Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:6.2.1.7 (BAL)
 1,977 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Choloyl-CoA synthetase (EC 6.2.1.7) was characterized for the first time under appropriated assay conditions. The p/ optimum for the reaction is pH 7.2.-7.3. The reaction has an absolute requirement for bivalent cation. Several different metal ions fulfil this requirement, but Mn2+ and Mg2+ were the most effective. The KAppm (apparent Km) for CoA, extrapolated from kinetic data, is 50 micronM, but in fact the rate of reaction is increased little by concentrations of CoA above 25 micronM. The KAppm for ATP is 600 micronM. High concentrations of ATP appear to cause substrate inhibition. The KAppm for cholate was 6 micronM. The enzyme was inhibited by treating the microsomal fraction with N-ethylmaleimide. The inclusion of various conjugated and unconjugated bile salts in the assay also inhibited the enzyme. Unconjugated bile salts were more potent inhibitors than the conjugated bile salts. High concentrations of oleic acid inhibited the enzyme. The properties of choloyl-CoA synthetase were not modified by alterations of the properties of the lipid phase of the microsomal membrane. Treatment with phospholipase A did not alter activity directly. Triton N-101 and Triton X-100 also were without effect on activity, and the enzyme was insensitive to temperature-induced phase transitions within the lipid portion of the membrane. The enzyme can be solubilized from the microsomal membrane in an active form by treatment with Triton N-101.
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PMID:Characterization of microsomal choloyl-coenzyme A synthetase. 1 1

The subcellular distribution and characteristics of trihydroxycoprostanoyl-CoA synthetase were studied in rat liver and were compared with those of palmitoyl-CoA synthetase and choloyl-CoA synthetase. Trihydroxycoprostanoyl-CoA synthetase and choloyl-CoA synthetase were localized almost completely in the endoplasmic reticulum. A quantitatively insignificant part of trihydroxycoprostanoyl-CoA synthetase was perhaps present in mitochondria. Peroxisomes, which convert trihydroxycoprostanoyl-CoA into choloyl-CoA, were devoid of trihydroxycoprostanoyl-CoA synthetase. As already known, palmitoyl-CoA synthetase was distributed among mitochondria, peroxisomes and endoplasmic reticulum. Substrate- and cofactor- (ATP, CoASH) dependence of the three synthesis activities were also studied. Cholic acid and trihydroxycoprostanic acid did not inhibit palmitoyl-CoA synthetase; palmitate inhibited the other synthetases non-competitively. Likewise, cholic acid inhibited trihydroxycoprostanic acid activation non-competitively and vice versa. The pH curves of the synthetases did not coincide. Triton X-100 affected the activity of each of the synthetases differently. Trihydroxycoprostanoyl-CoA synthetase was less sensitive towards inhibition by pyrophosphate than choloyl-CoA synthetase. The synthetases could not be solubilized from microsomal membranes by treatment with 1 M-NaCl, but could be solubilized with Triton X-100 or Triton X-100 plus NaCl. The detergent-solubilized trihydroxycoprostanoyl-CoA synthetase could be separated from the solubilized choloyl-CoA synthetase and palmitoyl-CoA synthetase by affinity chromatograpy on Sepharose to which trihydroxycoprostanic acid was bound. Choloyl-CoA synthetase and trihydroxycoprostanoyl-CoA synthetase could not be detected in homogenates from kidney or intestinal mucosa. The results indicate that long-chain fatty acids, cholic acid and trihydroxycoprostanic acid are activated by three separate enzymes.
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PMID:Subcellular distribution and characteristics of trihydroxycoprostanoyl-CoA synthetase in rat liver. 252 99

Liver peroxisomes from both rat and humans have previously been shown to contain enzymes that catalyze the oxidative cleavage of the C27-steroid side chain in the formation of bile acids. It has not been clear, however, whether the initial step, formation of the CoA-esters of the 5 beta-cholestanoic acids, also occurs in these organelles. In the present work the subcellular localization of 3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholestanoyl-CoA (THCA-CoA) ligase (THCA-CoA synthetase) and of 3 alpha,7 alpha-dihydroxy-5 beta-cholestanoyl-CoA (DHCA-CoA) ligase in rat liver has been investigated. Main subcellular fractions and peroxisome-rich density gradient fractions from rat liver were incubated with THCA or DHCA, CoA, ATP, and Mg2+. Formation of THCA-CoA and DHCA-CoA was determined after high pressure liquid chromatography of the incubation extracts. The microsomal fraction contained the highest specific (and also relative specific) activity both for the formation of THCA-CoA and DHCA-CoA. The rates of THCA-CoA formation were further increased from 124-159 nmol/mg.hr-1 in crude microsomal fractions to 184-220 nmol/mg.hr-1 when studied in purified rough endoplasmic reticulum fractions. Formation of THCA-CoA in peroxisomal fractions prepared in Nycodenz density gradients could be accounted for by a small contamination (3-7%) by microsomal protein. The distribution of THCA-CoA ligase was different from that of palmitoyl-CoA ligase that was found to be localized also to the peroxisomal fractions.
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PMID:Subcellular localization of 3 alpha, 7 alpha-dihydroxy- and 3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholestanoyl-coenzyme A ligase(s) in rat liver. 318 23

1. The subcellular location of enzymes conjugating bile acids with glycine or taurine was investigated by centrifugation of rat liver homogenates. 2. [14C]Cholic acid-conjugating activity was predominantly associated with the soluble-microsomal region of the gradient after centrifugation in a Ti-15 zonal rotor but the bulk of the conjugating activity sedimented with mitochondrial-lysosomal fractions in differential pelleting experiments. 3. Cholate: CoA ligase (EC 6.2.1.7) and cholyltransferase (EC 2.3.1) were not enriched in purified Golgi or plasma-membrane fractions. Cholate: CoA ligase was distributed evenly between rough- and smooth-surfaced microsomal subfractions but cholyltransferase showed a dual soluble-rough microsomal activity distribution. 4. Sedimentation of cholyltransferase in mitochondria-enriched fractions prepared by differential centrifugation appears to be an artefact of sedimentation of rough microsomal membranes in mitochondrial fractions. 5. The subcellular distribution of bile acid-conjugating enzymes is discussed with reference to hepatic processing of bile acids.
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PMID:Subcellular distribution of hepatic bile acid-conjugating enzymes. 617 37

Cholic acid:CoA ligase (EC 6.2.1.7, choloyl-CoA synthetase) and deoxycholic acid:CoA ligase catalyze the synthesis of choloyl-CoA and deoxycholoyl-CoA from their respective bile acids in rat liver. A modification of the phase partition assay was introduced which yields significantly (3-fold) higher specific activities for cholic acid:CoA ligase than previously reported. An independent method of separating choloyl-CoA from the substrates by high-pressure liquid chromatography was also developed and validates the modification. Both enzymic activities were found to be localized predominantly in the endoplasmic reticulum of rat liver. The level of either ligase in other purified, active subcellular fractions is consistent with the level of contamination by endoplasmic reticulum, estimated by using marker enzymes. Hence, the ligase assay can be used as a sensitive enzymic marker for endoplasmic reticulum in rat liver. The kinetic parameters of both enzymic activities were determined by using purified rough endoplasmic reticulum from rat liver. While the apparent maximal velocities for the two substrates are similar, the Michaelis constant for deoxycholate is significantly lower than that for cholate. Taurocholate and deoxycholate are shown to be competitive inhibitors of cholic acid:CoA ligase. The inhibition constant of deoxycholate is similar to its Michaelis constant for the deoxycholoyl-CoA-synthesizing reaction, suggesting that the same enzyme is responsible for both ligase activities.
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PMID:Subcellular distribution of cholic acid:coenzyme a ligase and deoxycholic acid:Coenzyme a ligase activities in rat liver. 663 41

We have examined the ability of HepG2 human hepatoblastoma cells and 7800 C1 Morris rat hepatoma cells to convert 3 alpha, 7 alpha, 12 alpha-trihydroxy-5 beta-cholestanoic acid (THCA) and 3 alpha, 7 alpha-dihydroxy-5 beta-cholestanoic acid (DHCA) to cholic acid and chenodeoxycholic acid, respectively. Cell extracts from both these cell lines could neither form cholic acid from THCA nor from the activated form, THCA-CoA. This suggests that both cell lines are defective in two enzyme activities involved in the pathway, the microsomal THCA-CoA ligase and the peroxisomal THCA-CoA oxidase. Furthermore, we show that the subsequent enzymes are active in the conversion to bile acids, because the product of the THCA-CoA oxidase, 3 alpha, 7 alpha, 12 alpha-trihydroxy-5 beta-cholest-24-enoyl-coenzyme A (delta 24-THCA-CoA) or delta 24-THCA in the presence of THCA-CoA ligase, are converted to cholic acid by both cell lines. HepG2 cells were able to slowly form chenodeoxycholic acid and cholic acid from 5 beta-cholestane-3 alpha, 7 alpha-diol and 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha-triol, respectively, in 24- and 96-h incubations. The rate of cholic acid formation was lower than the rate for chenodeoxycholic acid and there was a clear accumulation of THCA. 7800 C1 Morris cells had no ability to form cholic acid or chenodeoxycholic acid after 96 h incubation. We conclude that these two cell lines have defects in two enzyme activities involved in the peroxisomal oxidation in bile acid formation, the microsomal THCA-CoA ligase and the peroxisomal THCA-CoA oxidase.
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PMID:Human hepatoblastoma cells (HepG2) and rat hepatoma cells are defective in important enzyme activities in the oxidation of the C27 steroid side chain in bile acid formation. 830 Dec 25

The stability of rat hepatic trihydroxycoprostanoyl-CoA syntethase was studied in its native membrane environment and after solubilisation by Triton X-100, and compared to that of choloyl-CoA synthetase. The lability of both delipidated enzymes could be suppressed by high concentrations of polyols such as sucrose and glucose. Addition of phospholipids to the assay mixtures was necessary to restore the activity of the stabilized enzymes. For further chromatographic separations, the addition of the hydrotrope Triton H-66 to the glucose-stabilized Triton X-100 solubilised synthetases improved their recovery on different matrices. Gel filtration revealed a native molecular mass of the Triton X-100/Triton H-66/protein micelles of 212 and 207 kDa for choloyl-CoA synthetase and trihydroxycoprostanoyl-CoA synthetase respectively.
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PMID:Stabilisation and partial purification of Triton X-100 solubilised trihydroxycoprostanoyl-CoA synthetase from rat liver. 890 53

In the present study, using the C24 bile acid chenodeoxycholic acid as substrate, rat liver bile acid CoA ligase activity (rBAL) was purified 200-fold from detergent-solubilized microsomes using a combination of Q-Sepharose anion exchange, hydroxyapatite, and CM-Sepharose chromatography. Purified rBAL had a molecular weight of 65 kDa by SDS-PAGE analysis. Gel filtration of purified rBAL indicated that rBAL activity forms a complex with other proteins with an apparent aggregate molecular weight of 243 kDa. A monoclonal antibody raised against the 65-kDa protein and covalently coupled to 6B-Sepharose completely absorbed rBAL activity from a semipurified preparation of rat liver microsomes. Western blot analysis confirmed the elution of the 65-kDa protein from the affinity phase at low pH. Optimum rBAL activity was found at pH 8.5, and activity was dependent on the divalent cation Mg2+. In the presence of 50 microM CoA and 2.5 mM MgCl2, kinetic analysis revealed that the apparent K(m)s of ATP and chenodeoxycholic acid of the purified enzyme were 548 +/- 247 and 18.0 +/- 6.2 microM, respectively, and the apparent Vmax was 9.53 +/- 2.0 nmol min-1 mg protein-1. The formation of chenodeoxycholyl-CoA by rBAL was strongly inhibited by hydrophobic bile acids (the C24 monohydroxy bile acid lithocholic acid and 3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholestanoic acid, the C27 homolog of cholic acid), but only weakly by cholic acid. Chenodeoxycholyl-CoA and 3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholestan-27-oyl-CoA were confirmed as reaction products of purified rBAL by HPLC-electrospray ionization mass spectrometry.
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PMID:Purification and characterization of a rat liver bile acid coenzyme A ligase from rat liver microsomes. 939 Jan 70

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.
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PMID:The human liver-specific homolog of very long-chain acyl-CoA synthetase is cholate:CoA ligase. 1074 48

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 lambdaZAP 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.
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PMID:Rat liver bile acid CoA:amino acid N-acyltransferase: expression, characterization, and peroxisomal localization. 1295 68


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