Oxidation of primary bile acids by a 7 alpha-hydroxysteroid dehydrogenase elaborating Clostridium bifermentans soil isolate

A gram-positive, rod-shaped anaerobe (strain F-6) was isolated from soil. This organism was identified by cellular morphology as well as fermentative and biochemical data as Clostridium bifermentans. Strain F-6 formed 7-ketolithocholic acid from chenodeoxycholic acid and 7-ketodeoxycholic acid from cholic acid in whole cell cultures, but did not transform deoxycholic acid, ursodeoxycholic acid, or ursocholic acid. This reaction is reversible. The structures of 7-ketolithocholic acid and 7-ketodeoxycholic acid were verified by mass spectroscopy and by thin-layer chromatography using Komarowsky's spray reagent. When incubated with the strain F-6 glycine and taurine conjugates of the primary bile acids were partially hydrolyzed and transformed to 7-keto products. Optimal yields of 7-ketolithocholic acid and 7-ketodeoxycholic acid were obtained after 78 h of incubation. Culture pH changed with time and was characterized by an initial drop (1.1 pH units) and a gradual increase back to the starting pH (7.3). Corroborating these observations, an inducible, NADP-dependent, 7 alpha-hydroxysteroid dehydrogenase was demonstrated in cell extracts of strain F-6. A trace of NAD-dependent 7 alpha-hydroxysteroid dehydrogenase was also found. A substantial increase in the specific activity of the NADP-dependent 7 alpha-hydroxysteroid dehydrogenase was observed when either 7-ketolithocholic acid, chenodeoxycholic acid, or deoxycholic acid was included in the growth medium. Optimal induction of the NADP-dependent 7 alpha-hydroxysteroid dehydrogenase was achieved with 0.3-0.4 mM 7-ketolithocholic acid. Production of the enzyme(s) was optimal at 6-8 h of growth and the 7 alpha-hydroxysteroid dehydrogenases had a pH optimum of approximately 11.(ABSTRACT TRUNCATED AT 250 WORDS)     

Formation of ursodeoxycholic acid from chenodeoxycholic acid by a 7 beta-hydroxysteroid dehydrogenase-elaborating Eubacterium aerofaciens strain cocultured with 7 alpha-hydroxysteroid dehydrogenase-elaborating organisms.

A gram-positive, anaerobic, chain-forming, rod-shaped anaerobe (isolate G20-7) was isolated from normal human feces. This organism was identified by cellular morphology as well as fermentative and biochemical data as Eubacterium aerofaciens. When isolate G20-7 was grown in the presence of Bacteroides fragilis or Escherichia coli (or another 7 alpha-hydroxysteroid dehydrogenase producer) and chenodeoxycholic acid, ursodeoxycholic acid produced. Time course curves revealed that 3 alpha-hydroxy-7-keto-5 beta-cholanoic acid produced by B. fragilis or E. coli or introduced into the medium as a pure substance was reduced by G20-7 specifically to ursodeoxycholic acid. The addition of glycine- and taurine-conjugated primary bile acids (chenodeoxycholic and cholic acids) and other bile acids to binary cultures of B. fragilis and G20-7 revealed that (i) both conjugates were hydrolyzed to give free bile acids, (ii) ursocholic acid (3 alpha, 7 beta, 12 alpha-trihydroxy-5 beta-cholanoic acid) was produced when conjugated (or free) cholic acid was the substrate, and (iii) the epimerization reaction was at least partially reversible. Corroborating these observations, an NADP-dependent 7 beta-hydroxysteroid dehydrogenase (reacting specifically with 7 beta-OH-groups) was demonstrated in cell-free preparations of isolate G20-7; production of the enzyme was optimal at between 12 and 18 h of growth. This enzyme, when measured in the oxidative direction, was active with ursodeoxycholic acid, ursocholic acid, and the taurine conjugate of ursodeoxycholic acid (but not with chenodeoxycholic, deoxycholic, or cholic acids) and displayed an optimal pH range of 9.8 to 10.2     

Formation of ursodeoxycholic acid from chenodeoxycholic acid by a 7 beta-hydroxysteroid dehydrogenase-elaborating Eubacterium aerofaciens strain cocultured with 7 alpha-hydroxysteroid dehydrogenase-elaborating organisms

A gram-positive, anaerobic, chain-forming, rod-shaped anaerobe (isolate G20-7) was isolated from normal human feces. This organism was identified by cellular morphology as well as fermentative and biochemical data as Eubacterium aerofaciens. When isolate G20-7 was grown in the presence of Bacteroides fragilis or Escherichia coli (or another 7 alpha-hydroxysteroid dehydrogenase producer) and chenodeoxycholic acid, ursodeoxycholic acid produced. Time course curves revealed that 3 alpha-hydroxy-7-keto-5 beta-cholanoic acid produced by B. fragilis or E. coli or introduced into the medium as a pure substance was reduced by G20-7 specifically to ursodeoxycholic acid. The addition of glycine- and taurine-conjugated primary bile acids (chenodeoxycholic and cholic acids) and other bile acids to binary cultures of B. fragilis and G20-7 revealed that (i) both conjugates were hydrolyzed to give free bile acids, (ii) ursocholic acid (3 alpha, 7 beta, 12 alpha-trihydroxy-5 beta-cholanoic acid) was produced when conjugated (or free) cholic acid was the substrate, and (iii) the epimerization reaction was at least partially reversible. Corroborating these observations, an NADP-dependent 7 beta-hydroxysteroid dehydrogenase (reacting specifically with 7 beta-OH-groups) was demonstrated in cell-free preparations of isolate G20-7; production of the enzyme was optimal at between 12 and 18 h of growth. This enzyme, when measured in the oxidative direction, was active with ursodeoxycholic acid, ursocholic acid, and the taurine conjugate of ursodeoxycholic acid (but not with chenodeoxycholic, deoxycholic, or cholic acids) and displayed an optimal pH range of 9.8 to 10.2     

Bile acid induction of 7 alpha- and 7 beta-hydroxysteroid dehydrogenases in Clostridium limosum

When grown in the presence of bile acids, two strains of Clostridium limosum were found to contain significant amounts of NADP-dependent 7 alpha/7 beta-hydroxysteroid dehydrogenase and NAD-dependent 7 alpha-hydroxysteroid dehydrogenase which were active against conjugated and unconjugated bile acids. No measurable activity could be found when deoxycholic acid (3 alpha, 12 alpha-dihydroxy-5 beta-cholan-24-oic acid) was used as substrate. No 7 beta-hydroxysteroid dehydrogenase activity and only a trace of 7 alpha-hydroxysteroid dehydrogenase activity could be demonstrated when bile acid was deleted from the growth medium. If bile acid was added after the time of inoculation, the amounts of 7 alpha/7 beta-hydroxysteroid dehydrogenase were greatly reduced. Enzyme enhancement was blocked by addition of rifampicin. The 7 alpha/7 beta-hydroxysteroid dehydrogenase components had pH optima of approximately 10.5. Both the 7 alpha/7 beta-hydroxysteroid dehydrogenase activities were heat-labile, with the 7 beta-component being the more stable of the two. When ranked according to the level of enzymes induced, the order in increasing bile acid induction power on an equimolar scale (0.4 mM) was: 7-ketodeoxycholic acid, cholic acid, chenodeoxycholic acid, and deoxycholic acid. Both 7-ketolithocholic acid and ursodeoxycholic acid were ineffective as enzyme inducers. Optimal induction was achieved with high concentrations of cholic acid (5 mM) and a harvest time of 24 hr. Addition of ursodeoxycholic acid to medium containing optimal concentrations of deoxycholic acid suppressed enzyme induction.(ABSTRACT TRUNCATED AT 250 WORDS)     

Partial purification and characterization of 7 alpha-hydroxysteroid dehydrogenase from rat liver microsomes

An NADPH-dependent 7 alpha-hydroxysteroid dehydrogenase acting on 3 alpha-hydroxy-7-keto-5 beta-cholanoic acid was partially purified 160-fold with a yield of 13% from rat liver microsomes using DEAE-cellulose, hydroxyapatite and Affi-Gel Blue column chromatography. The specific activity of the purified enzyme was 91.3 nmol chenodeoxycholic acid formed/min per mg of protein. The reaction was reversible, and the optimum pH of the enzyme for the oxidation was about 8.5, whereas that for the reduction was about 5.0 A molecular weight of the enzyme was estimated to be about 130,000 by Superose 6TM gel filtration chromatography. The apparent Km value for 3 alpha-hydroxy-7-keto-5 beta-cholanoic acid was 35.7 microM and that for NADPH was 90.9 microM. The preferred substrate for the enzyme was 3 alpha-hydroxy-7-keto-5 beta-cholanoic acid rather than 3 alpha,12 alpha-dihydroxy-7-keto-5 beta-cholanoic acid, a 7-keto-bile acid analogue. The enzyme also preferred the unconjugated form to the conjugated forms. The enzyme activity was inhibited by p-chloromercuribenzoate; however, the inhibition was prevented by addition of reduced form of glutathione to the reaction mixture, indicating that the enzyme requires a sulfhydryl group for activity.     

3alpha-, 7alpha- and 12alpha-hydroxysteroid dehydrogenase activities from Clostridium perfringens.

25 strains of Clostridium perfringens were screened for hydroxysteroid dehydrogenase activity; 19 contained NADP-dependent 3alpha-hydroxysteroid dehydrogenase and eight contained NAD-dependent 12alpha-hydroxysteroid dehydrogenase active against conjugated and unconjugated bile salts. All strains containing 12alpha-hydroxysteroid dehydrogenase also contained 3alpha-hydroxysteroid dehydrogenase although 12alpha-hydroxysteroid dehydrogenase was invariably in lesser quantity than the 3alpha-hydroxysteroid dehydrogenase. In addition, 7alpha-hydroxysteroid dehydrogenase activity was evident only when 3alpha, 7alpha, 12alpha-trihydroxy-5beta-cholanoate was substrate but notably absent when 3alpha, 7alpha-dihydroxy-5beta-cholanoate was substrate. The oxidation product 12alpha-hydroxy-3, 7-diketo-5beta-cholanoate is rapidly further degraded to an unknown compound devoid of either 3alpha- or 7alpha-OH groups. Group specificity of these enzymes was confirmed by thin-layer chromatography studies of the oxidation products. These enzyme systems appear to be constitutive rather than inducible. In contrast to C. perfringens. Clostridium paraputrificum (five strains tested) contained no measurable hydroxysteroid dehydrogenase activity. pH studies of the C. perfringens enzymes revealed a sharp pH optimum at pH 11.3 and 10.5 for the 3alpha-OH- and 12alpha-OH-oriented activities, respectively. Kinetic studies gave Km estimates of approx. 5 X 10(-5) and 8 X 10(-4) M with 3alpha, 7a-dihydroxy-5beta-cholanoate and 3alpha, 12alpha-dihydroxy-5beta-cholanoate as substrates for two respective enzymes. 3alpha-hydroxysteroid dehydrogenase was active against 3alpha-OH-containing steroids such as androsterone regardless of the sterochemistry of the 5H (Both A/B cis and A/B trans steroides were substrates). There was no activity against 3beta-OH-containing steroids. The 3alpha- and 12alpha-hydroxysteroid dehydrogenase activities, although differing in cofactor requirements cannot be distinguished by their appearance in the growth curve, their mobility on disc gel electrophoresis, elution volume on passage through Sephadex G-200 or heat inactivation studies.     

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.     

Identification of 7 alpha,12 alpha-dihydroxy-5 beta-cholestan-3-one 3 alpha-hydroxysteroid dehydrogenase

A reductase catalyzing the reduction of the 3-ketone group of 7 alpha,12 alpha-dihydroxy-5 beta-cholestan-3-one and 7 alpha-hydroxy-5 beta-cholestan-3-one, which are the intermediates in the conversion of cholesterol to cholic acid and chenodeoxycholic acid, respectively, into the 3 alpha-hydroxyl group, was purified about 250-fold as judged by the activity from the 100,000 X g supernatant of rat liver homogenate. The purified enzyme was electrophoretically homogeneous, and its molecular weight determined by sodium dodecyl sulfate-polyacrylamide gel electrophoretography was 32,000. The absorption spectrum of the purified enzyme showed only a peak at 280 nm due to aromatic amino acids, precluding the presence of a chromophoric prosthetic group in the molecule. The enzyme showed activity toward a variety of substrates, including 3-oxo-5 beta-cholanoic acid, androsterone, 9,10-phenanthrenquinone, p-nitrobenzaldehyde, but not toward glucuronic acid, DL-glyceraldehyde, and glycolaldehyde. The optimal pH for the reduction of 7 alpha-hydroxy-5 beta-cholestan-3-one was 7.4, and the cofactor required was either NADPH or NADH, though the former gave the higher activity. Judging from the chromatography behavior as well as substrate specificity, the enzyme was identified as 3 alpha-hydroxysteroid dehydrogenase (3 alpha-hydroxysteroid:NAD(P)+ oxidoreductase, EC 1.1.1.50).     

D-Malic enzyme of Pseudomonas fluorescens

By the enrichment culture technique 14 gram-negative bacteria and two yeast strains were isolated that used D(+)-malic acid as sole carbon source. The bacteria were identified as Pseudomonas putida, Pseudomonas fluorescens, Pseudomonas aeruginosa and Klebsiella aerogenes. In cell-free extracts of P. fluorescens and P. putida the presence of malate dehydrogenase, D-malic enzyme (NAD-dependent) and L-malic enzyme (NADP-dependent) was demonstrated. D-Malic enzyme from P. fluorescens was purified. Stabilization of the enzyme by 50 mM ammonium sulphate an 1 mM EDTA was essential. Preparation of D-malic enzyme that gave one band with disc gel electrophoresis showed a specific activity of 4-5 U/mg. D-Malic enzyme requires divalent cations. The Km values were for malate Km = 0.3 mM and for NAD Km = 0.08 mM. The pH optimum for the reaction was found to be in the range of pH 8.1 to pH 8.8. D-Malic enzyme is partially inhibited by oxaloacetic acid, meso-tartaric acid, D-lactic acid and ATP. Determined by gel filtration and gradient gel electrophoresis, the molecular weight was approximately 175 000.     

NADP-dependent 3 beta-, 7 alpha- and 7 beta-hydroxysteroid dehydrogenase activities from a lecithinase-lipase-negative Clostridium species 25.11.c

A lecithinase-lipase-negative Clostridium sp. 25.11.c., not fitting in any of the species of Clostridia described so far as judged by morphological, physiological, and biochemical data, was shown to contain NADP-dependent 3 beta-, 7 alpha- and 7 beta-hydroxysteroid dehydrogenases. The three hydroxysteroid dehydrogenases could be demonstrated in the supernatant and in the membrane fraction after solubilization with Triton X-100, suggesting enzymes which were originally membrane bound. The 3 beta-hydroxysteroid dehydrogenase was synthesized constitutively, and the specific enzyme activity was significantly reduced by growth medium supplementation with 3-keto bile acids and trisubstituted bile acids. A pH optimum of 7.5 and a molecular weight of approx. 104,000 were estimated by molecular sieve chromatography. The enzyme reduced the 3-keto group of bile acids; an oxidation of a 3 beta-hydroxyl function could not be demonstrated. The lowest Km values were found for disubstituted bile acids, trisubstituted and conjugated bile acids having higher Km values. 7 alpha-Hydroxysteroid dehydrogenase, but not 7 beta-hydroxysteroid dehydrogenase, was already present in uninduced cells. The specific activities, however, were greatly enhanced when cells were grown in the presence of chenodeoxycholic acid or 3 alpha-hydroxy-7-keto-5 beta-cholanoic acid. Ursodeoxycholic acid with its 7 beta-hydroxyl group was ineffective as an inducer. Molecular weights of approx. 82,000 and 115,000 were found for the 7 alpha-hydroxysteroid dehydrogenase and the 7 beta-hydroxysteroid dehydrogenase, respectively. In contrast to the in vivo situation, the reaction could only be demonstrated in the reductive direction in vitro. Here, the pH optimum for the overall reaction was 8.5-8.7. 3 beta-, 7 alpha- and 7 beta-hydroxysteroid dehydrogenase activities were readily demonstrated for at least 48 h when preparations were stored at 4 degrees C, but were found to be heat-sensitive.     

Enzyme histochemical observations on the segmentation of the proximal tubules in the kidney of the female rat.

The segmentation of the proximal tubules in the kidney of the female rat was studied by means of enzyme histochemical reactions and the results compared with those observed in male and recently described by Jacobsen and J0rgensen (1973 a). Reactions were performed for the following soluble, coezyme-dependent oxido-reductases: glucose 6-phosphate dehydrogenase, alpha-glycerophosphate dehydrogenase, 3 alpha-hydroxysteroid dehydrogenase, NAD-as well as NADP-dependent isocitrate dehydrogenases, NAD-dependent malate dehydrogenase, NADP-dependent, decarboxylating malate dehydrogenase, uridine diphosphate glucose dehydrogenase. Measures were taken to reduce enzyme diffusion and eliminate interference from tissue tetrazolium reductases. Furthermore, reactions were performed for a number of less soluble or insoluble enzymes: glucose 6-phosphatase, mitochondrial alpha-glycerophosphate dehydrogenase, beta-hydroxybutyrate dehydrogenase, succinate dehydrogenase and tetrazolium reductases. In the proximal tubules of the female rat all enzymes studied--except beta-hydroxybutyrate dehydrogenase--showed segmental differences, most of them clearly revealing three segments. Sex differences were found concerning all enzymes except uridine diphosphate glucose dehydrogenase and NADP-dependent isocitrate dehydrogenase. The most pronounced sex-related differences were seen in the third segment in which part the male rat showed highest activity in respect to tetrazolium reductases, NAD-dependent isocitrate dehydrogenase, succinate dehydrogenase, beta-hydroxybutyrate dehydrogenase, 3 alpha-hydroxysteroid dehydrogenase and glucose 6-phosphate dehydrogenase and the female in respect to glucose 6-phosphatase, alpha-glycerophosphate dehydrogenases, and NADP-dependent, decarboxylating malate dehydrogenase. A few of the enzymes exhibited minor sex differences in the first two segments.     

Lyophilized Clostridium perfringens 3 alpha- and Clostridium bifermentans 7 alpha-hydroxysteroid dehydrogenases: two new stable enzyme preparations for routine bile acid analysis

Preparations of 3 alpha-hydroxysteroid dehydrogenase (EC 1.1.1.50) from Clostridium perfringens were successfully lyophilized into a stable powder form. Purification of the enzyme was achieved using triazine dye affinity chromatography. C. perfringens 3 alpha-hydroxysteroid dehydrogenase was purified 24-fold using Reactive Red 120 (Procion Red) -cross-linked agarose (70% yield). Quantitative measurement of bile acids with the purified enzymes, 3 alpha-hydroxysteroid dehydrogenase and 7 alpha-hydroxysteroid dehydrogenase (EC 1.1.1.159) from Clostridium bifermentans (strain F-6), was achieved spectrophotometrically. Standard curves with chenodeoxycholic acid (CDC) and cholic acid were linear within a concentration range of 20-100 microM. Analysis of mixtures of ursodeoxycholic acid and CDC showed the additive nature of the 3 alpha-hydroxysteroid dehydrogenase and showed also that 7 alpha-hydroxyl groups were independently quantified by the 7 alpha-hydroxysteroid dehydrogenase. Bile acids in Folch extracts of human bile samples were measured using purified preparations of Pseudomonas testosteroni 3 alpha-hydroxysteroid dehydrogenase, C. perfringens 3 alpha-hydroxysteroid dehydrogenase, Escherichia coli 7 alpha-hydroxysteroid dehydrogenase and C. bifermentans (strain F-6) 7 alpha-hydroxysteroid dehydrogenase. Statistical comparison validated the use of C. perfringens 3 alpha- and C. bifermentans 7 alpha-hydroxysteroid dehydrogenases for the quantification of bile acids in bile.     

Alcohol Dehydrogenases from Strawberry Seeds

Two alcohol dehydrogenases (alcohol: NAD oxidoreductase, EC 1.1.1.1 and alcohol: NADP oxidoreductase, EC 1.1.1.2) were partially purified from extracts of strawberry seeds by conventional methods. Some of physical, chemical and kinetic properties of the enzymes are described. On the basis of gel filtration, the molecular weights were estimated to be approximately 78,000 for NAD-dependent enzyme and 82,000 for NADP-dependent enzyme. Thiol-reacting compounds inhibited both enzymes. NAD-dependent alcohol dehydrogenase reacted only with aliphatic alcohols and aldehydes, while aromatic and terpene alcohols and aldehydes were the better substrates for NADP-dependent alcohol dehydrogenase than aliphatic alcohols and aldehydes.     

Purification and characterization of a microbial, NADP-dependent bile acid 7 alpha-hydroxysteroid dehydrogenase

A constitutively expressed 7 alpha-hydroxysteroid dehydrogenase (7 alpha-HSDH) has been purified over 1200-fold, to apparent homogeneity, from an intestinal anaerobic bacterium. The purified protein had a subunit molecular mass of 32 kDa as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Sepharose CL-6B gel filtration gave a native molecular mass estimate of 124 kDa, suggesting that this enzyme existed as a tetramer of identical subunits. Sulfhydryl reactive compounds were potent inhibitors of 7 alpha-HSDH activity, however, metal ion chelators had no effect upon catalytic activity. The purified enzyme was highly NADP-dependent. Bile acid substrate utilization studies revealed that the enzyme was specific for the oxidation of an unhindered 7 alpha-hydroxyl group. A wide variety of bile acids and analogs were used as substrates including glycine and taurine conjugates, and methyl esters, amines, and bile alcohols. The purified 7 alpha-HSDH obeyed Michaelis-Menten kinetics. Hanes plots of substrate saturation kinetics revealed that most bile acid substrates had Km values ranging from 4 to 20 microM, while Vmax was 601 and 674 mumol/min/mg in the direction of bile acid oxidation and reduction, respectively. Primary kinetic plots and product inhibition patterns were consistent with an ordered sequential mechanism, with NADP(H) binding first. The N-terminal amino acid sequence analysis of the purified enzyme revealed a striking homology to several short, non-zinc alcohol/polyol dehydrogenases and a putative, cholate-inducible, hydroxysteroid dehydrogenase from the same organism. The high specific activity together with the stability, substrate range, and ease of purification, make this enzyme an excellent candidate for use in quantitating primary bile acids both in laboratory and clinical samples. Spectrofluorometry allowed for the quantitation of as little as 10 nM of both free and conjugated primary bile acids.     

Genetic control of soluble NDA-dependent sorbitol dehydrogenase in Drosophila melanogaster

Experiments utilizing standard techniques of cell fractionation and disc electrophoresis have revealed the presence of three distinctly different enzymes which catalyze the oxidation of d-sorbitol in crude extracts of Drosophila melanogaster adults. These include (1) a soluble NAD-dependent sorbitol dehydrogenase (NAD-SoDH^s), (2) a mitochondrial NAD-dependent sorbitol dehydrogenase (NAD-SoDH^m), and (3) a soluble NADP-dependent sorbitol dehydrogenase (NADP-SoDH). The structural gene for NAD-SoDH^s has been mapped to a locus between 65.3 and 65.6 on the third chromosome by means of an electrophoretic variant and a low-activity allele. Through the use of segmental aneuploidy, this gene has been localized to the region limited by salivary bands 91B–93F. Because mutants which alter either the activity or electrophoretic mobility of the soluble NAD-dependent enzyme have no significant measurable effect on the mitochondrial or NADP-dependent forms, it is suggested that the enzymes in this system are coded for autonomously by different genes.     

NAD-dependent 3alpha- and 12alpha-hydroxysteroid dehydrogenase activities from Eubacterium lentum ATCC no. 25559.

Eubacterium lentum (ATCC No. 25559) was shown to contain 3alpha-and 12alpha-hydroxysteroid dehydrogenases both of which were NAD-dependent and active against conjugated and unconjugated bile salts. In addition, the 3alpha-hydroxysteroid dehydrogenase was active against members of the Androstan series containing a 3alpha-hydroxyl group regardless of the stereo-orientation of the 5-H-. No measurable activity against 7alpha-, 7beta-, 11beta-, or 17beta-hydroxyl groups was demonstrated. The growth of E. lentum and the production of 3alpha- and 12alpha-hydroxysteroid dehydrogenases were greatly enhanced by the addition of L-, D- or DL-arginine to the medium. Yields of hydroxysteroid dehydrogenase were optimal in the range of 0.50-0.75% arginine; however, the growth of the organisms was further enhanced at arginine concentrations greater than 0.75%. The 12alpha-hydroxysteroid dehydrogenase was heat labile and could be selectively inactivated by heating at 50 degrees C for 45 min. Both the heated enzyme preparation (containing only 3alpha-hydroxysteroid dehydrogenase) and the unheated enzyme preparation (containing 3alpha- and 12alpha-hydroxysteroid dehydrogenases) were useful in the spectrophotometric quantification of bile salts. The optimal pH values for 3alpha- and 12alpha-hydroxysteroid dehydrogenases were 11.3 and 10.2, respectively. Kinetic studies have Km estimates of 2.10(-5) M and 1.0.10(-4) M with 3alpha,7alpha-dihydroxy-5beta-cholanoyl glycine and 7alpha,12alpha-dihydroxy-5beta-cholanoate for the two respective enzymes.     

Kinetic properties of 7 alpha-hydroxysteroid dehydrogenase from Escherichia coli 080

Results on the kinetics of 7 alpha-hydroxysteroid dehydrogenase 7 alpha-HSDH showed that this enzyme could oxidize all bile acids having an -OH group at the C-7 position. Lineweaver-Burk plots showed Michaelis constant (Km) values of 0.83 and 0.12 mM for cholic acid and chenodeoxycholic acid, respectively. The effect of enzyme concentration on the reaction velocity showed a constant increase in the enzyme activity with increase in enzyme-protein concentration. 7 alpha-HSDH was activated by Na+, K+, Ca2+, and Mn2+ ions and by reducing agents having a thiol group (dithiothreitol, 2-mercaptoethanol). Co2+, Hg2+, Fe3+, Mg2+, Zn2+, Ba2+, and Cu2+ ions, chelating agents (potassium oxalate, heparin, EDTA) oxidizing agents (sodium perchlorate, sodium periodate, sodium persulphate), and detergents (Tween 20, Tween 40, Tween 80, Triton X-100, sodium lauryl sulphate) were inhibitory to 7 alpha-HSDH activity.     

The presence of an NADP-dependent alcohol dehydrogenase activity in Acinetobacter calcoaceticus

Abstract A soluble NADP-dependent alcohol dehydrogenase activity (EC 1.1.1.2) was found in all five strains of Acinetobacter calcoaceticus tested. In A. calcoaceticus NCIB8250, this dehydrogenase was not induced by growth on ethanol, but was present at approximately the same specific activity when this strain was grown on a variety of carbon sources. The specific activity of the NADP-dependent alcohol dehydrogenase is about 10% of the activity of the NAD-dependent alcohol dehydrogenase found in bacteria grown on ethanol. The distinct biochemical properties of the NADP-dependent dehydrogenase showed that this activity was not due to lack of nucleotide specificity of the NAD-dependent dehydrogenase.     

Isocholic acid formation from 7 alpha,12 alpha-dihydroxy-3-keto-5 beta-cholanoic acid with human liver enzyme

The formation of isocholic acid from 7 alpha, 12 alpha-dihydroxy-3-keto-5 beta-cholanoic acid by human liver preparations was examined in vitro. Liver preparations were incubated with 7 alpha, 12 alpha-dihydroxy-3-keto-5 beta-cholanoic acid at pH 7.4 in a phosphate buffer containing NADPH or NADH. The products formed were analyzed by gas chromatography and gas chromatography/mass spectrometry. Results showed that 7 alpha,12 alpha-dihydroxy-3-keto-5 beta-cholanoic acid was reduced mainly to isocholic acid and to cholic acid in a smaller amount in the presence of NADPH, while it was reduced only to cholic acid in the presence of NADH. The reducing enzyme participating in the formation of isocholic acid was localized largely in the cytosol and had more specificity to the unconjugated form as substrate than to the conjugated forms. 3-Keto bile acid analogues, 3-keto-5 beta-cholanoic and 7 alpha-hydroxy-3-keto-5 beta-cholanoic acids were not reduced to the corresponding iso-bile acids by the cytosol in the same conditions used in the isocholic acid formation and the activity of the enzyme catalyzing the reduction of 7 alpha,12 alpha-dihydroxy-3-keto-5 beta-cholanoic acid to isocholic acid was not inhibited by the addition of 3-keto-5 beta-cholanoic acid or 7 alpha-hydroxy-3-keto-5 beta-cholanoic acid to the reaction mixture. Furthermore, on column chromatography of Affi-Gel Blue, the peak of the enzyme catalyzing the reduction of 7 alpha,12 alpha-dihydroxy-3-keto-5 beta-cholanoic acid to isocholic acid was clearly distinguished from that of the enzyme catalyzing the reduction of 3-keto-5 beta-cholanoic acid to isolithocholic acid and that of alcohol dehydrogenase. These results indicate that this enzyme catalyzing the reduction of 7 alpha,12 alpha-dihydroxy-3-keto-5 beta-cholanoic acid to isocholic acid is different from the enzyme(s) catalyzing the reduction 3-keto-5 beta-cholanoic and 7 alpha-hydroxy-3-keto-5 beta-cholanoic acids to the corresponding iso-bile acids and from alcohol dehydrogenase, and has a stereospecific character for 7 alpha,12 alpha-dihydroxy-3-keto-5 beta-cholanoic acid.     

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.