Human liver steroid sulphotransferase sulphates bile acids.

The sulphation of bile acids is an important pathway for the detoxification and elimination of bile acids during cholestatic liver disease. A dehydroepiandrosterone (DHEA) sulphotransferase has been purified from male and female human liver cytosol using DEAE-Sepharose CL-6B and adenosine 3',5'-diphosphate-agarose affinity chromatography [Falany, Vazquez & Kalb (1989) Biochem. J. 260, 641-646]. Results in the present paper show that the DHEA sulphotransferase, purified to homogeneity, is also reactive towards bile acids, including lithocholic acid and 6-hydroxylated bile acids, as well as 3-hydroxylated short-chain bile acids. The highest activity towards bile acids was observed with lithocholic acid (54.3 +/- 3.6 nmol/min per mg of protein); of the substrates tested, the lowest activity was detected with hyodeoxycholic acid (4.2 +/- 0.01 nmol/min per mg of protein). The apparent Km values for the enzyme are 1.5 +/- 0.31 microM for lithocholic acid and 4.2 +/- 0.73 microM for taurolithocholic acid. Lithocholic acid also competitively inhibits DHEA sulphation by the purified sulphotransferase (Ki 1.4 microM). No evidence was found for the formation of bile acid sulphates by sulphotransferases different from the DHEA sulphotransferase during purification work. The above results suggest that a single steroid sulphotransferase with broad specificity encompassing neutral steroids and bile acids exists in human liver.     

6 alpha-glucuronidation of hyodeoxycholic acid by human liver, kidney and small bowel microsomes

Kinetic constants for the glucuronidation of hyodeoxycholic acid in man were determined using microsomal preparations of liver, kidney and small bowel. The affinity of hyodeoxycholic acid for the microsomal hepatic and extrahepatic enzymes was in the same range as previously observed for the monohydroxy bile acid lithocholic acid and about 3-14-times the affinity for the dihydroxy bile acids chenodeoxycholic, deoxycholic and ursodeoxycholic acids. The Vmax values for glucuronidation of hyodeoxycholic acid with hepatic microsomes were 10-30-times higher and with kidney microsomes 50-110-times higher than for the bile acids lacking a 6 alpha-hydroxy group. The site of glucuronidation was determined by gas chromatographic-mass spectrometric analysis of derivatives of products formed after periodate and chromic acid oxidation. Hyodeoxycholic acid glucuronides synthesized with microsomal preparations from the three organs were all found to be conjugated at the 6 alpha position. This has previously been shown to be the site of glucuronidation of endogenous hyodeoxycholic acid glucuronide excreted in urine.     

A rapid and sensitive method for the assay of UDP-glucuronosyltransferase activity toward bile acids

A rapid and sensitive procedure is described for the assay of rat liver microsomal UDP-glucuronosyltransferase activity toward the bile acids chenodeoxycholic acid, deoxycholic acid, ursodeoxycholic acid, and lithocholic acid using the radioactively labeled bile acids as substrates. The unreacted bile acids were separated from the bile acid glucuronides formed as products of the enzymatic reactions by extraction with chloroform, leaving the bile acid glucuronides in the aqueous phases. The bile acid glucuronides were characterized by their mobilities in thin-layer chromatography and identified by their sensitivity to hydrolysis with β-glucuronidase and inhibition of hydrolysis by the specific β-glucuronidase inhibitor d-saccharic acid-1,4-lactone. Enzyme activities were optimal at pH 6.8 and were maximally stimulated about fourfold by the addition of the nonionic detergent Brij 58 at a concentration of 0.3 mg/mg microsomal protein. The kinetic parameters for the various bile acids as substrates were determined.     

Formation of hyodeoxycholic acid from muricholic acid and hyocholic acid by an unidentified gram-positive rod termed HDCA-1 isolated from rat intestinal microflora.

From the rat intestinal microflora we isolated a gram-positive rod, termed HDCA-1, that is a member of a not previously described genomic species and that is able to transform the 3alpha,6beta, 7beta-trihydroxy bile acid beta-muricholic acid into hyodeoxycholic acid (3alpha,6alpha-dihydroxy acid) by dehydroxylation of the 7beta-hydroxy group and epimerization of the 6beta-hydroxy group into a 6alpha-hydroxy group. Other bile acids that were also transformed into hyodeoxycholic acid were hyocholic acid (3alpha, 6alpha,7alpha-trihydroxy acid), alpha-muricholic acid (3alpha,6beta, 7alpha-trihydroxy acid), and omega-muricholic acid (3alpha,6alpha, 7beta-trihydroxy acid). The strain HDCA-1 could not be grown unless a nonconjugated 7-hydroxylated bile acid and an unidentified growth factor produced by a Ruminococcus productus strain that was also isolated from the intestinal microflora were added to the culture medium. Germfree rats selectively associated with the strain HDCA-1 plus a bile acid-deconjugating strain and the growth factor-producing R. productus strain converted beta-muricholic acid almost completely into hyodeoxycholic acid.     

Purification and characterization of bile salt sulfotransferase from human liver

Bile salt sulfotransferase, the enzyme responsible for the formation of bile salt sulfate esters, was purified extensively from normal human liver. The purification procedure included DEAE-Sephadex chromatography, taurocholate-agarose affinity chromatography, and preparative isoelectrofocusing. The final preparation had a specific activity of 18 nmol min-1 mg protein-1, representing a 760-fold purification from the cytosol fraction with a overall yield of 15%. The human enzyme has a Mr of 67,000 and a pI of 5.2. DEAE-Sephadex chromatography of the cytosol fraction revealed only a single species of activity. The limiting Km for the sulfuryl donor, 3'-phosphoadenosine-5'-phosphosulfate (PAPS), is 0.7 microM. The limiting Km for the sulfuryl acceptor, glycolithocholate (GLC), is 2 microM. Reciprocal plots were intersecting. Product inhibition studies established that adenosine 3',5'-diphosphate (PAP) was competitive with PAPS (Ki = 0.2 microM) and noncompetitive with respect to GLC. GLC sulfate was competitive with GLC (Ki = 2.2 microM) and noncompetitive with respect to PAPS. Also, 3-ketolithocholate, a dead-end inhibitor, was competitive with GLC (Ki = 0.6 microM) and noncompetitive with respect to PAPS. Iso-PAP (the 2' isomer of PAP) was competitive with PAPS (Ki = 0.3 microM) and noncompetitive with GLC. The cumulative results of the steady-state kinetics experiments point to a random mechanism for the binding of substrates and release of products. The purified enzyme displays no activity toward estrone, testosterone, or phenol. Among the reactive substrates tested, the Vmax/Km values are in the order GLC greater than 3-beta OH-5-cholenic acid greater than glycochenodeoxycholate greater than glycocholate. p-Chloromercuribenzoate inactivated the enzyme. Either PAPS or GLC protected against inactivation, suggesting the presence of a sulfhydryl group at the active site.     

Human cecal bile acids: concentration and spectrum

To obtain information on the concentration and spectrum of bile acids in human cecal content, samples were obtained from 19 persons who had died an unnatural death from causes such as trauma, homicide, suicide, or drug overdose. Bile acid concentration was measured via an enzymatic assay for 3alpha-hydroxy bile acids; bile acid classes were determined by electrospray ionization mass spectrometry and individual bile acids by gas chromatography mass spectrometry and liquid chromatography mass spectrometry. The 3alpha-hydroxy bile acid concentration (mumol bile acid/ml cecal content) was 0.4 +/- 0.2 mM (mean +/- SD); the total 3-hydroxy bile acid concentration was 0.6 +/- 0.3 mM. The aqueous concentration of bile acids (supernatant after centrifugation) was identical, indicating that most bile acids were in solution. By liquid chromatography mass spectrometry, bile acids were mostly in unconjugated form (90 +/- 9%, mean +/- SD); sulfated, nonamidated bile acids were 7 +/- 5%, and nonsulfated amidated bile acids (glycine or taurine conjugates) were 3 +/- 7%. By gas chromatography mass spectrometry, 10 bile acids were identified: deoxycholic (34 +/- 16%), lithocholic (26 +/- 10%), and ursodeoxycholic (6 +/- 9), as well as their primary bile acid precursors cholic (6 +/- 9%) and chenodeoxycholic acid (7 +/- 8%). In addition, 3beta-hydroxy derivatives of some or all of these bile acids were present and averaged 27 +/- 18% of total bile acids, indicating that 3beta-hydroxy bile acids are normal constituents of cecal content. In the human cecum, deconjugation and dehydroxylation of bile acids are nearly complete, resulting in most bile acids being in unconjugated form at submicellar and subsecretory concentrations.     

Characterization of NADP-dependent 7 beta-hydroxysteroid dehydrogenases from Peptostreptococcus productus and Eubacterium aerofaciens.

Peptostreptococcus productus strain b-52 (a human fecal isolate) and Eubacterium aerofaciens ATCC 25986 were found to contain NADP-dependent 7 beta-hydroxysteriod dehydrogenase activity. The enzyme was synthesized constitutively by both organisms, and the enzyme yields were suppressed by the addition of 0.5 mM 7 beta-hydroxy bile acid to the growth medium. Purification of the enzyme by chromatography resulted in preparations with 3.5 (P. productus b-52, on Sephadex G-200) and 1.8 (E. aerofaciens, on Bio-Gel A-1.5 M) times the activity of the crude cell extracts. A pH optimum of 9.8 and a molecular weight of approximately 53,000 were shown for the enzyme of strain b-52, and an optimum pH at 10.5 and a molecular weight of 45,000 was shown for that from strain ATCC 25986. Kinetic studies revealed that both enzyme preparations oxidized the 7 beta-hydroxy group in unconjugated and conjugated bile acids, a lower Km value being demonstrated with free bile acid than with glycine and taurine conjugates. No measureable activity against 3 alpha-, 7 alpha-, or 12 alpha-hydroxy groups was detected in either enzyme preparation. When tested with strain ATCC 25986, little 7 beta-hydroxy-steroid dehydrogenase activity was detected in cells grown in the presence of glucose in excess. The enzyme from strain b-52 was found to be heat labile (90% inactivation at 50 degrees C for 3 min) and highly sensitive to sulfhydryl inhibitors.     

Purification and properties of S-adenosyl-L-methionine:nicotinic acid-N-methyltransferase from cell suspension cultures of Glycine max L

A soluble enzyme which catalyzes the transfer of the methyl group from S-adenosyl-L-methionine to the nitrogen atom of pyridine-3-carboxylic acid (nicotinic acid) could be detected in protein preparations from heterotrophic cell suspension cultures of soybean (Glycine max L.). Enzyme activity was enriched nearly 100-fold by ammonium sulfate precipitation, gel filtration, and ion-exchange chromatography to study kinetic properties. S-adenosyl-L-methionine:nicotinic acid-N-methyltransferase (EC 2.1.1.7) showed a pH optimum at pH 8.0 and a temperature optimum between 35 and 40 degrees C. The apparent KM values were determined to be 78 microM for nicotinic acid and 55 microM for the cosubstrate. S-Adenosyl-L-homocysteine was a competitive inhibitor of the methyltransferase with a KI value of 95 microM. The native enzyme had a molecular mass of about 90 kDa. The catalytic activity was inhibited by reagents blocking SH groups, whereas other divalent cations did not significantly influence of the enzyme reaction. The purified methyltransferase revealed a remarkable specificity for nicotinic acid. No other pyridine derivative was a suitable methyl group acceptor. To study a potential methyltransferase activity with nicotinamide as substrate, an additional purification step was necessary to remove nicotinamide amidohydrolase activity from the enzyme preparation. This was achieved by affinity chromatography on S-adenosyl-L-homocysteine-Sepharose thus leading to a 580-fold purified enzyme which showed no methyltransferase activity toward nicotinamide as substrate.     

Suitability of primary monolayer cultures of adult rat hepatocytes for studies of cholesterol and bile acid metabolism

Monolayer cultures of hepatocytes isolated from cholestyramine-fed rats and incubated in serum-free medium converted exogenous [4-14C]cholesterol into bile acids at a 3-fold greater rate than did cultures of hepatocytes prepared from untreated rats. Cholic acid and beta-muricholic acid identified and quantitated by gas-liquid chromatography and thin-layer chromatography were synthesized by cultured cells for at least 96 h following plating. The calculated synthesis rate of total bile acids by hepatocytes prepared from cholestyramine-fed animals was approximately 0.058 micrograms/mg protein/h. beta-Muricholic acid was synthesized at approximately a 3-fold greater rate than cholic acid in these cultures. Cultured hepatocytes rapidly converted the following intermediates of the bile acid pathway; 7 alpha-hydroxy[7 beta-3H]cholesterol, 7 alpha-hydroxy-4-[6 beta-3H] cholesten-3-one, and 5 beta-[7 beta-3H]cholestane-3 alpha, 7 alpha, 12 alpha-triol into bile acids. [24-14C]Chenodeoxycholic acid and [3H]ursodeoxycholic acid were rapidly biotransformed to beta-muricholic acid. 3-Hydroxy-3-methylglutaryl-coenzyme A reductase activity measured in microsomes of cultured hepatocytes decreased during the initial 48 h following plating, but remained relatively constant for the next 72 h. In contrast, cholesterol 7 alpha-hydroxylase activity appeared to decrease during the first 48 h, followed by an increase over the next 48 h. Despite the apparent changes in enzyme activity in vitro, the rate of bile acid synthesis by whole cells during this time period remained constant. It is concluded that primary monolayer cultures of rat hepatocytes can serve as a useful model for studying the interrelationship between cholesterol and bile acid metabolism.     

Novel inhibitors and substrates of bilirubin: UDP-glucuronosyltransferase. Arylalkylcarboxylic acids

The in vitro inhibitory potency of 20 structurally related alkanoic and arylalkanoic acids has been investigated on rat liver UDP-glucuronosyltransferase. These compounds were tested on the microsomal and purified enzyme, and a cloned cDNA expressed in COS 7 cell cultures. Among all the acids tested, 7,7,7-triphenylheptanoic acid was the most powerful inhibitor of bilirubin:UDP-glucuronosyltransferase with a lower effect on 1-naphtol, androsterone and testosterone glucuronidation. The inhibition was competitive towards the microsomal and purified bilirubin:UDP-glucuronosyltransferases with Kiapp values of 12.0 microM and 1.6 microM, respectively. Twenty analogues were examined, and the results showed that their inhibitory potency on bilirubin:UDP-glucuronosyltransferase activity was a function of at least three structural features (a) the presence of a hydrophobic triphenyl moiety; (b) the length of the aliphatic chain and (c) the presence of a carboxylic group. These inhibitors were also tested as possible substrates of UDP-glucuronosyltransferases. The strongest inhibitors were poor substrates of rat liver microsomal UDP-glucuronosyltransferases. However, 7,7,7-triphenylheptanoic acid was actively glucuronidated by purified bilirubin:UDP-glucuronosyltransferase, in contrast to its analogues with decreasing alkyl chain length. In addition, glucuronidation of this molecule was enhanced by clofibrate treatment but could not be detected in Gunn rats, which are deficient in bilirubin:UDP-glucuronosyltransferase, further indicating that the glucuronidation of this compound was catalysed by bilirubin:UDP-glucuronosyltransferase. The results suggest that 7,7,7-triphenylheptanoic acid may be a useful structural probe to investigate the molecular basis of glucuronidation of bilirubin and carboxylic acids.     

Bile acid: CoASH ligases from guinea pig and porcine liver microsomes. Purification and characterization

A procedure for the purification of the enzyme bile acid:CoA ligase from guinea pig liver microsomes was developed. Activity toward chenodeoxycholate, cholate, deoxycholate, and lithocholate co-purified suggesting that a single enzyme form catalyzes the activation of all four bile acids. Activity toward lithocholate could not be accurately assayed during the earlier stages of purification due to a protein which interfered with the assay. The purified ligase had a specific activity that was 333-fold enriched relative to the microsomal cell fraction. The purification procedure successfully removed several enzymes that could potentially interfere with assay procedures for ligase activity, i.e. ATPase, AMPase, inorganic pyrophosphatase, and bile acid-CoA thiolase. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis the purified ligase gave a single band of approximately 63,000 Mr. A molecular size of 116,000 +/- 4,000 daltons was obtained by radiation inactivation analysis of the ligase in its native microsomal environment, suggesting that the functional unit of the ligase is a dimer. The purified enzyme was extensively delipidated by adsorption to alumina. The delipidated enzyme was extremely unstable but could be partially stabilized by the addition of phospholipid vesicles or detergent. However, such additions did not enhance enzymatic activity. Kinetic analysis revealed that chenodeoxycholate, cholate, deoxycholate, and lithocholate were all relatively good substrates for the purified enzyme. The trihydroxy bile acid cholate was the least efficient substrate due to its relatively low affinity for the enzyme. Bile acid:CoA ligase could also be solubilized from porcine liver microsomes and purified 180-fold by a modification of the above procedure. The final preparation contains three polypeptides as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The three peptides range in size from 50,000 to 59,000, somewhat smaller than the guinea pig enzyme. The functional size of the porcine enzyme in its native microsomal environment was determined by the technique of radiation inactivation analysis to be 108,000 +/- 5,000 daltons. Thus, the functional form of the porcine enzyme also appears to be a dimer.     

Metabolism of two hydroxy-7-oxocholanic acids in isolated perfused rat liver

The metabolism of 7-oxolithocholic acid and 7-oxodeoxycholic acid in isolated perfused rat livers was compared. The metabolites extracted from the bile of perfused livers were analysed by gas chromatography. The amount of bile acids excreted in bile was greater after infusion with 7-oxolithocholic acid than with 7-oxodeoxycholic acid. When 7-oxolithocholic acid was infused almost all of the bile acids excreted in bile were taurine conjugates; with 7-oxodeoxycholic acid about 10 percent remained unconjugated. 7-Oxolithocholic acid was more susceptible to reduction than 7-oxodeoxycholic acid. 7-Oxolithocholic acid was preferably reduced to 7 beta-hydroxy rather than to 7 alpha-hydroxy metabolites. In contrast, 7-oxodeoxycholic acid was reduced predominantly to the 7 alpha-hydroxy rather than to the 7 beta-hydroxy metabolite.     

Effect of indomethacin on the uptake, metabolism and excretion of 3-oxocholic acid: studies in isolated hepatocytes and perfused rat liver

3 alpha-Hydroxysteroid dehydrogenase catalyzes the reduction of 3-oxo-bile acids and binds 3 alpha-hydroxy bile acids. Indomethacin is a competitive inhibitor of the enzyme. In incubations of isolated rat hepatocytes, indomethacin delayed the intracellular reduction and the initial uptake of 3-oxocholic acid. Following a tracer dose of 3-oxocholic acid in perfused rat liver, rapid biliary excretion was observed mainly as taurocholic acid. Only 1.1% of the dose was recovered in the caval outflow and nearly all appeared in the first 5 min collection. When the tracer dose was given after initiating a constant infusion of indomethacin (50 microM), a dramatic decrease in biliary excretion was observed, still mainly as taurocholic acid, and 14% of the dose was recovered in the caval effluent: 10% in the first 5 min collection, mainly as 3-oxocholic acid, followed by a steady, slow release of mainly taurocholic acid. The increased intrahepatic retention of bile acids and slow release into perfusate and bile in response to indomethacin are consistent with displacement of bile acids from cytosolic protein.     

Expression of a human liver cDNA encoding a UDP-glucuronosyltransferase catalysing the glucuronidation of hyodeoxycholic acid in cell culture

A cDNA encoding a human liver UDPGT (HLUG 25) transcribed and translated in vitro showed that the encoded protein was synthesized as a precursor and was cleaved and glycosylated when dog pancreatic microsomes were present during translation. The UDPGT cDNA was transiently expressed in mammalian cell culture (COS-7 cells) resulting in the biosynthesis of a polypeptide of 52 kDa. This expressed UDPGT glycoprotein catalysed the glucuronidation of hyodeoxycholic acid forming an ether glucuronide. These results suggest that this UDPGT isoenzyme may be responsible for the glucuronidation of 6 alpha-hydroxy bile acids in human liver.     

Biosynthesis of hydroxyl-linked glucuronides of short-chain bile acids by rat liver 3-hydroxysteroid UDP-glucuronosyltransferase

Microsomal preparations from livers of Sprague-Dawley rats catalyze the glucuronidation of 3 alpha-hydroxy-5 beta-H (3 alpha, 5 beta) short-chain bile acids (C20-C23), predominantly at the hydroxyl group, while the glucuronidation of 3 beta, 5 beta short-chain bile acids occurs exclusively at the carboxyl group. A similar pattern of conjugation was also observed in Wistar rats having normal levels of 3-hydroxysteroid UDP-glucuronosyltransferase. Significant reductions of formation rates for hydroxyl-linked, but not carboxyl-linked, short-chain bile acid glucuronides were observed in hepatic microsomes from Wistar rats with low 3-hydroxysteroid UDP-glucuronosyltransferase activity. 3-Hydroxysteroid UDP-glucuronosyltransferase, purified to homogeneity from Sprague-Dawley liver microsomes, catalyzed the 3-O-glucuronidation of 3 alpha, 5 beta C20-23 bile acids, as well as of lithocholic and isolithocholic acids (C24). The apparent Michaelis constants (KM) for short-chain bile acids were similar to the value obtained for androsterone. 3 alpha, 5 beta-C20 and 3 beta, 5 beta-C20 competitively inhibited glucuronidation of androsterone by the purified 3-hydroxysteroid UDP-glucuronosyltransferase. Purified 17 beta-hydroxysteroid and p-nitrophenol UDP-glucuronosyltransferases did not catalyze the glucuronidation of bile acids. In addition, none of the purified transferases catalyzed the formation of carboxyl-linked bile acid glucuronides. The results show that 3-hydroxysteroid UDP-glucuronosyltransferase, an enzyme specific for 3-hydroxyl groups of androgenic steroids and some conventional bile acids, also catalyzes the glucuronidation of 3 alpha-hydroxyl (but not carboxyl) groups of 3 alpha, 5 beta short-chain bile acids.     

Strain differences in purified rat hepatic 3 alpha-hydroxysteroid UDP-glucuronosyltransferase.

Qualitative and quantitative differences of purified hepatic 3 alpha-hydroxysteroid UDP-glucuronosyltransferase were investigated in Wistar and Sprague-Dawley rats. Individual differences in the glucuronidation rate of androsterone and chenodeoxycholic acid were observed in hepatic microsomal fractions from Wistar but not Sprague-Dawley rats. No individual variation was observed in the glucuronidation of testosterone, p-nitrophenol or oestrone. The 3 alpha-hydroxysteroid UDP-glucuronosyltransferases from livers of Wistar and Sprague-Dawley rats were isolated and highly purified by using Chromatofocusing and affinity chromatography. The amount of 3 alpha-hydroxysteroid UDP-glucuronosyltransferase in the liver of Wistar rats exhibiting low rates for androsterone glucuronidation is about 10% or less than that found in hepatic microsomal fractions obtained from Wistar rats having high rates for androsterone glucuronidation. The apparent Km for androsterone with purified 3 alpha-hydroxysteroid UDP-glucuronosyltransferase from Wistar rats with high glucuronidation activity (6 microM) was not different from that observed for the enzyme purified from Sprague-Dawley animals, whereas that for the enzyme purified from Wistar rats with low glucuronidation activity was substantially higher (120 microM). Despite the differences in apparent Km values for androsterone, the apparent Km for UDP-glucuronic acid (0.3 mM) was not different in the different populations of rats.     

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.     

Lithocholate binding by Y and Y' proteins in bovine small intestine.

Cytosolic proteins may play an important role in the intracellular transport of bile acids in enterocytes. The lithocholate binding properties of cytosolic protein from bovine small intestine were studied. Lithocholate binding was observed in the Y (45-50 kDa), Y' (30-35 kDa), and Z fractions (10-15 kDa) following gel filtration of cytosol. A Y protein with glutathione S-transferase activity (46 kDa) was purified by S-octyl-glutathione affinity chromatography and chromatofocusing (eluted at pH 7.5) of the Y fraction. Two Y' bile acid binding proteins with dihydrodiol dehydrogenase activity were partially purified from the Y' fraction by chromatofocusing and hydroxyapatite-HPLC. The lithocholate binding affinity of Y' protein (Kd < 0.35 microM) was higher than that of Y protein (Kd = 2 microM) and was comparable to that of Z protein (Kd = 0.2 microM). The binding affinity of Y protein was higher for bilirubin (Kd = 2.5 microM) than that for BSP (Kd = 200 microM). This was comparable to the binding affinity of bovine hepatic Y protein. These data indicate that Y' and Z proteins participate in the intracellular transport of bile acids from the brush border to the basolateral pole in enterocytes.     

Hyodeoxycholate-6-O-glucuronide cannot be quantitated with 3 alpha-hydroxysteroid dehydrogenase

The reaction of the 6-hydroxylated bile acid, hyodeoxycholic acid, and its 6-O-glucuronide conjugate with 3 alpha-hydroxysteroid dehydrogenase was examined. A standard end-point assay and determination of the initial rates of reaction showed only minimal activity of the enzyme toward hyodeoxycholate-6-glucuronide in spite of the presence of a free 3 alpha-hydroxyl group. It was established that 6-hydroxylation itself did not significantly affect the enzyme reaction. It is concluded that the 6-glucuronide either blocks or hinders enzyme access to the 3-hydroxyl group.     

7 alpha-Dehydroxylation of bile acids by resting cells of an unidentified, gram-positive, nonsporeforming anaerobic bacterium.

Transformation of bile acids by washed whole cells of strain HD-17, an unidentified gram-positive anaerobic bacterium isolated from human feces, was studied. 7 alpha-Dehydroxylase was produced only during adaptive growth on medium containing 7 alpha-hydroxy bile acids. Both the extent of hydroxylation and the state of conjugation of the bile acids had marked effects on the induction of the enzyme, and the order of the enzyme induction was conjugated cholic acid much greater than cholic acid greater than taurochenodeoxycholic acid greater than or equal to chenodeoxycholic acid. The addition of excess glucose to the growth medium appreciably reduced the enzyme level. The induced enzyme required strict anaerobic conditions for activity and had an optimal pH range of 6.5 to 7.5. In contrast with the induction of the enzyme, the induced enzyme showed a low degree of substrate specificity between cholic acid and chenodeoxycholic acid, with some preference for the former. In addition, the organism contained 3 alpha-, 7 alpha-, and 12 alpha-hydroxysteroid dehydrogenases, and the addition of bile acids to the medium somewhat enhanced the production of the oxidoreductases. The dehydrogenations were obviously stimulated by oxygen as a terminal electron acceptor. The organism also contained bile salt hydrolase.