 cytosolic protein was assayed for rBAT activity under standard conditions, utilizing either 0.25 mM taurine or 0.25 mM glycine as substrate (4Falany C.N. Johnson M. Barnes S. Diasio R.B. Molecular cloning and expression of human liver bile acid CoA:amino acid:N-acyltransferase.J. Biol. Chem. 1994; 266: 19375-19379Abstract Full Text PDF Google Scholar, 5Falany C.N. Fortinberry H. Leiter E.H. Barnes S. Cloning and expression of mouse liver bile acid CoA: Amino acid N-acyltransferase.J. Lipid Res. 1997; 38: 86-95Abstract Full Text PDF PubMed Google Scholar). There was no detectable BAT activity with cholyl-CoA and either taurine or glycine in control Sf9 cytosol. However, BAT activity was present with both amino acid substrates in cytosol from Sf9 cells infected with rBAT baculovirus. To confirm that BAT activity was due to successful expression of rBAT in Sf9 cells, immunoblot analysis with the rabbit anti-mBAT antibody was carried out (Fig. 1). Figure 1A shows a Coomassie blue-stained gel containing an aliquot of purified native rBAT as well as rat liver cytosol, control Sf9 cytosol, and cytosol from Sf9 expressing rBAT. Immunoblot analysis of Sf9 cytosol detects rBAT protein in Sf9 cells transfected with rBAT-baculovirus but not in control Sf9 cells (Fig. 1B). Two bands in transfected Sf9 cytosol are detected that correspond to rBAT with the 6-His tag (upper band) and without the 6-His tag (lower band) due to proteolytic cleavage. The molecular mass of the lower band corresponds to that of purified rBAT (lane 4, Fig. 1B) from which the 6-His tag has been proteolytically removed. Time-dependent TEV protease digestion of Ni-NTA-purified 6-His-BAT protein resulted in generation of a protein with the same molecular mass as that observed with native rBAT (data not shown). Native rBAT expressed in Sf9 cells was capable of forming both taurine and glycine conjugates. Therefore, the rates of bile acid conjugation with both taurine and glycine were determined using purified expressed rBAT (Fig. 2). The Km values for glycine (4.4 ± 0.1 mM) and taurine (2.0 ± 0.1 mM) cholyl CoA conjugation with expressed purified rBAT were similar to the values obtained with expressed hBAT (5.6 mM and 1.8 mM, respectively) (4Falany C.N. Johnson M. Barnes S. Diasio R.B. Molecular cloning and expression of human liver bile acid CoA:amino acid:N-acyltransferase.J. Biol. Chem. 1994; 266: 19375-19379Abstract Full Text PDF Google Scholar). The Vmax/Km value for taurine is 3.3-fold greater than that of glycine, suggesting that the expressed enzyme is more efficient at taurine conjugation of cholyl CoA than glycine conjugation. To evaluate the tissue-specific expression of rBAT, cytosolic fractions of heart, liver, spleen, lung, kidney, brain, and testis tissue from a young adult male Sprague-Dawley rat were analyzed to evaluate expression of rBAT protein and message. Figure 3Ashows an immunoblot analysis of cytosol prepared from these tissues with the specific rabbit anti-mBAT antibody. Of the tissues tested, only the liver had detectable levels of immunoreactive rBAT protein. A corresponding Northern blot was carried out with the rBAT cDNA as a probe using total RNA prepared from these same tissues (Fig. 3B). Of the tissues examined, only the liver had detectable rBAT mRNA. In subsequent experiments, the expression of BAT could not be detected in intestinal tissue by either immunoblot analysis or by RT-PCR amplification of BAT message (data not shown). Thus, results from both immunoblot and Northern blot analysis indicate that rBAT expression is specific to the liver and is not readily detectable in other tissues. To evaluate the subcellular distribution of rBAT and rBAL, immunoblot analysis of the fractions from the self-generated OptiPrep gradient designed to purify peroxisomes was performed with mBAT and rBAL antibodies. To identify fractions enriched in peroxisomes, immunoblot analysis of PMP70 was performed using a rabbit anti-PMP70 antibody. As shown in Fig. 4, fractions 1–4 had high levels of both rBAT and PMP70 immunoreactivity as compared with fractions 5–8. Immunoblot analysis of an aliquot of the cytosolic fraction of liver also detected both rBAT and PMP70, indicating that these proteins have a similar distribution pattern. rBAL immunoreactive protein was localized to the microsomal fraction with no obvious immunoreactivity in cytosol or peroxisomal fractions. Also, no rBAT or PMP70 immunoreactivities were detectable in liver microsomes. Subcellular fractions from the OptiPrep peroxisomal purification gradient were also assayed for rBAL and rBAT enzymatic activities (Fig. 5). rBAT activity was concentrated in fractions 1–4 with little or no activity in fractions 5–8, corresponding to the pattern of immunoreactive rBAT distribution. Consistent with the lack of rBAL immunoreactivity, rBAL activity was not found in any of the peroxisomal gradient fractions; however, rBAL activity was readily detectable in the liver microsomal fraction (data not shown). The effect of clofibrate treatment on cytosolic rBAT and microsomal rBAL activities in rat liver was examined to determine the relationship of their expression to peroxisomal induction. For both control and clofibrate
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