A comparative study of bile acid CoA:amino acid:N-acyltransferase (BAT) from four mammalian species.

1. Bile acid CoA:amino acid:N-acyltransferase (BAT) was partially purified from dog, human, pig and rat livers. The interspecies variation in substrate specificity and kinetics were determined for glycine and taurine. 2. BAT activity from dog liver formed bile acid conjugates with taurine exclusively, whereas BAT activity from each of the other species formed conjugates with both taurine and glycine. 3. Biliary composition of glycine and taurine bile acid conjugates could partly be accounted for by substrate affinity (Km) and turnover number (Vmax) of BAT activity. 4. A monospecific anti-human BAT polyclonal antibody reacted on Western blot analysis with a 40 kDa band in a 100,000 g supernatant fraction from rat liver. 5. Immunoabsorption chromatography using an anti-human BAT antibody-Sepharose affinity column showed that both the immunoreactive protein band and BAT activity were removed from the 100,000 g supernatant fraction from human and rat livers.     

Quantification and regulation of the subcellular distribution of bile acid coenzyme A:amino acid N-acyltransferase activity in rat liver

Bile acid coenzyme A:amino acid N-acyltransferase (BAT) is responsible for the amidation of bile acids with the amino acids glycine and taurine. To quantify total BAT activity in liver subcellular organelles, livers from young adult male and female Sprague-Dawley rats were fractionated into multiple subcellular compartments. In male and female rats, 65-75% of total liver BAT activity was found in the cytosol, 15-17% was found in the peroxisomes, and 5-10% was found in the heavy mitochondrial fraction. After clofibrate treatment, male rats displayed an increase in peroxisomal BAT specific activity and a decrease in cytosolic BAT specific activity, whereas females showed an opposite response. However, there was no overall change in BAT specific activity in whole liver homogenate. Treatment with rosiglitazone or cholestyramine had no effect on BAT activity in any subcellular compartment. These experiments indicate that the majority of BAT activity in the rat liver resides in the cytosol. Approximately 15% of BAT activity is present in the peroxisomal matrix. These data support the novel finding that clofibrate treatment does not directly regulate BAT activity but does alter the subcellular localization of BAT.     

Radioassay of bile acid coenzyme A:glycine/taurine: N-acyltransferase using an n-butanol solvent extraction procedure

A rapid, specific, and sensitive radioassay for measuring bile acid CoA:glycine/taurine: N-acyltransferase (EC 2.3.1) has been developed. In this assay, 3H-labeled amino acids (glycine or taurine) are conjugated with unlabeled bile acid CoA derivatives to form 3H-labeled bile acid amidates. Following incubation, the 3H-labeled bile acid amidate is separated from the unreacted amino acid by an n-butanol extraction method. The extraction procedure was developed by evaluating the effects of buffer concentration and pH on the recovery of radiolabeled bile acid amidate standards in the presence of human hepatic cytosol. Highest recovery (greater than 90%) of bile acid amidate standards occurred under acidic conditions (pH 2) in the presence of 1% (w/v) SDS. When the radioassay and accompanying n-butanol extraction procedure were utilized to study the amidation of glycine or taurine with cholic acid in human hepatic cytosol, a single peak of radioactivity corresponding with either authentic glycocholate or taurocholate was detected in the n-butanol phase by high-performance liquid chromatography. This assay for bile acid CoA:glycine/taurine: N-acyltransferase activity was linear with incubation time and protein concentration. This assay should be useful in the biochemical studies of this enzyme, as well as in the examination of bile acid amidation in clinical liver specimens.     

Purification and characterization of bile acid-CoA:amino acid N-acyltransferase from rat liver

An in vitro study of bile acid-CoA:amino acid N-acyltransferase activity of rat liver was undertaken in order to determine whether separate amino acid-specific enzymes catalyzed the formation of glycine and taurine conjugates of bile acids as postulated by others. Polyacrylamide gel electrophoresis of 200-fold purified enzyme localized the glycine- and taurine-dependent activities to a single band. Both activities were optimal at pH 7.8 and showed similar loss of activity at pH 6.0, pH 9.0, in the presence of 5,5'-dithiobis(2-nitrobenzoic acid), and at temperatures exceeding 50 degrees. With the purified fraction, Km for glycine was 31 mM and Km for taurine was 0.8 mM. Km for several bile acid-CoA substrates was approximately 20 micron and independent of the amino acid acceptor. Only amino acids with terminal alpha- or beta-amino groups were active as acyl acceptors. Acyl donors were limited to bile acid-CoA derivatives. The data support the conclusion that the rat has a single bile acid-CoA:amino acid N-acyltransferase. The substrate kinetics are consistent with previous observations that taurine conjugates predominate in rat bile at normal hepatocellular concentrations of glycine and taurine.     

Conserved residues in the putative catalytic triad of human bile acid Coenzyme A:amino acid N-acyltransferase

Human bile acid-CoA:amino acid N-acyltransferase (hBAT), an enzyme catalyzing the conjugation of bile acids with the amino acids glycine or taurine has significant sequence homology with dienelactone hydrolases and other alpha/beta hydrolases. These enzymes have a conserved catalytic triad that maps onto the mammalian BATs at residues Cys-235, Asp-328, and His-362 of the human sequence, albeit that the hydrolases contain a serine instead of a cysteine. In the present study, the function of the putative catalytic triad of hBAT was examined by chemical modification with the cysteine alkylating reagent N-ethylmaleimide (NEM) and by site-directed mutagenesis of the triad residues followed by enzymology studies of mutant and wild-type hBATs. Treatment with NEM caused inactivation of wild-type hBAT. However, preincubation of wild-type hBAT with the substrate cholyl-CoA before NEM treatment prevented loss of N-acyltransferase activity. Substitution of His-362 or Asp-328 with alanine results in inactivation of hBAT. Although substitution of Cys-235 with serine generated an hBAT mutant with lower N-acyltransferase activity, it substantially increased the bile acid-CoA thioesterase activity compared with wild type. In summary, data from this study support the existence of an essential catalytic triad within hBAT consisting of Cys-235, His-362, and Asp-328 with Cys-235 serving as the probable nucleophile and thus the site of covalent attachment of the bile acid molecule.     

Measurement and subcellular distribution of choloyl-CoA synthetase and bile acid-CoA:amino acid N-acyltransferase activities in rat liver

An improved method for assaying choloyl-CoA synthetase activity (E.C. 6.2.1.7) and two methods for specific measurement of bile acid-CoA:amino acid N-acyltransferase activity (E.C. 2.3.1) are described. The methods are shown to be reproducible, linear with respect to time and enzyme protein, and result in estimates of enzymic activity that conform to the theoretical stoichiometry of the individual reactions. Utilizing these methods, the subcellular distribution of the rat liver enzymic activity catalyzing the formation of glycine and taurine conjugates of bile acids is shown. Choloyl-CoA synthetase is associated with the microsomal membranes and bile acid-CoA:amino acid N-acyltransferase activity with the postmicrosomal supernatant. No significant amino acid N-acyltransferase activity is present in the lysosome fraction. These studies provide methods that will permit further study of the individual enzymic reactions involved in the intrahepatic conjugation of bile acids with amino acids.     

Peroxisomal bile acid-CoA:amino-acid N-acyltransferase in rat liver

Bile acid-CoA:amino-acid N-acyltransferase activity was measured in subcellular fractions of rat liver homogenate. The conversion of [14C]choloyl-CoA and [14C]chenodeoxycholoyl-CoA into the corresponding [14C]tauro- and glyco-bile acids was calculated after isolation of the product by high performance liquid chromatography. There was an enrichment of bile acid-CoA:amino-acid N-acyltransferase activity in the light mitochondrial (L) fraction and to a lesser extent in the microsomal fraction. Surprisingly, no enrichment was found in the cytosolic fraction. Subfractionation of the L-fraction by Nycodenz gradient centrifugation, showed that the activity of the N-acyltransferase had a bimodal distribution and co-sedimented with peroxisomes (particulate catalase) and microsomes (esterase). The highest specific amidation activity of both choloyl-CoA and chenodeoxycholoyl-CoA was always found in the most peroxisome-rich fractions. [14C]Taurocholate formation in the peroxisomal fraction was 2.2 mumol/mg of protein/min. Striking differences were observed in the Km values and the saturation concentrations for glycine and taurine. The peroxisomal amidation of [14C]choloyl-CoA had a Km for taurine of 0.9 x 10(-3) M and for glycine of 17 x 10(-3) M. The results are consistent with the possibility that most of de novo synthesized bile acids conjugate to taurine by a peroxisomal bile acid-taurine N-acyltransferase in rat liver. The bile acids deconjugated in the gut and recirculating to the liver may be activated and amidated by the microsomal enzyme system prior to biliary secretion.     

Purification and characterization of the enzymes of bile acid conjugation from fish liver

1. The two steps in bile acid conjugation have been studied in subcellular fractions of liver from three species of fish; vermillion rockfish, canary rockfish and ling codfish. 2. The bile acid: coenzyme A (CoA) ligase activity in homogenates and isolated microsomes is undetectable due to indeterminate factors. 3. A purification scheme is presented which eliminates the interfering factors. The purified ligase was found to have a lower affinity for bile acids as compared to the mammalian form and to be present in much lower titer. 4. Since it appears to be the rate controlling enzyme in all species, it is expected that the rate of bile acid conjugation is much slower in non-mammalian liver as compared to mammalian liver. 5. The bile acid-CoA:taurine N-acyltransferase was found to exist as a dimer of molecular weight 100,000, in contrast to the monomeric mammalian forms. 6. The only major kinetic difference is that the fish liver forms have rates of glycine conjugation which are only 1-2% of the rate with taurine, in part due to a very high Km for glycine.     

Hepatocyte nuclear factor 4alpha is a central regulator of bile acid conjugation

Hepatocyte nuclear factor 4alpha (HNF4alpha) has an important role in regulating the expression of liver-specific genes. Because bile acids are produced from cholesterol in liver and many enzymes involved in their biosynthesis are preferentially expressed in liver, the role of HNF4alpha in the regulation of bile acid production was examined. In mice, unconjugated bile acids are conjugated with taurine by the liver-specific enzymes, bile acid-CoA ligase and bile acid-CoA:amino acid N-acyltransferase (BAT). Mice lacking hepatic HNF4alpha expression exhibited markedly decreased expression of the very long chain acyl-CoA synthase-related gene (VLACSR), a mouse candidate for bile acid-CoA ligase, and BAT. This was associated with markedly elevated levels of unconjugated and glycine-conjugated bile acids in gallbladder. HNF4alpha was found to bind directly to the mouse VLACSR and BAT gene promoters, and the promoter activities were dependent on HNF4alpha-binding sites and HNF4alpha expression. In conclusion, HNF4alpha plays a central role in bile acid conjugation by direct regulation of VLACSR and BAT in vivo.     

Molecular cloning and expression of rat liver bile acid CoA ligase

Bile acid CoA ligase (BAL) is responsible for catalyzing the first step in the conjugation of bile acids with amino acids. Sequencing of putative rat liver BAL cDNAs identified a cDNA (rBAL-1) possessing a 51 nucleotide 5'-untranslated region, an open reading frame of 2,070 bases encoding a 690 aa protein with a molecular mass of 75,960 Da, and a 138 nucleotide 3'-nontranslated region followed by a poly(A) tail. Identity of the cDNA was established by: 1) the rBAL-1 open reading frame encoded peptides obtained by chemical sequencing of the purified rBAL protein; 2) expressed rBAL-1 protein comigrated with purified rBAL during SDS-polyacrylamide gel electrophoresis; and 3) rBAL-1 expressed in insect Sf9 cells had enzymatic properties that were comparable to the enzyme isolated from rat liver. Evidence for a relationship between fatty acid and bile acid metabolism is suggested by specific inhibition of rBAL-1 by cis-unsaturated fatty acids and its high homology to a human very long chain fatty acid CoA ligase. In summary, these results indicate that the cDNA for rat liver BAL has been isolated and expression of the rBAL cDNA in insect Sf9 cells results in a catalytically active enzyme capable of utilizing several different bile acids as substrates.     

Biological properties of the 2-fluoro-beta-alanine conjugates of cholic acid and chenodeoxycholic acid in the isolated perfused rat liver

The isolated perfused rat liver was used to examine the hepatic extraction, biliary secretion and effect on bile flow of the 2-fluoro-beta-alanine conjugates of cholic acid and chenodeoxycholic acid. The naturally occurring taurine and glycine conjugates of these bile acids were used for comparisons. The 2-fluoro-beta-alanine conjugates were extracted by the liver to a similar extent as the taurine and glycine conjugates. The biliary secretion rate and increase in bile flow were similar for all the cholic acid conjugates. On the other hand, the maximal biliary secretion rate of the 2-fluoro-beta-alanine conjugate of chenodeoxycholate was similar to that of the glycochenodeoxycholate, but 47% lower than that of taurochenodeoxycholate. In addition, the 2-fluoro-beta-alanine conjugate of chenodeoxycholate produced a decrease in bile flow that was comparable to that observed with the glycochenodeoxycholate (54% vs. 74%), but which was greater than that produced by the taurochenodeoxycholate (12%). In summary, these data demonstrate that the biological properties of the 2-fluoro-beta-alanine conjugates of cholic acid and chenodeoxycholic acid are not markedly different from those of the naturally occurring taurine and glycine conjugates. These data also suggest that the amino acid moiety can influence the biliary secretion and cholestatic properties of chenodeoxycholic acid conjugates.     

Purification and characterization of bile acid-CoA:amino acid N-acyltransferase from human liver

The bile acid-conjugating enzyme, bile acid-CoA: amino acid N-acyltransferase, was purified 480-fold from the soluble fraction of homogenized frozen human liver. Purification was accomplished by a combination of anion exchange chromatography, chromatofocusing, glycocholate-AH-Sepharose affinity chromatography, and high performance liquid chromatography (HPLC) gel filtration. Following purification, the reduced, denatured enzyme migrated as a single 50-kDa protein band by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A similar molecular mass was obtained for the native enzyme by HPLC gel filtration. Elution from the chromatofocusing column suggested an apparent isoelectric point of 6.0 (+/- 0.2). Using a rabbit polyclonal antibody raised against the purified enzyme, Western blot analysis using 100,000 x g human liver supernatant confirmed that the affinity-purified polyclonal antibody was specific for human liver bile acid-CoA:amino acid N-acyltransferase. The purified enzyme utilized glycine, taurine, and 2-fluoro-beta-alanine (a 5-fluorouracil catabolite), but not beta-alanine, as substrates. Kinetic studies revealed apparent Km values for taurine, 2-fluoro-beta-alanine, and glycine of 1.1, 2.2, and 5.8 mM, respectively, with corresponding Vmax values of 0.33, 0.19, and 0.77 mumol/min/mg protein. These data demonstrate that a single monomeric enzyme is responsible for the conjugation of bile acids with glycine or taurine in human liver.     

The human bile acid-CoA:amino acid N-acyltransferase functions in the conjugation of fatty acids to glycine

Bile acid-CoA:amino acid N-acyltransferase (BACAT) catalyzes the conjugation of bile acids to glycine and taurine for excretion into bile. By use of site-directed mutagenesis and sequence comparisons, we have identified Cys-235, Asp-328, and His-362 as constituting a catalytic triad in human BACAT (hBACAT) and identifying BACAT as a member of the type I acyl-CoA thioesterase gene family. We therefore hypothesized that hBACAT may also hydrolyze fatty acyl-CoAs and/or conjugate fatty acids to glycine. We show here that recombinant hBACAT also can hydrolyze long- and very long-chain saturated acyl-CoAs (mainly C16:0-C26:0) and by mass spectrometry verified that hBACAT also conjugates fatty acids to glycine. Tissue expression studies showed strong expression of BACAT in liver, gallbladder, and the proximal and distal intestine. However, BACAT is also expressed in a variety of tissues unrelated to bile acid formation and transport, suggesting important functions also in the regulation of intracellular levels of very long-chain fatty acids. Green fluorescent protein localization experiments in human skin fibroblasts showed that the hBACAT enzyme is mainly cytosolic. Therefore, the cytosolic BACAT enzyme may play important roles in protection against toxicity by accumulation of unconjugated bile acids and non-esterified very long-chain fatty acids.     

Differential regulation of cytosolic and peroxisomal bile acid amidation by PPAR alpha activation favors the formation of unconjugated bile acids

In human liver, unconjugated bile acids can be formed by the action of bile acid-CoA thioesterases (BACTEs), whereas bile acid conjugation with taurine or glycine (amidation) is catalyzed by bile acid-CoA:amino acid N-acyltransferases (BACATs). Both pathways exist in peroxisomes and cytosol. Bile acid amidation facilitates biliary excretion, whereas the accumulation of unconjugated bile acids may become hepatotoxic. We hypothesized that the formation of unconjugated and conjugated bile acids from their common substrate bile acid-CoA thioesters by BACTE and BACAT is regulated via the peroxisome proliferator-activated receptor alpha (PPARalpha). Livers from wild-type and PPARalpha-null mice either untreated or treated with the PPARalpha activator WY-14,643 were analyzed for BACTE and BACAT expression. The total liver capacity of taurochenodeoxycholate and taurocholate formation was decreased in WY-14,643-treated wild-type mice by 60% and 40%, respectively, but not in PPARalpha-null mice. Suppression of the peroxisomal BACAT activity was responsible for the decrease in liver capacity, whereas cytosolic BACAT activity was essentially unchanged by the treatment. In both cytosol and peroxisomes, the BACTE activities and protein levels were upregulated 5- to 10-fold by the treatment. These effects caused by WY-14,643 treatment were abolished in PPARalpha-null mice. The results from this study suggest that an increased formation of unconjugated bile acids occurs during PPARalpha activation.     

Inactivation of human liver bile acid CoA:amino acid N-acyltransferase by the electrophilic lipid, 4-hydroxynonenal

The hepatic enzyme bile acid CoA:amino acid N-acyltransferase (BAT) catalyzes the formation of amino acid-conjugated bile acids. In the present study, protein carbonylation of BAT, consistent with modification by reactive oxygen species and their products, was increased in hepatic homogenates of apolipoprotein E knock-out mice. 4-Hydroxynonenal (4HNE), an electrophilic lipid generated by oxidation of polyunsaturated long-chain fatty acids, typically reacts with the amino acids Cys, His, Lys, and Arg to form adducts, some of which (Michael adducts) preserve the aldehyde (i.e., carbonyl) moiety. Because two of these amino acids (Cys and His) are members of the catalytic triad of human BAT, it was proposed that 4HNE would cause inactivation of this enzyme. As expected, human BAT (1.6 microM) was inactivated by 4HNE in a dose-dependent manner. To establish the sites of 4HNE's reaction with BAT, peptides from proteolysis of 4HNE-treated, recombinant human BAT were analyzed by peptide mass fingerprinting and by electrospray ionization-tandem mass spectrometry using a hybrid linear ion trap Fourier transform-ion cyclotron resonance mass spectrometer. The data revealed that the active-site His (His362) dose-dependently formed a 4HNE adduct, contributing to loss of activity, although 4HNE adducts on other residues may also contribute.     

The utilization of alanine, glutamic acid, and serine as amino acid substrates for glycine N-acyltransferase

The conjugation of benzoyl-CoA with the aliphatic and acidic amino acids by glycine N-acyltransferase, as well as the amides of the latter group, was investigated. Bovine and human liver benzoyl-amino acid conjugation were investigated using electrospray ionization tandem mass spectrometry (ESI-MS-MS). Bovine glycine N-acyltransferase catalyzed conjugation of benzoyl-CoA with Gly (Km(Gly) = 6.2 mM), Asn (Km(Asn) = 129 mM), Gln (Km(Gln) = 353 mM), Ala (Km(Ala) = 1573 mM), Glu (Km(Glu) = 1148 mM) as well as Ser in a sequential mechanism. In the case of the human form, conjugation with Gly (Km(Gly) = 6.4 mM), Ala (Km(Ala) = 997 mM), and Glu was detected. The presence of these alternative conjugates did not inhibit bovine glycine N-acyltransferase activity significantly. Considering the relatively low levels at which these conjugates are formed, it is unlikely that they will have a significant contribution to acyl-amino acid conjugation under normal conditions in vivo. However, their cumulative contribution to acyl-amino acid conjugation under metabolic disease states may prove to have a useful contribution to detoxification of elevated acyl-CoAs.     

Identification of an amino acid transporter associated with the cystinuria-related type II membrane glycoprotein.

We identified an amino acid transporter that is associated with the cystinuria-related type II membrane glycoprotein, rBAT (related to b(0,+) amino acid transporter). The transporter designated BAT1 (b(0, +)-type amino acid transporter 1) from rat kidney was found to be structurally related to recently identified amino acid transporters for system L, system y(+)L, and system x(-)C, which are linked, via a disulfide bond, to the other type II membrane glycoprotein, 4F2hc (4F2 heavy chain). In the nonreducing condition, a 125-kDa band, which seems to correspond to the heterodimeric complex of BAT1 and rBAT, was detected in rat kidney with anti-BAT1 antibody. The band was shifted to 41 kDa in the reducing condition, confirming that BAT1 and rBAT are linked via a disulfide bond. The BAT1 and rBAT proteins were shown to be colocalized in the apical membrane of the renal proximal tubules where massive cystine transport had been proposed. When expressed in COS-7 cells with rBAT, but not with 4F2hc, BAT1 exhibited a Na(+)-independent transport of cystine as well as basic and neutral amino acids with the properties of system b(0,+). The results from the present investigation were used to establish a family of amino acid transporters associated with type II membrane glycoproteins.     

A coupled assay for bile acid:CoA ligase

A new assay for the enzyme bile acid:CoA ligase is presented. The new assay is designed to supplant the existing radiometric assays which require radiolabeled bile acids. The new assay couples the formation of bile acid-CoA to its glycination in a reaction catalyzed by bile acid-CoA:glycine N-acyltransferase. The coupling reaction utilizes [14C]glycine and the bile acid-CoA is quantitatively converted to [14C]glycobile acid. The [14C]glycobile acid is isolated by solvent extraction and quantitated by liquid scintillation counting. The method is shown to be accurate, highly sensitive, and applicable to a wide variety of bile acids.     

The human liver-specific homolog of very long-chain acyl-CoA synthetase is cholate:CoA ligase

Unconjugated bile acids must be activated to their CoA thioesters before conjugation to taurine or glycine can occur. A human homolog of very long-chain acyl-CoA synthetase, hVLCS-H2, has two requisite properties of a bile acid:CoA ligase, liver specificity and an endoplasmic reticulum subcellular localization. We investigated the ability of this enzyme to activate the primary bile acid, cholic acid, to its CoA derivative. When expressed in COS-1 cells, hVLCS-H2 exhibited cholate:CoA ligase (choloyl-CoA synthetase) activity with both non-isotopic and radioactive assays. Other long- and very long-chain acyl-CoA synthetases were incapable of activating cholate. Endogenous choloyl-CoA synthetase activity was also detected in liver-derived HepG2 cells but not in kidney-derived COS-1 cells. Our results are consistent with a role for hVLCS-H2 in the re-activation and re-conjugation of bile acids entering liver from the enterohepatic circulation rather than in de novo bile acid synthesis.