Purification and reconstitution of the bile acid transport system from hepatocyte sinusoidal plasma membranes

The taurocholic acid transport system from hepatocyte sinusoidal plasma membranes has been studied using proteoliposome reconstitution procedures. Membrane proteins were initially solubilized in Triton X-100. Following detergent removal, the resultant proteins were incorporated into lipid vesicles prepared from soybean phospholipids (asolectin) using sonication and freeze-thaw procedures. The resultant proteoliposomes demonstrated Na+-dependent transport of taurocholic acid which could be inhibited by bile acids. Greatly reduced amounts of taurocholic acid were associated with the phospholipid or membrane proteins alone prior to proteoliposome formation. Membrane proteins were fractionated on an anionic glycocholate-Sepharose 4B affinity column which was prepared by coupling (3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholan-24-oyl)-N alpha-lysine to activated CH-Sepharose 4B via the epsilon-amino group of lysine resulting in the retention of a free carboxyl group. The adsorbed proteins enriched in components in the 54 kDa zone, which were originally identified by photoaffinity labeling to be components of the bile acid transport system, were also incorporated into liposomes. This vesicle system showed almost a 4-fold increase in Na+-dependent taurocholic acid uptake when compared to proteoliposomes formed from total membrane protein, as well as sensitivity to inhibition by bile acids. These results demonstrate that the bile acid carrier system can be reconstituted in proteoliposomes and that utilizing proteins in the 54 kDa zone leads to a significant enhancement in the transport capacity of the reconstituted system, consistent with the role of 54 kDa protein(s) as component(s) of the bile acid carrier system.     

牛磺胆酸作用下副溶血弧菌全基因组比较转录谱的表达分析

目的利用DNA芯片技术研究副溶血弧菌对牛磺胆酸刺激反应的全局性基因转录变化概况,找出其中的表达调控变化规律,为副溶血弧菌基因转录调控网络的构建提供实验和理论依据。方法副溶血弧菌分别在正常和添加了50mmol／L牛磺胆酸的培养基中孵育至对数中期,收集菌体,提取RNA,利用全基因组DNA芯片分析比较两者基因转录变化。并应用聚类分析比较其中的变化规律。结果比较转录谱分析证实一共有255个基因的转录表达发生显著性变化,和对照组相比,上调的基因明显占主导优势。而在这些变化的基因中,关于蛋白合成和硫代谢以及谷氨酸合成相关的基因均呈现明显的转录上调变化。结论我们利用DNA芯片技术描绘出了副溶血弧菌在添加牛磺胆酸后全部基因转录水平变化的概图,并发现了蛋白合成,硫代谢和谷氨酸合成相关的基因的变化规律,这给我们下一步的转录调控网络研究提供了良好的靶标。     

Characterization and purification of bile salt hydrolase from Lactobacillus sp. strain 100-100.

We have characterized and purified the bile salt hydrolase from Lactobacillus sp. strain 100-100. Bile salt hydrolase from cells of the strain was purified with column and high-performance liquid chromatography. The activity was assayed in whole cells and cell-free extracts with either a radiochemical assay involving [14C]taurocholic acid or a nonradioactive assay involving trinitrobenzene sulfonate. The activity was detectable only in stationary-phase cells. Within 20 min after conjugated bile acids were added to stationary-phase cultures of strain 100-100, the activity in whole cells increased to levels three- to fivefold higher than in cells from cultures grown in medium free of bile salts. In cell-free extracts, however, the activity was about equal, 1.41 and 1.53 mumol/min per mg of protein, respectively, whether or not the cells have been grown with bile salts present. When supernatant solutions from cultures grown in medium containing taurocholic acid were used to suspend cells grown in medium free of the bile salt, the bile salt hydrolase activity detected in whole cells increased two- to threefold. Two forms of the hydrolase were purified from the cells and designated hydrolases A and B. They eluted from anion-exchange high-performance liquid chromatography in two sets of fractions, A at 0.15 M NaCl and B at 0.18 M NaCl. Their apparent molecular weights in nondenaturing polyacrylamide gel electrophoresis were 115,000 and 105,000, respectively. However, discrepancies existed in the apparent molecular weights and number of peptides detected in sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the two forms. Both had similar substrate specificities, highest on taurodeoxycholic and glycocholic acid, and pH optima between 3.8 and 4.5. The kinetic properties were also similar, with Vmaxs of 17 and 53 micromoles/min per mg of protein and Kms of 0.76 and 0.95 mM taurocholic acid for A and B, respectively. Therefore, whether the enzyme exists in two forms in the cells remains to be determined.     

Influence of dietary bile acids on formation of bile acids in rat

Various taurine-conjugated bile acids were fed to rats at the 1%-level in the diet for 3 or 7 days and the effect on several hydroxylations involved in the biosynthesis and metabolism of bile acids was studied. The hydroxylations studied were all catalyzed by the microsomal fraction of liver homogenate fortified with NADPH. The 7α-hydroxylation of cholesterol was inhibited by feeding taurocholic acid, taurocheno-deoxycholic acid and taurodeoxycholic acid for 3 as well as 7 days. No marked inhibition was obtained with taurohyodeoxycholic acid or taurolithocholic acid. The 12α-hydroxylation of 7α-hydroxy-4-cholesten-3-one was inhibited after 3 as well as 7 days by all bile acids except taurohyodeoxycholic acid. With this acid a marked stimulation of 12α-hydroxylation was observed. The effects of the different bile acids on the 7α-hydroxylation of taurodeoxycholic acid were not very marked. The 6β-hydroxylation of lithocholie acid and taurochenodeoxycholic acid was stimulated by taurocholic acid and taurodeoxycholic acid. The reaction was inhibited by taurochenodeoxycholic acid, at least after 7 days. Taurohyodeoxycholic acid inhibited the 6β-hydroxylation slightly and taurolithocholic acid had no effect. The results were discussed in the light of present knowledge concerning mechanisms of regulation of formation and metabolism of bile acids and it was suggested that the mechanisms may be more complex than previously thought.     

Newly identified bile acid binders in rat liver cytosol. Purification and comparison with glutathione S-transferases

Gel filtration of male rat liver cytosol preincubated with radiolabeled lithocholic, chenodeoxycholic, and glycochenodeoxycholic acids, and taurocholic acid revealed two major peaks of radioactivity, one co-eluting with the glutathione S-transferases and the other with a separate fraction, respectively. Chromatofocusing of the pooled fractions containing the new bile acid binding activity resulted in a separation of bile acid binding from the previously described organic anion binding activity in this fraction. Two binding peaks for lithocholic acid (pI 5.6, Binder I, and pI 5.5, Binder II) were identified on chromatofocusing and were further purified to apparent homogeneity by hydroxyapatite chromatography. The two Binders were monomers having identical molecular weight (33,000) and similar amino acid compositions. Bile acid binding to purified Binders I and II and glutathione S-transferases A, B, and C was studied by inhibition of the fluorescence of bound 1-anilino-8-naphthalenesulfonate (ANS). Confirmatory experiments using equilibrium dialysis produced comparable results. Glutathione S-transferase B had greater affinity for bile acids than transferases A or C. Binder II, which had greater affinity than Binder I for most bile acids, had greater affinity for chenodeoxycholic acid than transferase B but comparable or lower affinities for the other bile acids. All bile acids studied diminished ANS fluorescence with Binder II. Taurocholic and cholic acids increased ANS fluorescence with Binder I without affecting KANS, whereas lithocholic and chenodeoxycholic acids diminished ANS fluorescence with Binder I. In summary, we have identified and isolated two proteins (Binders I and II) which, along with glutathione S-transferase B, are the major hepatic cytosol bile acid binding proteins; these proteins have overlapping but distinct specificities for various bile acids.     

广东眼镜蛇蛇胆的鉴别和质量控制的的研究Ⅲ．眼镜蛇蛇胆的薄层层析

眼镜蛇蛇胆的薄层层析结果表明蛇胆中至少可见到８个显色斑点,其中的特征性成分是牛磺胆酸（Ｒｆ为０．２５９７）。层析结果的重现性和分辨率均高。     

Identification,release and olfactory detection of bile salts in the intestinal fluid of the Senegalese sole (<Emphasis Type="Italic">Solea senegalensis</Emphasis>)

Olfactory sensitivity to bile salts is wide-spread in teleosts; however, which bile salts are released in sufficient quantities to be detected is unclear. The current study identified bile salts in the intestinal and bile fluids of Solea senegalensis by mass spectrometry–liquid chromatography and assessed their olfactory potency by the electro-olfactogram. The main bile salts identified in the bile were taurocholic acid (342 mM) and taurolithocholic acid (271 mM) plus a third, unidentified, bile salt of 532.3 Da. These three were also present in the intestinal fluid (taurocholic acid, 4.13 mM; taurolithocholic acid, 0.4 mM). In sole-conditioned water, only taurocholic acid (0.31 μM) was released in sufficient quantities to be measured (release rate: 24 nmol kg⁻¹ min⁻¹). Sole had high olfactory sensitivity to taurocholic acid but not to taurolithocholic acid. Furthermore, olfactory sensitivity was higher in the upper (right) olfactory epithelium than the lower (left). These two bile acids contribute about 40% of the olfactory potency of intestinal fluid and account for the difference in potency at the two epithelia. Taurocholic acid (but not taurolithocholic acid), and possibly other types of bile acid not tested, could be used as chemical signals and the upper olfactory epithelium is specialised for their detection.     

Convenient Non-Chromatographic Assays for the Microbial Deconjugation and 7α-OH Bioconversion of Taurocholate

We described two convenient assay methods to estimate bile acid deconjugation and bile acid bioconversion at the 7alpha-OH position by individual microorganisms grown in media containing taurocholic acid. The methods are based on (i) a selective chemical assay for taurine conjugates previously described and (ii) the use of a cell-free preparation of 7alpha-hydroxysteroid dehydrogenase from Escherichia coli to directly quantify 7alpha-OH groups. These non-chromatographic approaches have been applied to the study of three model strains of intestinal organisms, E. coli, Bacteroides fragilis, and Clostridium perfringens, grown in standard media in the presence of purified tritiated taurocholate. Assay results were confirmed by thin-layer chromatography solvent systems designed to separate conjugated from unconjugated bile acid and unmodified cholic acid nucleus from 7alpha-OH bioconversion product(s) (primarily 3alpha, 12alpha dihydroxy, 7-keto-cholanoic acid). In addition, 7alpha-hydroxysteroid dehydrogenase activity was demonstrated in cell-free extracts of all three organisms. Of the three organisms, only C. perfringens was demonstrated to (i) deconjugate taurocholic acid, (ii) contain 3alpha-hydroxysteroid dehydrogenase activity, (iii) convert cholic acid into at least five labeled metabolites visible on thin-layer chromatography, and (iv) catalyze significant tritium exchange with water in the medium.     

The effect of dibutyryl cyclic AMP on the excretion of taurocholic acid from isolated rat liver cells

Dibutyryl cyclic AMP (50-1000 microM) was found to increase the initial rate of efflux of taurocholic acid from isolated rat hepatocytes. Efflux of the bile acid was inhibited by sodium, and in the absence of sodium dibutyryl cyclic AMP failed to stimulate the rate. Increasing the concentration of calcium from 0 to 1.2 mM had no effect on the initial rate of taurocholic acid efflux from the cells, but the absence of calcium markedly reduced the effect of dibutyryl cyclic AMP. The results suggest that changes in the fluxes of sodium and calcium are involved in the effect of the cyclic nucleotide on taurocholic acid efflux from the cells.     

Preparative separation and amino acid composition of neurofilament triplet proteins

Abstract: Intact neurofilaments were isolated from bovine spinal cord white matter, washed by sedimentation in 0.1 m -NaCl, and extracted with 8 m -urea. Solubilized neurofilament triplet proteins of molecular weights approximately 68,000 (P68), 150,000 (P150), and 200,000 (P200) were purified by preparative electrophoresis, using an LKB 7900 Uniphor apparatus. The method provides for an enhanced yield of purified protein and has markedly reduced admixture of electrophoresed protein with acrylamide and associated protein contaminants. Amino acid compositions of the purified neurofilament triplet proteins are reported and compared.     

The availability of different sources of cholesterol for bile acid synthesis by cultured chick embryo hepatocytes

The availability of different sources of cholesterol for bile acid synthesis by cultured chick embryo hepatocytes was studied. Mevalonolactone was taken up by the cells and converted to cholesterol, cholesterol ester and tauroconjugates of bile acids. The addition of mevalonolactone had little effect on the conversion of endogenous cholesterol to taurocholic acid; however, taurochenodeoxycholic acid synthesis was stimulated. 25-30% of the cholesterol synthesized from mevalonolactone was converted to taurochenodeoxycholic, taurocholic and two so-far unidentified bile acids. All bile acids were secreted into the incubation medium. When cholesterol was added as mixed liposomes with phosphatidylcholine, it was taken up by the cells and converted to bile acids. At low concentrations of liposomes, the greater part of the cholesterol which was taken up by the cells was converted to bile acids. At higher concentrations, considerable amounts of cholesterol and cholesterol ester accumulated inside the cells. When mevalonolactone and cholesterol liposomes was added together, both substrates were used simultaneously for bile acids synthesis. HDL cholesterol was the best substrate tested, yielding large amounts of two, so-far, unidentified bile acids (possibly allo-bile acids) and smaller amounts of taurocholic and taurochenodeoxycholic acid. Addition of HDL suppressed the conversion of endogenous cholesterol to taurocholic acid; taurochenodeoxycholic acid synthesis, however, was stimulated.     

Internal sequence analysis of proteins eluted from polyacrylamide gels

We have developed an elution-digestion-sequencing (EDS) method, which yields the internal amino acid sequence of partially purified proteins. The overall yield for the method was greater than 60%. The method yielded peptide peaks that could be sequenced on HPLC for all tested proteins with masses from 45 to 200·10³ and yielded internal amino acid sequence information when as little as 10 pmol of partially purified protein was used as the starting material. The EDS method was extremely reliable and gave sequence information for each of 25 proteins tested, including high-molecular-mass proteins (M_r>100·10³) that were difficult to sequence by other methods.     

广东眼镜蛇蛇胆的鉴别及质量控制的研究Ⅳ：广东眼镜蛇蛇胆中牛磺酸、甘氨酸、牛磺胆酸和甘氨胆酸的测定

本文采用日立８３５－５０型氨基酸自动分析仪测定比较了广东眼镜蛇蛇胆及牛、猪、兔、鸡胆中牛磺酸、甘氨酸和其他氨基酸的成分与含量,并间接测定了牛磺胆酸与甘氨胆酸的含量和比值。结果表明广东眼镜蛇蛇胆中牛磺酸（６．６６７ｇ／１００ｇ干重）与牛磺胆酸（２７．４７ｇ／１００ｇ干重）的含量最高,而甘氨酸（０．５９８ｇ／１００ｇ干重）与甘氨胆酸（３．７０ｇ／１００ｇ干重）的含量最低,其他氨基酸的含量甚微。实验结果为蛇胆的真伪鉴别及内在质量控制提供了有实用价值的科学依据,同时也提供了一个准确、灵敏、微量的测定方法。     

Olfactory transduction pathways in the Senegalese sole Solea senegalensis

This study tested whether differences in sensitivity between the upper and lower olfactory epithelia of Solea senegalensis are associated with different odorant receptors and transduction pathways, using the electro‐olfactogram. Receptor mechanisms were assessed by cross‐adaptation with amino acids (L‐cysteine, L‐phenylalanine and 1‐methyl‐L‐tryptophan) and bile acids (taurocholic acid and cholic acid). This suggested that relatively specific receptors exist for 1‐methyl‐L‐tryptophan and L‐phenylalanine (food‐related odorants) in the lower epithelium, and for taurocholic acid (conspecific‐derived odorant) in the upper. Inhibition by U73122 [a phospholipase C (PLC) inhibitor] suggested that olfactory responses to amino acids were mediated mostly, but not entirely, by PLC‐mediated transduction (IC₅₀; 15–55 nM), whereas bile acid responses were mediated by both PLC and adenylate cyclase–cyclic adenosine monophosphate (AC–cAMP) (using SQ‐22536; an AC inhibitor). Simultaneous application of both drugs rarely inhibited responses completely, suggesting possible involvement of non‐PLC and non‐AC mediated mechanisms. For aromatic amino acids and bile acids, there were differences in the contribution of each transduction pathway (PLC, AC and non‐PLC and non‐AC) between the two epithelia. These results suggest that differences in sensitivity of the two epithelia are associated with differences in odorant receptors and transduction mechanisms.     

A new bile acid conjugate, ciliatocholic acid, from bovine gall bladder bile.

This study was carried out to investigate the occurrence of ciliatocholic acid in bovine gall bladder bile. Ciliatocholic acid was synthesized according to the method described by Bergstr?m and Norman for the synthesis of taurocholic acid. Elemental analysis, melting point, and the infrared spectrum of this substance were determined. An isolation procedure for ciliatocholic acid was established by stepwise elution with an HCl-ethanol solvent system using a Dowex-1 anion exchange resin column chromatographic technique. Ciliatocholic acid amounting to 158 mug (as ciliatine) per 100 ml of gall bladder bile was found in the fraction eluted with 0.01 N HCl in 50% ethanol. This coumpound was purified by preparative thin-layer chromatography and confirmed to be ciliatocholic acid from the hydrolytic stability, phosphorus determination, and chromatographic behavior. Thus, bovine gall bladder bile contains a small amount of ciliatocholic acid.     

Partial purification and characterization of NAD-dependent 7alpha-hydroxysteroid dehydrogenase from Bacteroides thetaiotaomicron.

A NAD-dependent 7alpha-hydroxysteroid dehydrogenase was purified 18-fold over the activity in crude cell extracts prepared from Bacteroides thetaiotaomicron NCTC 10852 using Bio-Gel A 1.5-M column chromatography. A molecular weight of 320 000 was estimated for the partially purified intact enzyme. Substrate saturation kinetics were performed using the 18-fold purified enzyme and the lowest Km values were obtained for 3alpha,7alpha-dihydroxy bile acid and bile salt substrates including chenodeoxycholic acid (Km 0.048 mM), glycochenodeoxycholic acid (Km 0.083 mM) and taurochenodeoxycholic acid (Km 0.059 mM). In contrast, 3alpha,7alpha,12alpha-trihydroxy bile acid and bile salts had higher Km values, i.e. cholic acid (Km 0.22 mM), glycoholic acid Km 0.32 mM) and taurocholic acid Km 0.26 mM). NAD had a Km value of 0.20 mM. The possible physiological significance of 7alpha-hydroxy bile acid oxidation to intestinal bacteroides strains was accessed by determining the rate of conversion of [14C]-cholic acid to 7-ketodeoxy[14C]cholic acid by whole cell suspensions under different incubation conditions. The rate of biotransformation of bile acid to keto-bile acid incubated anaerobically under N2 gas increased markedly when potential electron acceptors such as fumarate (10 mM) or menadione (4 mM) was added exogenously. These results suggest that bile acid oxidation reactions may be linked to energy-generating systems in this bacterium.     

Characterization of proteins from arthrodial membranes of the lobster, Homarus americanus

A total of six proteins from the abdominal arthrodial membrane (intersegmental membrane) of the lobster, Homarus americanus, were purified and their amino acid sequences were determined by a combination of mass spectrometry and Edman degradation. The proteins are acidic with pI-values close to 4 and they all have molecular masses ≈12 kDa. The sequences of five of the proteins differ in only a few residues, while the sixth protein differs from the others in more than half of the positions. Only little similarity is observed between the sequences of the arthrodial membrane proteins and those of proteins purified from the calcified parts of the exoskeleton of H. americanus. The arthrodial membrane proteins contain the Rebers-Riddiford consensus sequence common in proteins from insect cuticles. Comparison of the complete sequences to the sequences available in databases shows that the lobster membrane proteins are more closely related to proteins from insect pliant cuticles than to proteins derived from cuticles destined for sclerotization. Characteristic features in the protein sequences are discussed, and it is suggested that the various sequence regions have specific roles in determining the mechanical properties of arthrodial membranes.     

Effects of ethanol in vitro on rat intestinal brush-border membranes

Ethanol, at concentrations found in the intestinal lumen after moderate drinking, has been shown to inhibit carrier-mediated intestinal transport processes. This inhibition could occur by direct interaction with membrane transporters, dissipation of the energy producing Na⁺ electrochemical gradient and/or nonspecific alteration of membrane integrity. The latter alteration may be reflected by changes in membrane fluidity, chemical composition or vesicular size. These possibilities were examined with studies in purified brush border membrane vesicles of rat intestine. Ethanol inhibited concentrative Na⁺-dependent d-glucose uptake in a dose-dependent manner. In contrast, ethanol did not inhibit concentrative d-glucose uptake under conditions of d-glucose trans-stimulation in the absence of a Na⁺ electrochemical gradient. Ethanol also inhibited initial, concentrative Na⁺-dependent taurocholic acid uptake, as well as equilibrium uptake. That ethanol exerted a dual effect on transport by increasing membrane conductance for Na⁺ while decreasing intravesicular space was supported by direct studies of Na⁺ uptake. Morphometric analysis confirmed that ethanol-treated membranes had a decreased intravesicular size when compared to untreated membranes. Finally, membrane fluidity measured by EPR showed that ethanol had a significant fluidizing effect without producing qualitative changes in membrane proteins, as determined by SDS gel electrophoresis. These results suggest that ethanol inhibits carrier-mediated transport by dissipation of the Na⁺ electrochemical gradient and alteration of membrane integrity rather than by direct interaction with membrane transporters.     

Lysis of Halobacteria in Bacto-Peptone by Bile Acids

All tested strains of halophilic archaebacteria of the genera Halobacterium, Haloarcula, Haloferax, and Natronobacterium lysed in 1% Bacto-Peptone (Difco) containing 25% NaCl, whereas no lysis was observed with other strains belonging to archaebacteria of the genera Halococcus, Natronococcus, and Sulfolobus, methanogenic bacteria, and moderately halophilic eubacteria. Substances in Bacto-Peptone which caused lysis of halobacteria were purified and identified as taurocholic acid and glycocholic acid. High-performance liquid chromatography analyses of peptones revealed that Bacto-Peptone contained nine different bile acids, with a total content of 9.53 mg/g, whereas much lower amounts were found in Peptone Bacteriological Technical (Difco) and Oxoid Peptone. Different kinds of peptones can be used to distinguish halophilic eubacteria and archaebacteria in mixed cultures from hypersaline environments.     

American eels produce and release bile acid profiles that vary across life stage

The American eel (Anguilla rostrata) is an imperilled fish hypothesized to use conspecific cues, in part, to coordinate long-distance migration during their multistage life history. Here, holding water and tissue from multiple American eel life stages was collected and analysed for the presence, profile and concentration of bile acids. Distinct bile acid profiles were identified in glass, elver, yellow eel and silver eel holding waters using ultraperformance liquid chromatography high-resolution mass spectrometry and principal component analysis. Taurochenodeoxycholic acid, taurodeoxycholic acid, cholic acid, deoxycholic acid, taurolithocholic acid and taurocholic acid were detected in whole tissue of American glass eels and elvers, and in liver, intestine and gallbladder samples of late-stage yellow eels. Bile acids were not a major component of silver eel washings or tissue. This study is novel because little was previously known about bile acids produced and emitted into the environment by American eels. Future behavioural studies could evaluate whether any bile acids produced by American eels influence conspecific migratory behaviour.