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PubMed1977

Calf thymus alkaline phosphatase. II. Interaction with detergents.

1. A number of detergents were used to dissolve calf thymus plasma membranes rich in alkaline phosphatase (orthophosporic-monoester phosphohydrolase (alkaline optimum), EC 3.1.3.1) activity. 2. The Stokes' radius (r) of alkaline phosphatase in each detergent was measured by gel filtraton. The size of the solubilized enzyme varied from r = 6.2 nm in sodium cholate to r = 8.3 nm in Berol EMU-043. With N-alkylsulphates, the apparent size increased with alkyl chain length, with r = 6.4 nm (C9) and r = 7.3 nm (C12). Tween 20 failed to solubilise the enzyme. 3. The effect of each detergent on the catalytic activity of alkaline phosphatase was determined. The non-ionic detergents Triton X-100, Nonidet P-40, Berol EMU-043, Tween 20 and the zwitterionic detergent Empigen BB increased V by 10--50% without substantially altering the Km for p-nitrophenylphosphate. The bile salts sodium deoxycholate and sodium cholate decreased V and increased the apparent affinity of the enzyme for nitrophenylphosphate. Inhibition was concentration-dependent up to the critical micellar concentration, above which it remained constant (deoxycholate, 33% cholate, 76%). Alkylsulphates (C8-12) had no significant inhibitory effect during 24 h at 23 degrees C. 4. Exchanging one detergent for another altered alkaline phosphatase activity to a state characteristic for the second detergent, e.g. the activity of cholate-inhibited alkaline phosphatase was restored to normal levels by excess of Triton X-100 and vice versa. The inhibitory effect of deoxycholate and cholate therefore result primarily from interactions between detergent and alkaline phosphate, rather than from selective removal of lipids from the enzyme. 5. Pure lecithin, lysolecithin and an ether-deoxylysolecithin each reactivated cholate-inhibited alkaline phosphatase in a concentration-dependent fashion. Cholesterol had no effect. 6. The half-life (t1/2) of membrane-bound alkaline phosphatase at 55 degrees C was 64 min. With the exception of Berol, solubilisation in non-ionic detergents caused no marked change in this sensitivity. The enzyme became more labile in deoxycholate (t1/2) = 31 min), but less labile in cholate (t1/2 = 99 min). Alkylsulphates, which are strong denaturants, markedly increased the sensitivity of the enzyme to heat-inactivation (C8, t1/2 = 13 min; C9--12, t1/2 less than 2 min). 7. It is concluded that membrane-bound alkaline phosphatase is separated from most if not all of its neighbouring lipid moieties by these detergents, which bind to the solubilised enzyme. The number and character of molecules binding to the enzyme influence its size and shape, its susceptibility to inactivation and its catalytic activity.

Alkaline Phosphatase↗
PubMed1987

Metabolism of the bile acid analogues 7 beta-methyl-cholic acid and 7 alpha-methyl-ursocholic acid.

The metabolism of two new bile acid analogues, 7 beta-methyl-cholate and 7 alpha-methyl-ursocholate, was compared with that of cholate in the hamster. After intraduodenal administration of 14C-labeled compounds into bile fistula hamsters, radioactivity was exclusively recovered in bile; the more hydrophobic bile acid was absorbed more rapidly. Hepatic extraction of intravenously infused compounds was efficient and administered analogues became major biliary bile acids. Amidation of cholate was essentially complete, whereas 39% of 7 beta-methyl-cholate and 65% of 7 alpha-methyl-ursocholate were secreted in unconjugated form. After intragastric administration of the compounds, radioactivity was quantitatively recovered in feces. Cholate was 7-dehydroxylated to deoxycholate, whereas 31% of 7 beta-methyl-cholate and 78% of 7 alpha-methyl-ursocholate were recovered unchanged. Fifty percent of 7 beta-methyl-cholate and 15% of 7 alpha-methyl-ursocholate were transformed into ketonic derivatives, without loss of the 7-hydroxyl group. It is concluded that the introduction of the 7-methyl group did not interfere with intestinal absorption, hepatic extraction, and biliary secretion but did affect enzymatic amidation and bacterial 7-dehydroxylation of the analogues.

Animals↗
PubMed1997

Size of the catalytically active unit of rat hepatic carboxylester lipase in the presence and absence of bile salt.

Carboxylester lipase (CEL) catalyzes the hydrolysis of cholesteryl esters, retinyl esters, and triacylglycerols. CEL monomer has a MW of approximately 70000. Hydrolysis of these esters is stimulated by millimolar trihydroxy bile salts such as cholate, that also induce aggregation. Liver cytosols from 12 rats were frozen and irradiated at -135 degrees C with high energy electrons. In several experiments, paired samples of cytosol were adjusted to 20 mM cholate before irradiation. All samples were assayed for CEL using cholesteryl oleate as substrate. In untreated cytosols, CEL activity surviving radiation exposure could be fit to a single exponential function, the slope of which yielded a target size of 91 +/- 18 kDa. In a subset of these cytosols irradiated in the presence of cholate the calculated target size was 100 +/- 19 kDa, a value indistinguishable from that obtained for untreated cytosols. Some samples were also assayed using retinyl palmitate and triolein as substrates. With retinyl palmitate the mean target sizes were 96 and 108 kDa in the absence and presence of cholate, respectively, approximately the same as those observed when using cholesteryl oleate. When triolein was used as substrate the target sizes in the absence of cholate were smaller than for the other two esters (67 +/- 18 kDa) and closer to the known monomer molecular weight, but again cholate had no significant effect on this size. The structure responsible for CEL activity contains no more than one 70000 MW monomer and the results show that cholate-induced oligomerization is not required for catalytic activity.

Animals↗
PubMed1988

Stability of mixed micellar bile models supersaturated with cholesterol.

The maximal equilibrium solubility of cholesterol in mixtures of phosphatidylcholine (PC)1 and bile salts depends on the cholesterol/PC ratio (Rc) and on the effective ratio (Re) between nonmonomeric bile salts and the sum (CT) of PC and cholesterol concentrations (Carey and Small, 1978; Lichtenberg et al., 1984). By contrast, the concentration of bile salts required for solubilization of liposomes made of PC and cholesterol does not depend on Rc (Lichtenberg et al., 1984 and 1988). Thus, for Rc greater than 0.4, solubilization of the PC-cholesterol liposomes yields PC-cholesterol-bile salts mixed micellar systems which are supersaturated with cholesterol. In these metastable systems, the mixed micelles spontaneously undergo partial revesiculation followed by crystallization of cholesterol. The rate of the latter processes depends upon Rc, Re, and CT. For any given Rc and Re, the rate of revesiculation increases dramatically with increasing the lipid concentration CT, reflecting the involvement of many mixed micelles in the formation of each vesicle. The rate also increases, for any given CT and Re, upon increasing the cholesterol to PC ratio, Rc, probably due to the increasing degree of supersaturation. Increasing the cholate to lipid effective ratio, Re, by elevation of cholate concentration at constant Rc and CT has a complex effect on the rate of the revesiculation process. As expected, cholate concentration higher than that required for complete solubilization at equilibrium yields stable mixed micellar systems which do not undergo revesiculation, but for lower cholate concentrations decreasing the degree of supersaturation (by increasing [cholate]) results in faster revesiculation. We interpret these results in terms of the structure of the mixed micelles; micelles with two or more PC molecules per one molecule of cholesterol are relatively stable but increasing the bile salt concentration may cause dissociation of such 1:2 cholesterol:PC complexes, hence reducing the stability of the mixed micellar dispersions. The instability of PC-cholesterol-cholate mixed systems with intermediary range of cholate to lipids ratio may be significant to gallbladder stone formation as: (a) biliary bile contains PC-cholesterol vesicles which may be, at least partially, solubilized by bile salts during the process of bile concentration in the gallbladder, resulting in mixtures similar to our model systems; and (b) the bile composition of cholesterol gallstone patients is within an intermediary range of bile salts to lipids ratio.

Cholesterol↗
PubMed1991

Effect of bile salts on monolayer curvature of a phosphatidylethanolamine/water model membrane system.

A partial phase diagram of the ternary system dioleoylphosphatidylethanolamine (DOPE)/sodium cholate/water has been determined using 31P Nuclear Magnetic Resonance (NMR) spectroscopy. In the absence of cholate, it is well known that the DOPE/water system forms a reversed hexagonal (HII) phase. We have found that addition of even small amounts of cholate to the DOPE/water system leads to a transition to a lamellar (L alpha) phase. At higher cholate concentrations, a cubic (I) phase (low water content) or a micellar solution (L1) phase (high water content) is present. Thus, cholate molecules have a strong tendency to alter the lipid monolayer curvature. Increasing the concentration of cholate changes the curvature of DOPE from negative (HII phase), through zero (L alpha phase), and finally to a phase of positive curvature (micellar solution). This observation can be rationalized in terms of the molecular structure of cholate, which is amphipathic and has one hydrophobic and one hydrophilic side of the steroid ring system. The cholate molecules have a tendency to lie flat on the lipid aggregate surface, thereby increasing the effective interfacial area of the polar head groups, and altering the curvature free energy of the system.

Cholic Acid↗
PubMed1991

Effect of competing anions on binding of bile salts by cholestyramine.

The binding of bile salts by cholestyramine may be influenced by other anions, as the Langmuir adsorption coefficients for three bile salts tested were similar to the model anion, citrate. However, the selectivity coefficient indicated preferential binding of cholate anion in comparison to citrate anion. Binding experiments confirmed cholestyramine's preference for bile salts as the presence of other anions reduced but did not prevent the binding of cholate anion. Binding of cholate anion was reduced in direct relationship to the citrate anion concentration. Prior exposure of cholestyramine to citrate anion caused the binding of cholate anion to decrease slightly. Sequential exposure of cholestyramine to simulated gastric fluid and simulated intestinal fluid containing cholate anion resulted in a decrease in cholate binding which was attributed to competition with anions present in the gastrointestinal fluids. Components of tomato juice and orange juice, fluids, commonly used to enhance ingestion of cholestyramine, also reduced the binding of cholate anion.

Adsorption↗
PubMed1980

Effects of cholic acid, chenodeoxycholic acid, and their related bile acids on cholesterol, phospholipid, and bile acid levels in serum, liver, bile, and feces of rats.

Effects of sodium cholate, deoxycholate, chenodeoxycholate, and lithocholate on serum and liver cholesterol levels, bile flow, biliary cholesterol, phospholipids, and bile acids, and fecal sterols and bile acids were examined in Wistar strain male rats fed either an ordinary diet or a 2% cholesterol diet. Cholate and deoxycholate increased serum and liver cholesterol levels, serum pre beta-lipoprotein, bile flow, and biliary secretion of cholesterol, phospholipids, and bile acids, but chenodeoxycholate and lithocholate did not. The total amounts of sterols and of bile acids in the feces did not differ between the cholate and the chenodeoxycholate groups. All the bile acids except lithocholate decreased fecal coprostanol when the diet included cholesterol. Cholate and deoxycholate produced similar bile acid compositions in the bile and feces, as was the case between chenodeoxycholate and lithocholate, though chenodeoxycholate slightly increased the amount of muricholic acids, and lithocholate that of hyodeoxycholic acid, in the feces. The effects of cholate and deoxycholate are similar to each other but different from that of chenodeoxycholate or lithocholate in rats. Cholate causes marked accumulation of cholesterol in tissues, increased bile flow and biliary lipid secretion but chenodeoxycholate does not. Cholate is absorbed much more efficiently than chenodeoxycholate.

Animals↗
PubMed1988

Phospholipid requirement for expression of ice nuclei in Pseudomonas syringae and in vitro.

Delipidation of partially purified outer membranes of Pseudomonas syringae by various delipidating agents resulted in a significant loss of ice nucleation activity associated with the cell envelopes of this and other ice nucleation active bacteria. Lipopolysaccharide depletion of such membranes caused no reduction in ice nucleation activity. Both phospholipid content and ice nucleation activity of membranes were decreased by a similar fractional amount with time after treatment with phospholipase A2. A proportional quantitative relationship between loss of ice nucleation activity and lipid removal with increasing concentrations of sodium cholate and sodium dodecyl sulfate (SDS) was also observed. Significant linear relationships between the amount of lipid removed by phospholipase A2, sodium cholate, and SDS and the loss of ice nucleation activity in P. syringae outer membranes were observed. However, the slopes of these linear relationships for membranes treated with phospholipase A2 (m = 0.80), SDS (m = 0.94), and sodium cholate (m = 0.53) differed. The lower slope value for cholate-treated membranes indicated a partial substitution of sodium cholate for the phospholipids removed. The ice nucleation activity of delipidated outer membranes was restored by reconstitution with various phospholipids in a cholate dialysis procedure. Lipid classes differed in their ability to restore ice nucleation activity to sodium cholate-treated outer membranes. These results suggest that a hydrophobic environment provided either by lipids or certain detergent micelles is required for proper assembly and structural organization of an oligomeric ice protein complex enabling its expression as an ice nucleus.

Cell Membrane↗
PubMed1999

Complex feedback regulation of bile acid synthesis in the hamster: the role of newly synthesized cholesterol.

Hepatic bile acid synthesis is regulated by recirculating bile acids, possibly by modulating the availability of newly synthesized and preformed cholesterol. Because data in the hamster on this mechanism are lacking, we fitted these animals with an extracorporeal bile duct and administered tritiated water intraperitoneally to label newly formed cholesterol. After interruption of the enterohepatic circulation, physiological and double-physiological doses of conjugated cholate (25 or 50 micromol/100 g. h) or of unconjugated deoxycholate (6 or 12 micromol) were infused intraduodenally for 54 hours and compared with controls. De novo and preformed cholesterol directly secreted into bile or used for cholate and chenodeoxycholate synthesis were quantitated by high-pressure liquid chromatography (HPLC)-liquid scintillation. Directly after depletion of the bile acid pool (6-9 hours) at nearly physiological conditions, chenodeoxycholate synthesis was significantly reduced by cholate and deoxycholate by up to 45% to 51%, whereas cholate formation decreased by approximately 22% during deoxycholate. This short-term effect was mainly mediated by reduced synthesis from preformed cholesterol. After long-term bile depletion (30-54 hours), bile acid synthesis returned to control levels during 25 micromol of cholate and of both deoxycholate doses. In contrast, only 50 micromol of cholate prevented derepression of bile acid synthesis. This long-term effect was mainly attributed to a diminished formation from de novo cholesterol exceeding the reduced synthesis from preformed cholesterol. In summary, short- and long-term regulation of bile acid synthesis in hamsters differs with respect to availabilities of preformed and de novo cholesterol.

Amino Acids↗
PubMed1987

Comparison of bile acid binding to sinusoidal and bile canalicular membranes isolated from rat liver.

Simon et al. (J. Clin. Invest., 70 (1982) 401) studied cholate binding to crude liver plasma membrane vesicles and suggested that the binding may represent mainly the binding to the receptor (carrier) on the canalicular membrane. This hypothesis was supported by finding a good correlation between the number of cholate binding sites on liver plasma membrane and the maximal rate of biliary secretion (Tm) for taurocholate. We studied bile acid binding to sinusoidal and canalicular membrane vesicles isolated from rat liver by a rapid filtration technique. Scatchard analysis of the saturation kinetics showed both [3H]cholate and [3H]chenodeoxycholate bind to two classes of binding site on each membrane. However, little difference was observed between the binding to sinusoidal and canalicular membrane vesicles for each bile acid (cholate, Kd1 = 10.4 and 19.8 microM, n1 = 31.0 23.6 pmol/mg protein, Kd2 = 1.32 and 1.73 mM, n2 = 13.1 and 23.4 nmol/mg protein; and chenodeoxycholate, Kd1 = 0.207 and 0.328 microM, n1 = 36.7 and 27.4 pmol/mg protein, Kd2 = 1.16 and 2.26 mM, and n2 = 20.6 and 24.2 nmol/mg protein; numbers show the mean values sinusoidal and canalicular membrane vesicles, respectively). Chenodeoxycholate binding to sinusoidal membrane vesicles was markedly inhibited by cholate but not by Rose bengal, an organic anion dye. These studies indicate that both membranes (sinusoidal and canalicular membrane vesicles) have two kinds of binding site for bile acids, although no clear difference in the binding properties was observed between the two membranes. Consequently, the cholate binding Simon detected may represent the binding not only to canalicular membrane vesicles but also to sinusoidal membrane vesicles.

Animals↗
PubMed1980

Solubilization and partial characterization of rat liver iodothyronine deiodinases.

Rat liver cells contain iodothyronine deiodinating enzymes (iodothyronine-5 and 5'-deiodinases), which are associated with the endoplasmic reticulum. In the present study, the iodothyronine deiodinases have been solubilized from the microsomal fraction of rat liver with 1.0% cholate and 0.25% of the polyoxyethylene ether W-1. Cholate can be effectively removed from the cholate extract with a mixture of the polystyrene beads XAD-2 and XAD-7. However, after some time, aggregation of proteins occurred. Cholate solubilized iodothyronine-5'-deiodinase has an apparent molecular weight of 65,000 and a Stokes radius of 36-37 A. The sedimentation coefficient is 4.3 S in 0.4-0.6% cholate, 7.6 S in 0.05% W-1 ether and 12.8 S in the absence of detergent. The enzyme solubilized with W-1 ether has an apparent molecular weight of approx. 200,000 and a Stokes radius of 52-56 A in 0.025% W-1 ether. In the latter extract, the sedimentation coefficient of the deiodinase is 4.3-5.2 S under different conditions. On DEAE-Sepharose chromatography, 70% of the bound deiodinases eluted with 0.1 M NaCl. The purification of this fraction was only 2-fold. Covalent chromatography, using activated thiol-Sepharose, resulted in approximately 3-fold purification of the deiodinases solubilized with W-1 ether, whereas in case of the cholate extract, no purification at all was obtained. Glutathione-Sepharose affinity chromatography resulted in no enrichment of the deiodinases.

Animals↗
PubMed1992

Interaction of sulfonylureas with the transport of bile acids into hepatocytes.

The sulfonylurea compounds glisoxepide and glibenclamide inhibit the uptake of bile acids into isolated rat hepatocytes. The Ki values for the inhibition of cholate uptake was 9 microM with glibenclamide and 200 microM with glisoxepide. The inhibition of cholate uptake by both sulfonylureas was noncompetitive. Uptake of the conjugated bile acid taurocholate was inhibited by glibenclamide, Ki = 75 microM. Again the inhibition was noncompetitive. Glisoxepide inhibited taurocholate uptake only in the absence of sodium ions. Under sodium-free conditions glisoxepide also strongly inhibited cholate uptake. The inhibition was competitive, Ki = 42 microM. Both bile acids interfered with the hepatocellular uptake of [3H]glisoxepide, with IC50 values of 375 and 467 microM for cholate and taurocholate, respectively. The uptake of [3H]glibenclamide was inhibited by cholate, IC50 = 328 microM, but not by taurocholate. Glisoxepide uptake was further inhibited by blockers of the hepatocellular monocarboxylate transporter, by the loop diuretic bumetanide, by 4,4'-diisothiocyano-2,2'-stilbenedisulfonate (DIDS) and by sulfate. Glibenclamide uptake was weakly inhibited by DIDS and by anthracene-9-carboxylic acid (A-9-C) but not by bumetanide and sulfate. Neither bromosulfophthalein nor the fatty acid oleate inhibited glisoxepide or glibenclamide uptake. These results are consistent with the transport of glisoxepide via the transport system for the unconjugated bile acid cholate. Glibenclamide uptake is mediated by a still unknown hepatocellular transport system.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1986

Interaction of the inhibitory GTP regulatory component with soluble cerebral cortical adenylate cyclase.

Functional interaction of the inhibitory GTP regulatory component (Ni) with the adenylate cyclase catalytic subunit has not previously been demonstrated after detergent solubilization. The present report describes a sodium cholate-solubilized preparation of rat cerebral cortical membrane adenylate cyclase that retains guanine nucleotide-mediated inhibition of activity. Methods of membrane preparation, cholate extraction, and assay conditions were manipulated such that guanosine-5'-(beta-gamma-imido)triphosphate [Gpp(NH)p] inhibited basal activity 40-60%. The rank order of potency among various GTP analogs was similar in cholate extracts and in membranes: guanosine-5'-0-(3-thiotriphosphate) greater than Gpp(NH)p greater than GTP. Inclusion of 0.1 mM EGTA reduced basal activity 70-90% and abolished Gpp(NH)p inhibition of basal activity in both membranes and cholate extracts. Forskolin-stimulated activity was also inhibited by Gpp(NH)p. Treatment of either membranes or cholate extracts with N-ethylmaleimide abolished Gpp(NH)p inhibition. Gel filtration of the cholate extract over a Sepharose 6B column in 0.1% Lubrol PX partially resolved the adenylate cyclase components. However, Gpp(NH)p inhibition of basal activity (60% of the control) was maintained in select column fractions. Sucrose gradient centrifugation totally resolved the catalytic subunit from both functional Ni and stimulatory GTP regulatory component (Ns) activities. The sedimentation of functional Ni activity was detected by assaying the ability of sucrose gradient fractions to confer Gpp(NH)p inhibition of the resolved catalytic activity. Labeling of gradient or column fractions with pertussis toxin and [32P]NAD revealed that both the 39,000- and 41,000-dalton substrates comigrated with the functional Ni activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenylyl Cyclase Inhibitors↗
PubMed1996

Pancreatic carboxyl ester lipase: a circulating enzyme that modifies normal and oxidized lipoproteins in vitro.

Pancreatic carboxyl ester lipase (CEL) hydrolyzes cholesteryl esters (CE), triglycerides (TG), and lysophospholipids, with CE and TG hydrolysis stimulated by cholate. Originally thought to be confined to the gastrointestinal system, CEL has been reported in the plasma of humans and other mammals, implying its potential in vivo to modify lipids associated with LDL, HDL (CE, TG), and oxidized LDL (lysophosphatidylcholine, lysoPC). We measured the concentration of CEL in human plasma as 1.2+/-0.5 ng/ml (in the range reported for lipoprotein lipase). Human LDL and HDL3 reconstituted with radiolabeled lipids were incubated with purified porcine CEL without or with cholate (10 or 100 microM, concentrations achievable in systemic or portal plasma, respectively). Using a saturating concentration of lipoprotein-associated CE (4 microM), with increasing cholate concentration there was an increase in the hydrolysis of LDL- and HDL3-CE; at 100 microM cholate, the present hydrolysis per hour was 32+/-2 and 1.6+/-0.1, respectively, indicating that CEL interaction varied with lipoprotein class. HDL3-TG hydrolysis was also observed, but was only approximately 5-10% of that for HDL3-CE at either 10 or 100 microM cholate. Oxidized LDL (OxLDL) is enriched with lysoPC, a proatherogenic compound. After a 4-h incubation with CEL, the lysoPC content of OxLDL was depleted 57%. Colocalization of CEL in the vicinity of OxLDL formation was supported by demonstrating in human aortic homogenate a cholate-stimulated cholesteryl ester hydrolytic activity inhibited by anti-human CEL IgG. We conclude that CEL has the capability to modify normal human LDL and HDL composition and structure and to reduce the atherogenicity of OxLDL by decreasing its lysoPC content.

Animals↗
PubMed1994

Bile salt-dependent and bile salt-independent cholesteryl ester hydrolase activities in rat liver cytosol.

These studies report on the relationship between the bile salt-dependent and -independent cholesteryl ester hydrolase (CEH) activities found in rat liver cytosol. The two activities show very similar Michaelis-Menten substrate kinetics and pH dependence. After gel filtration of cytosol, the bile salt-independent activity elutes much earlier than the bile salt-dependent activity, suggesting that the two activities are associated with entities of different molecular size. However, when gel filtration is carried out in the presence of bile salt, the bile salt-dependent activity elutes as a large aggregate, similar to the bile salt-independent activity's behavior in the absence of bile salt. Both activities coelute after cytosol is passed through an ion exchange column. After each chromatographic procedure the recovery of the bile salt-dependent activity was substantially higher than the recovery of the bile salt-independent activity. When cytosol is incubated with anti-rat pancreatic CEH in the absence of cholate, the bile salt-dependent activity is inhibited more than 90% whereas bile salt-independent activity remains unaffected even at high antibody concentrations. When cytosol is incubated with anti-rat pancreatic CEH in the presence of cholate both CEH activities remain unaffected. The prevention of immunoinhibition by cholate seems to be specific for this detergent since CHAPS, a cholate analog, does not prevent immunoinhibition of the bile salt-dependent activity by anti-CEH. The experimental results are consistent with a model for CEH activity in liver cytosol in which there is only one enzyme that can exist in a monomeric, inactive form (that can be activated by addition of cholate to the assay and represents the bile salt-dependent activity) and in an active complex comprising several enzyme monomers as well as cholate micelles (that accounts for the bile salt-independent activity).

Animals↗
PubMed1976

[The significance of the bacterial steroid degradation for the etiology of large bowel cancer. IV. Deconjugation of glycocholic acid, oxidation, and reduction of cholic acid by saccharolytic Bacteroides species (author's transl)].

A total of 36-38 strains (depending on the test series) of the strictly anaerobic Bacteroides species V. bulgatus, B. fragilis, B. thetaiotamicron, and B. distasonis was tested for the ability of splitting the acide amide linkage of glycocholate and for a further degradation of cholate. We found 23 of 38 strains (60 per cent) to be able to deconjugate glycocholate, but as many as 18 (46 per cent) resulted in an intensive to complete degradation. On the other hand 32 of 36 strains (89 per cent) were able to degrade cholate when cultivated anaerobically. They normally formed 1 transformation product, however, occasionally 2-3. Using the aerobic incubation of resting cells as test system we observed 30 active strains of 37 (81 per cent), which as well produced 1 degradation product generally, but sometimes 2-4 compounds. 3 strains transformed cholate during growth only. Thin layer-, gas chromatography, and combined gas chromatography-mass spectrometry were used for the identification of transformation products. We proved the main degradation product of all active strains to be 3alpha,12alpha-dihydroxy-7-oxo-5beta-cholanoate, by anaerobic cultivation as well as by aerobic incubation of cell suspensions. Deoxycholate (3alpha,12alpha-dihydroxy-5beta-cholanoate), in vivo the main product of cholate transformation, was hitherto found to be formed by 3 strains only as main degradation product, it may probably be a by-product of the degradation with other strains. All these strains, however, may also oxidise the 7alpha-hydroxyl group beside dehydroxylating cholate. For this reason we assume that the conditions for the formation of deoxycholate are in general not existent in our experiments. The third degradation product, formed under anaerobic conditions, is probably a monohydroxy-monooxo-cholanoate, derived from deoxycholate. Structures of additional transformation products, occasionally found under aerobic incubation, which are all less polar than 3alpha,12alpha-dihydroxy-7-oxo-5beta-cholanoic acid, could not be elucidated on account of minor amounts available. Bacteroides species are unable to transform the side chain of cholate, either as anaerobically growing cultures, or as aerobically incubated resting cells.

Aerobiosis↗
PubMed2003

Preparation and characterization of reactive and stable glucose oxidase-containing liposomes modulated with detergent.

Glucose oxidase-containing liposomes (GOL) as well as detergent-modulated glucose oxidase-containing liposomes were prepared and characterized, focusing not only on the reactivity of the liposomes upon external addition of glucose but also on the leakage of the entrapped glucose oxidase (GO) from the liposomes with the aim of developing a reactive and stable liposomal GO system. The membranes of the GOL prepared were composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and modulated with either Triton X-100 or cholate. In the absence of added detergent, no GO leakage from the GOL was observed while its enzymatic activity was very low (low glucose permeability). As detergent-modulated liposomes, mixed POPC/Triton X-100 and mixed POPC/cholate liposomes (abbreviated as TL and CL, respectively) were prepared at different effective detergent/POPC molar ratios (R(e)) ranging from R(e) = 0 to R(e) = R(e) (sat) (R(e) (sat) is the critical value of R(e) at which the liposome membrane is saturated with detergent). The reactivity of GO-loaded TL (abbreviated as GOTL) or GO-loaded CL (GOCL) increased drastically with increase in the respective detergent content in the liposomes. In the case of GOTL, at R(e) (sat) = 0.40, a high reactivity was measured with a simultaneous high extent of GO leakage, suggesting that the observed enzymatic reaction was catalyzed mainly by leaked GO, caused by the interaction of Triton X-100 with the POPC membrane. On the other hand, GOCL prepared at R(e) (sat) = 0.43 showed relatively high reactivity with only a small extent of GO leakage, suggesting that most of the enzyme reaction was limited by the glucose permeation across the bilayers of GOCL. The GO leakage from GOCL was found to occur mostly during the rearrangement of the liposomal membrane during the preparation of the GOCL (mixing the GOL and cholate). Fluorescence polarization measurements of membrane-associated DPH (1,6-diphenyl-1,3,5-hexatriene) indicated that CL prepared by modifying POPC with cholate did not lead to a drastic change in membrane fluidity, indicating that the interacting cholate molecules did not penetrate deeply into the POPC bilayers. In summary, it was clearly shown that the membrane permeability of GOL can be quite simply modulated by mixing it with a certain amount of cholate to form highly reactive and stable GOCL with minimal enzyme leakage.

Cholates↗
PubMed2004

Expression of ABCG5 and ABCG8 is required for regulation of biliary cholesterol secretion.

The major pathway for elimination of cholesterol in mammals is via secretion into bile. Biliary cholesterol secretion is mediated by the ATP-binding cassette (ABC) transporters ABCG5 (G5) and ABCG8 (G8) and is stimulated by cholesterol and by the non-cholesterol steroids cholate and diosgenin. To define the relationship between G5G8 expression and biliary cholesterol secretion, we measured G5 and G8 mRNA levels and biliary cholesterol concentrations in genetically manipulated mice expressing 0, 1, 2, 5, 10, or 16 copies of the two genes. Biliary cholesterol levels varied directly with G5G8 copy number and hepatic mRNA levels over a >16-fold range. Thus neither delivery of cholesterol to the transporter nor levels of cholesterol acceptors in bile were limiting under these conditions. In wild-type mice, cholate and diosgenin both increased biliary cholesterol concentrations 2-3-fold. The increase in biliary cholesterol content was dependent on expression of G5 and G8; neither steroid increased biliary cholesterol levels in G5G8-/- mice. Cholate treatment was associated with a farnesoid X receptor (FXR)-dependent increase in hepatic mRNA and protein levels of G5 and G8. In contrast to cholate, diosgenin treatment did not affect G5G8 expression. Diosgenin increased the expression of several pregnane X receptor (PXR) target genes and the choleretic effect of diosgenin was reduced by approximately 70% in PXR knock-out mice. Thus G5 and G8 are required to modulate biliary cholesterol secretion in response to cholate and diosgenin, but the choleretic effects of these two steroids are mediated by different mechanisms requiring FXR and PXR, respectively.

ATP Binding Cassette Transporter, Subfamily G, Mem↗