Further studies on the bile salt induction of 7 alpha- and 7 beta-hydroxysteroid dehydrogenases in Clostridium absonum

Optimal induction of 7 alpha- and 7 beta-hydroxysteroid dehydrogenase in 100-ml cultures grown to stationary phase was achieved by the addition of metabolizable bile salt inducers: chenodeoxycholate, 7-ketolithocholate or cholate at 2.5-3 h after inoculation. Bile salt addition prior to or after this period markedly reduced the enzyme levels induced. However, when the non-metabolizable inducers deoxycholate and 12-ketolithocholate were similarly added, no significant differences in enzyme levels were observed between addition at 2.5-3 h or at earlier times. The ability of both metabolizable and non-metabolizable bile salts to induce the enzymes fell markedly when additions were made later than approximately 3.5 h. Kinetic studies using 1-l cultures suggest that in a larger culture a somewhat earlier inducer addition period is optimal. When ranked according to the level of enzymes induced the order in decreasing induction power was: chenodeoxycholate, 7-ketolithocholate, deoxycholate, 12-ketolithocholate and cholate. Mixtures of cholate and suboptimal concentrations of deoxycholate induced the culture better than the sum of the two concentrations individually. The end product, ursodeoxycholate, was very effective in blocking the induction by chenodeoxycholate or deoxycholate. Ursocholate (3 alpha, 7 beta, 12 alpha-trihydroxy-5 beta-cholanoate) was less effective. Cultures when grown for 3 h with various bile salts or none, then centrifuged and recultured for a further 3 h in fresh medium containing chenodeoxycholate, all yielded identical enzyme levels within experimental error. We conclude that exposure of the organism to bile salt inducer in the last 3 h of culture was important, while the history of the culture prior to this time was unimportant in the induction process.     

Bile-salt inhibition of sodium ion-coupled D-glucose and L-alanine accumulation by brush-border-membrane vesicles from hamster jejunum.

The effects of bile salts on Na+-coupled accumulation of D-glucose and L-alanine by brush-border-membrane vesicles isolated from hamster jejunum were investigated. The approximate percentage inhibition of Na+-coupled D-glucose accumulation produced by various bile salts at a concentration of 1 mM were: deoxycholate and chenodeoxycholate, 60%; glycine and taurine conjugates of deoxycholate and chenodeoxycholate, 40--50%; lithocholate, 45%; cholate and its glycine and taurine conjugates, less than 10%. Inhibition of Na+-coupled accumulation of D-glucose was rapid, reversible and not due to dissolution of the vesicles. Na+-coupled accumulation of L-alanine was also inhibited by deoxycholate. Deoxycholate but not cholate enhanced (1) the rate of Na+ influx, (2) the rate of influx of D-glucose and L-alanine in the absence of a Na+ gradient and (3) the rate of efflux of D-glucose and L-alanine from vesicles preloaded with this sugar or amino acid. Deoxycholate-stimulated efflux of D-glucose was not blocked by phlorizin, which completely prevented efflux in the absence of this bile salt. These results suggest that selected bile salts inhibit Na+-coupled accumulation of D-glucose and L-alanine by enhancing the rate of dissipation of the Na+ gradient required for substrate accumulation. In addition, bile salts may also decrease D-glucose and L-alanine accumulation by increasing the rate of efflux of these substrates across the brush-border plasma membrane.     

Influence of various bile salts on ββ-glucuronidase activity of intestinal bacteria

While the decrease of the β-glucuronidase activity of sonicated cells of Clostridium perfringens and Escherichia coli was obvious for sodium deoxycholate (DC), it was not so obvious for other bile salts (sodium glycocholate and sodium cholate). The enzyme activity of intact cells of these bacteria was significantly enhanced by the presence of DC, but not by the other bile salts in the buffer. These results suggest that the permeability of the bacterial cells is increased more by the presence of DC than by other bile salts.     

Partial purification and characterization of an NAD-dependent 3 beta-hydroxysteroid dehydrogenase from Clostridium innocuum.

In nine strains of Clostridium innocuum, 3 beta-hydroxysteroid-dehydrogenating activities were detected. 3 beta, 7 alpha, 12 alpha-Trihydroxy- and 3 beta-hydroxy-12-keto-5 beta-cholanoic acids were identified as reduction products of the respective 3-keto bile acids by gas-liquid chromatography and gas-liquid chromatography-mass spectrometry. One strain was shown to contain a NAD-dependent 3 beta-hydroxysteroid dehydrogenase. Enzyme production was constitutive in the absence of added bile acids. The specific enzyme activity was significantly reduced by growth medium supplementation with 3-keto bile acids, with trisubstituted acids being more effective than disubstituted ones. A pH optimum of 10.0 to 10.2 was found after partial purification by DEAE-cellulose chromatography. A molecular weight of about 56,000 was established. 3 beta-hydroxysteroid dehydrogenase activity was also found in the membrane fraction after solubilization with Triton X-100, suggesting that the enzyme was originally membrane bound. The enzyme reduced a 3-keto group in unconjugated and conjugated bile acids, lower Km values being demonstrated with disubstituted than with trisubstituted bile acids. Keto functions at C-7 and C-12 further reduced the Km value. The enzyme was found to be partially heat labile (86% inactivation at 50 degrees C for 10 min).     

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.     

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.     

Isolation of a rat intestinal Clostridium strain producing 5 alpha- and 5 beta-bile salt 3 alpha-sulfatase activity

An unnamed sporeforming microorganism, termed Clostridium sp. strain S2, possessing bile salt sulfatase activity was isolated from rat intestinal microflora. The microorganism was a strictly anaerobic, nonmotile, gram-negative, asaccharolytic, sporeforming rod requiring CO2, vitamin K, and taurine; the guanine-plus-cytosine content of the DNA was 40.8 mol% (Tm), and the strain was tentatively classified as an atypical Clostridium species. Sulfatase activity was specific for 3 alpha-sulfate esters of 5 alpha- and 5 beta-bile salts, leaving the 3 beta-, 7 alpha-, and 12 alpha-sulfates unchanged. Strain S2 also deconjugated tauro- and glyco-conjugated bile salts and partially reduced into the corresponding 6 alpha-hydroxy bile salts. By these reactions, alpha-muricholate and beta-muricholate were more than 80% converted into hyocholate and omega-muricholate, respectively. In addition, strain S2 produced 12 alpha-hydroxysteroid dehydrogenase converting deoxycholate into 3 alpha-hydroxy-12-oxo-5 beta-cholanoate. When strain S2 was associated with gnotobiotic rats, the fecal bile salts were more than 90% desulfated and the fecal excretion of allochenodeoxycholate was five times lower than in control rats.     

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.     

The order of hepatic cytotoxicity of bile salts in vitro does not agree with that examined in vivo in rats

Using an enzyme release from isolated rat hepatocytes incubated with a bile salt as a marker, the cytotoxic order of bile salts was found to be taurochenodeoxycholate, glycochenodeoxycholate greater than tauroursodeoxycholate, glycoursodeoxycholate, cholate greater than taurocholate, glycocholate. Thus, the cytotoxicity of conjugates of ursodeoxycholate was greater than that of conjugates of cholate. However, these data do not agree with the order of cytotoxicity of these bile salts previously studied in vivo by the authors which demonstrated the least cytotoxic nature of conjugates of ursodeoxycholate.     

Effect of free and conjugated bile salts on alpha-amylase activity.

The effect of individual bile salts on alpha-amylase hydrolysis of Cibachron Blue starch was studied at pH 6.0. With sodium cholate, taurocholate and taurodeoxycholate, enzyme activity was increased to 150-160 percent of the control value, at a concentration of similar to 1 mmol/l bile salt. The increased activity extended up to 4 mmol/l. The bile salts sodium deoxycholate and taurochenodeoxycholate exerted activation and inhibition depending on the concentration. With deoxycholate (0.75 mmol/l), activation (150 percent) was evident, while inhibition was apparent above 2.5 mmol/l. With taurochenodeoxycholate maximum activity (135 percent) was observed at 0.25 mmol/l, while inhibition was evident above 1.5 mmol/l. Chenodeoxycholate and lithocholate exerted marked inhibition at concentrations as low as 0.5 mmol/l. Inhibition of alpha-amylase by chenodeoxycholate was competitive with both soluble and insoluble starch substrates. Since the pH of the jejunum is in the region of 6.0 the phenomenon of activation and inhibition of alpha-amylase by bile salts at this pH could be of physiological significance.     

Phospholipid fatty acid and lipopolysaccharide fatty acid signature lipids in methane-utilizing bacteria

The effect of human bile juice and bile salts (sodium cholate, sodium taurocholate, sodium glycochenodeoxycholate and sodium chenodeoxycholate) on growth, sporulation and enterotoxin production by enterotoxin-positive and enterotoxin-negative strains of Clostridium perfringens was determined. Each bile salt inhibited growth to a different degree. A mixture of bile salts completely inhibited the growth of enterotoxin-positive strains of this organism. Human bile juice completely inhibited the growth of all the strains at a dilution of 1:320. A distinct stimulatory effect of the bile salts on sporulation was observed in the case of C. perfringens strains NCTC 8239 and NCTC 8679. The salts also increased enterotoxin concentrations in the cell extracts of the enterotoxin-positive strains tested. No effect on enterotoxin production was detected when an enterotoxin-negative strain was examined.     

Partial purification and characterization of an NAD-dependent 3 beta-hydroxysteroid dehydrogenase from Clostridium innocuum

In nine strains of Clostridium innocuum, 3 beta-hydroxysteroid-dehydrogenating activities were detected. 3 beta, 7 alpha, 12 alpha-Trihydroxy- and 3 beta-hydroxy-12-keto-5 beta-cholanoic acids were identified as reduction products of the respective 3-keto bile acids by gas-liquid chromatography and gas-liquid chromatography-mass spectrometry. One strain was shown to contain a NAD-dependent 3 beta-hydroxysteroid dehydrogenase. Enzyme production was constitutive in the absence of added bile acids. The specific enzyme activity was significantly reduced by growth medium supplementation with 3-keto bile acids, with trisubstituted acids being more effective than disubstituted ones. A pH optimum of 10.0 to 10.2 was found after partial purification by DEAE-cellulose chromatography. A molecular weight of about 56,000 was established. 3 beta-hydroxysteroid dehydrogenase activity was also found in the membrane fraction after solubilization with Triton X-100, suggesting that the enzyme was originally membrane bound. The enzyme reduced a 3-keto group in unconjugated and conjugated bile acids, lower Km values being demonstrated with disubstituted than with trisubstituted bile acids. Keto functions at C-7 and C-12 further reduced the Km value. The enzyme was found to be partially heat labile (86% inactivation at 50 degrees C for 10 min).     

Sodium cholate-induced changes in the conformation and activity of rat pancreatic cholesterol esterase

Pancreatic cholesterol esterase (CEase) regulates dietary cholesterol absorption and is activated in the presence of trihydroxy bile salts while remaining inactive monohydroxy bile salts. CEase from rat pancreas has been purified by ammonium sulfate precipitation, hydroxylapatite chromatography, and gel filtration on Sephacryl S-200/S-300 columns connected in series, and its homogeneity and Mr (55,418 +/- 288) have been determined by sedimentation equilibrium centrifugation. The effects of tri-, di-, and monohydroxy bile salts on the conformation of the purified enzyme in buffer solution and in an in vitro assay system were studied by circular dichroism spectropolarimetry. The CD spectrum of the enzyme in solution shows a curve shape suggestive of an alpha-helicity, but low mean residue ellipticity (MRE) values may indicate an important beta-turn contribution. Sodium cholate, a trihydroxy bile salt, induces a decrease in the negative MRE values of the enzyme in solution at bile salt concentrations of 70-100 nM, with no further spectral changes at concentrations as high as 1 mM. Sodium cholate concentrations higher than 1 microM also induce an increase in the enzyme's negative MRE values under activity assay conditions, which reverts toward its original value once the reaction reaches equilibrium. These latter changes are interpreted as induced by substrate binding to the enzyme followed by partial substrate depletion after the reaction reaches equilibrium. Sodium deoxycholate, a dihydroxy bile salt, induces unstable transient increases and decreases in the MRE values of CEase in buffer solution and under activity assay conditions. These changes are bile salt concentration-dependent and may reflect self-association of the protein. Sodium taurolithocholate, a monohydroxy bile salt, does not affect the CD spectrum of CEase, and neither the di- or the monohydroxy bile salt activates the enzyme.     

Solubilization of lipids from hamster bile-canalicular and contiguous membranes and from human erythrocyte membranes by conjugated bile salts.

We have demonstrated in vitro the efficacy of the taurine-conjugated dihydroxy bile salts deoxycholate and chenodeoxycholate in solubilizing both cholesterol and phospholipid from hamster liver bile-canalicular and contiguous membranes and from human erythrocyte membrane. On the other hand, the dihydroxy bile salt ursodeoxycholate and the trihydroxy bile salt cholate solubilize much less lipid. The lipid solubilization by the four bile salts correlated well with their hydrophobicity: glycochenodeoxycolate, which is more hydrophobic than the tauro derivative, also solubilized more lipid. All the dihydroxy bile salts have a threshold concentration above which lipid solubilization increases rapidly; this correlates approximately with the critical micellar concentration. The non-micelle-forming bile salt dehydrocholate solubilized no lipid at all up to 32 mM. All the dihydroxy bile acids are much more efficient at solubilizing phospholipid than cholesterol. Cholate does not show such a pronounced discrimination. Lipid solubilization by chenodeoxycholate was essentially complete within 1 min, whereas that by cholate was linear up to 5 min. Maximal lipid solubilization with chenodeoxycholate occurred at 8-12 mM; solubilization by cholate was linear up to 32 mM. Ursodeoxycholate was the only dihydroxy bile salt which was able to solubilize phospholipid (although not cholesterol) below the critical micellar concentration. This similarity between cholate and ursodeoxycholate may reflect their ability to form a more extensive liquid-crystal system. Membrane specificity was demonstrated only inasmuch as the lower the cholesterol/phospholipid ratio in the membrane, the greater the fractional solubilization of cholesterol by bile salts, i.e. the total amount of cholesterol solubilized depended only on the bile-salt concentration. On the other hand, the total amount of phospholipid solubilized decreased with increasing cholesterol/phospholipid ratio in the membrane.     

Characterization of NADP-dependent 12 beta-hydroxysteroid dehydrogenase from Clostridium paraputrificum

Clostridium paraputrificum D 762-06 was found to contain an NADP-dependent 12 beta-hydroxysteroid dehydrogenase, already present in uninduced cells. Its specific activity could, however, be enhanced up to about 3-fold by the inclusion of bile acids with a 12-keto group or a 12 beta-hydroxy group in the growth medium. 3 alpha-Hydroxy-12-keto-5 beta-cholanoic acid was the most effective inducer. A pH optimum of 10.0 and a molecular weight of 126,000 were estimated by molecular sieve chromatography. The enzyme preparation reduced 12-keto groups in conjugated and unconjugated bile acids and oxidized a 12 beta-hydroxy function, but oxidative activity was only about 25% of the reductive one. Disubstituted bile acids showed lower Km values than the corresponding trisubstituted ones, the lowest Km values being those observed for 3,12- and 7,12-5 beta-cholanoic acids. No measurable activity against 12 alpha-hydroxyl groups could be detected. The enzyme was found to be heat-labile (95% inactivation at 50 degrees C for 10 min), but the activity was maintained for about 4 weeks when lyophilized preparations were stored at -20 degrees C. 12 beta-Hydroxysteroid dehydrogenase activity was also demonstrated in the membrane fraction after solubilization with Triton X-100, suggesting that it was originally a membrane-bound enzyme.     

Modulation of gamma-glutamyl transpeptidase activity by bile acids

The free bile acids (cholate, chenodeoxycholate, and deoxycholate) stimulate the hydrolysis and transpeptidation reactions catalyzed by gamma-glutamyl transpeptidase, while their glycine and taurine conjugates inhibit both reactions. Kinetic studies using D-gamma-glutamyl-p-nitroanilide as gamma-glutamyl donor indicate that the free bile acids decrease the Km for hydrolysis and increase the Vmax; transpeptidation is similarly activated. The conjugated bile acids increase the Km and Vmax of hydrolysis and decrease both of these for transpeptidation. This mixed type of modulation has also been shown to occur with hippurate and maleate (Thompson, G.A., and Meister, A. (1980) J. Biol. Chem. 255, 2109-2113). Glycine conjugates are substantially stronger inhibitors than the taurine conjugates. The results with free cholate indicate the presence of an activator binding domain on the enzyme with minimal overlap on the substrate binding sites. In contrast, the conjugated bile acids, like maleate and hippurate, may overlap on the substrate binding sites. The results suggest a potential feedback role for bile ductule gamma-glutamyl transpeptidase, in which free bile acids activate the enzyme to catabolize biliary glutathione and thus increase the pool of amino acid precursors required for conjugation (glycine directly and taurine through cysteine oxidation). Conjugated bile acids would have the reverse effect by inhibiting ductule gamma-glutamyl transpeptidase.     

Vectorial transport of unconjugated and conjugated bile salts by monolayers of LLC-PK1 cells doubly transfected with human NTCP and BSEP or with rat Ntcp and Bsep

Na(+)-taurocholate-cotransporting peptide (NTCP)/SLC10A1 and bile salt export pump (BSEP)/ABCB11 synergistically play an important role in the transport of bile salts by the hepatocyte. In this study, we transfected human NTCP and BSEP or rat Ntcp and Bsep into LLC-PK1 cells, a cell line devoid of bile salts transporters. Transport by these cells was characterized with a focus on substrate specificity between rats and humans. The basal to apical flux of taurocholate across NTCP- and BSEP-expressing LLC-PK1 monolayers was 10 times higher than that in the opposite direction, whereas the flux across the monolayer of control and NTCP or BSEP single-expressing cells did not show any vectorial transport. The basal to apical flux of taurocholate was saturated with a K(m) value of 20 microM. Vectorial transcellular transport was also observed for cholate, chenodeoxycholate, ursodeoxycholate, their taurine and glycine conjugates, and taurodeoxycholate and glycodeoxycholate, whereas no transport of lithocholate was detected. To evaluate the respective functions of NTCP and BSEP and to compare them with those of rat Ntcp and Bsep, we calculated the clearance by each transporter in this system. A good correlation in the clearance of the examined bile salts (cholate, chenodeoxycholate, ursodeoxycholate, and their taurine or glycine conjugates) was observed between transport by human and that of rat transporters in terms of their rank order: for NTCP, taurine conjugates > glycine conjugates > unconjugated bile salts, and for BSEP, unconjugated bile salts and glycine conjugates > taurine conjugates. In conclusion, the substrate specificity of human and rat NTCP and BSEP appear to be very similar at least for monovalent bile salts under physiological conditions.     

An active type IV secretion system encoded by the F plasmid sensitizes Escherichia coli to bile salts

F(+) strains of Escherichia coli infected with donor-specific bacteriophage such as M13 are sensitive to bile salts. We show here that this sensitivity has two components. The first derives from secretion of bacteriophage particles through the cell envelope, but the second can be attributed to expression of the F genes required for the formation of conjugative (F) pili. The latter component was manifested as reduced or no growth of an F(+) strain in liquid medium containing bile salts at concentrations that had little or no effect on the isogenic F(-) strain or as a reduced plating efficiency of the F(+) strain on solid media; at 2% bile salts, plating efficiency was reduced 10(4)-fold. Strains with F or F-like R factors were consistently more sensitive to bile salts than isogenic, plasmid-free strains, but the quantitative effect of bile salts depended on both the plasmid and the strain. Sensitivity also depended on the bile salt, with conjugated bile salts (glycocholate and taurocholate) being less active than unconjugated bile salts (deoxycholate and cholate). F(+) cells were also more sensitive to sodium dodecyl sulfate than otherwise isogenic F(-) cells, suggesting a selectivity for amphipathic anions. A mutation in any but one F tra gene required for the assembly of F pili, including the traA gene encoding F pilin, substantially restored bile salt resistance, suggesting that bile salt sensitivity requires an active system for F pilin secretion. The exception was traW. A traW mutant was 100-fold more sensitive to cholate than the tra(+) strain but only marginally more sensitive to taurocholate or glycocholate. Bile salt sensitivity could not be attributed to a generalized change in the surface permeability of F(+) cells, as judged by the effects of hydrophilic and hydrophobic antibiotics and by leakage of periplasmic beta-lactamase into the medium.     

Cholate-induced dimerization of detergent- or phospholipid-solubilized bovine cytochrome C oxidase

Bovine heart cytochrome c oxidase (CcO), solubilized by either nonionic detergents or phospholipids, completely dimerizes upon the addition of bile salts, e.g., sodium cholate, sodium deoxycholate, or CHAPS. Bile salt induced dimerization occurs whether dodecyl maltoside, decyl maltoside, or Triton X-100 is the primary solubilizing detergent or the enzyme is dispersed in phosphatidylcholine, phosphatidylethanolamine, or mixtures thereof. In each case, complete CcO dimerization can be verified by sedimentation velocity and sedimentation equilibrium after correction for bound detergent and/or phospholipid. The relative concentration of the bile salt is critical for production of homogeneous, dimeric CcO. For example, enzyme solubilized by 2 mM detergent requires an equal molar concentration of sodium cholate. Similarly, enzyme dispersed in 20 mM phospholipid requires 50 mM sodium cholate, concentrations that are commonly used to reconstitute CcO into small unilamellar vesicles. Bile salts do more than just stabilize dimeric CcO and prevent detergent-induced dissociation into monomers. They are able to completely reverse detergent-induced monomerization and cause completely monomeric CcO to reassociate. Dimeric CcO so generated is no more stable than the original complex and easily dissociates into monomers if the bile salt is removed. The dimerization process is dependent upon a full complement of subunits; e.g., if subunits VIa and VIb are removed, the resulting monomeric CcO will not reassociate upon the addition of sodium cholate. These results support four important consequences: (1) dissociation of dimeric CcO into monomers is reversible; (2) stable dimers can be produced under solution conditions; (3) dimers can be stabilized even at relatively high pH and low enzyme concentration; and (4) subunits VIa and VIb are required for dimerization.     

Taurocholate is more potent than cholate in suppression of bile salt synthesis in the rat

Synthesis of bile salts is regulated through negative feedback inhibition by bile salts returning to the liver. Individual bile salts have not been distinguished with regard to inhibitory potential. We assessed inhibition of bile salt synthesis by either cholate or its taurine conjugate in bile fistula rats. After allowing synthesis to maximize, baseline synthesis was determined by measuring bile salt output in four consecutive 6-hr periods. Next, sodium cholate (+[(14)C]cholate) or taurocholate (+[(14)C]taurocholate) was infused into the jugular vein for 36 hr and bile was collected in 6-hr aliquots. Hepatic flux of exogenous bile salt was determined by measuring output of radioactivity in bile divided by specific activity of the infusate. Synthesis was determined during the last four 6-hr periods of infusion by subtracting exogenous bile salt secretion from the total bile salt output. Thirteen studies using cholate and 13 using taurocholate were performed. Hepatic flux of infused bile salt varied from 1 to 12 micro mol/100 g per rat per hr. Percent suppression of synthesis varied directly with hepatic flux of exogenous bile salt for both cholate and taurocholate in a linear fashion (r = 0.66, P < 0.01 and r = 0.87, P < 0.0005, respectively). Slope of the taurocholate line was 7.82 (% suppression/ micro mol per 100 g per hr), while slope of the cholate line was 3.66 (P < 0.05), indicating that taurocholate was approximately twice as potent as cholate in suppression of synthesis. At fluxes of 10-12 micro mol/100 g per hr, taurocholate suppressed synthesis 84 +/- 8 (SEM) % while cholate suppressed synthesis only 42 +/- 12% (P < 0.02). The x-intercept of the taurocholate line was 0.65 ( micro mol/100 g per hr), while that of the cholate line was -1.01 (NS) suggesting that the threshold for initial suppression of synthesis did not differ for these two bile salts. We conclude that taurocholate is a more effective inhibitor of hepatic bile salt synthesis than cholate, and that intestinal deconjugation of bile salts may play a role in the regulation of synthesis.-Pries, J. M., A. Gustafson, D. Wiegand, and W. C. Duane. Taurocholate is more potent than cholate in suppression of bile salt synthesis in the rat.