The apoprotein fraction of the bile lipoprotein complex: isolation, partial characterization and phospholipid binding properties

A bile apoprotein fraction (Apo BLC) was isolated by preparative isoelectric focusing (I.E.F.) from the detergent-free form of the bile lipoprotein complex (BLC). Analytical I.E.F. of Apo BLC yields a characteristic and reproducible pattern of two narrow acidic bands (pI 4,8-5,0). This apoprotein presents a strong tendency to undergo self-aggregation in aqueous buffer. A low molecular weight constituent of Apo BLC has been isolated after gel filtration, its mean Mw is estimated by SDS-PAGE at 7,500 daltons. The binding capacity of Apo BLC for phospholipids was investigated on dimyristoylphosphatidylcholine liposomes by gel filtration and zone electrophoresis. The resulting structures, larger than the original single-shelled vesicles, acquire and anodic electrophoretic mobility. Apo BLC has a weaker affinity for lysophosphatidylcholines: these phospholipids decrease the degree of aggregation of the apoprotein. These studies contribute additional data concerning the high affinity of Apo BLC for phosphatidylcholines, which are the major phospholipid constituents of bile. The discussion deals with the fact that association of Apo BLC with bile phosphatidylcholines may present some implications in the pathogeny of LpX and in the process of intestinal fat absorption.     

Hepatocellular transport and secretion of biliary lipids.

Bile is the route for elimination of cholesterol from the body. Recent studies have begun to elucidate hepatocellular, molecular and physical-chemical mechanisms whereby bile salts stimulate biliary secretion of cholesterol together with phospholipids, which are enriched (up to 95%) in phosphatidylcholines. Active translocation of bile salts and phosphatidylcholines across the hepatocyte's canalicular plasma membrane provides the driving force for biliary lipid secretion. This facilitates physical-chemical interactions between detergent-like bile salt molecules and the ectoplasmic leaflet of the canalicular membrane, which result in biliary secretion of cholesterol and phosphatidylcholines as vesicles. Within the hepatocyte, separate molecular pathways function to resupply bile salts, phosphatidylcholines and cholesterol to the canalicular membrane for ongoing biliary lipid secretion.     

Structural basis for bile salt inhibition of pancreatic phospholipase A2

Bile salt interactions with phospholipid monolayers of fat emulsions are known to regulate the actions of gastrointestinal lipolytic enzymes in order to control the uptake of dietary fat. Specifically, on the lipid/aqueous interface of fat emulsions, the anionic portions of amphipathic bile salts have been thought to interact with and activate the enzyme group-IB phospholipase A2 (PLA2) derived from the pancreas. To explore this regulatory process, we have determined the crystal structures of the complexes of pancreatic PLA2 with the naturally occurring bile salts: cholate, glycocholate, taurocholate, glycochenodeoxycholate, and taurochenodeoxycholate. The five PLA2-bile salt complexes each result in a partly occluded active site, and the resulting ligand binding displays specific hydrogen bonding interactions and extensive hydrophobic packing. The amphipathic bile salts are bound to PLA2 with their polar hydroxyl and sulfate/carboxy groups oriented away from the enzyme's hydrophobic core. The impaired catalytic and interface binding functions implied by these structures provide a basis for the previous numerous observations of a biphasic dependence of the rate of PLA2 catalyzed hydrolysis of zwitterionic glycerophospholipids in the presence of bile salts. The rising or activation phase is consistent with enhanced binding and activation of the bound PLA2 by the bile salt induced anionic charge in a zwitterionic interface. The falling or inhibitory phase can be explained by the formation of a catalytically inert stoichiometric complex between PLA2 and any bile salts in which it forms a stable complex. The model provides new insight into the regulatory role that specific PLA2-bile salt interactions are likely to play in fat metabolism.     

Evidence for the synthesis and secretion of APF--a bile lipid associated protein--by isolated rat hepatocytes

Bile lipids are thought to be secreted in a lipoprotein complex in which they are associated with cholesterol and a protein called the anionic polypeptidic fraction (APF). APF is present in both bile and serum HDL. The association of APF with both bile and lipoprotein strongly suggests that hepatocytes may be responsible for the synthesis and secretion of this protein. In the present work we attempted to verify this by studying the incorporation of [14C]leucine into APF in isolated rat hepatocytes and by immunolocalization in cell cultures. Results obtained showed that synthesis of APF by cells follows the same kinetic pattern as albumin and that it was the third most abundant protein in the bile secretion. Immunolocalization confirmed that APF is synthesized in the endoplasmic reticulum of hepatocytes. This protein which appears to be rapidly secreted could be of great value for the specific detection of the lipids destined for bile secretion.     

Dietary soybean phosphatidylcholines lower lipidemia: mechanisms at the levels of intestine, endothelial cell, and hepato-biliary axis

The beneficial metabolic effects of dietary soybean lecithin on lipid metabolism are now more clearly established. The intestinal absorption of cholesterol is decreased by soybean phosphatidylcholine-enriched diet and results in a cholesterol-lowering effect. There is an enhancement of the cholesterol efflux by endothelial cells incubated with soybean phosphatidylcholines, and a stimulation of the reverse cholesterol transport by high density lipoprotein-phosphatidylcholines. As a result of all these processes, phosphatidylcholines provided by the soybean lecithin metabolism appear to be key molecules controlling the biodynamic exchanges of lipids. They regulate homeostasis of cholesterol and fatty acids by decreasing their synthesis and promoting cholesterol oxidation into bile salts. Finally, the outcome is the increase in bile secretion of these lipids and/or their metabolite forms. Such findings constitute promising goals in the field of nutritional effects of soybean lecithin in the treatment or prevention of hyperlipidemia and related atherosclerosis.     

Role of 57-72 loop in the allosteric action of bile salts on pancreatic IB phospholipase A(2): regulation of fat and cholesterol homeostasis

Mono- and biphasic kinetic effects of bile salts on the pancreatic IB phospholipase A2 (PLA2) catalyzed interfacial hydrolysis are characterized. This novel phenomenon is modeled as allosteric action of bile salts with PLA2 at the interface. The results and controls also show that these kinetic effects are not due to surface dilution or solubilization or disruption of the bilayer interface where in the mixed-micelles substrate replenishment becomes the rate-limiting step. The PLA2-catalyzed rate of hydrolysis of zwitterionic dimyristoylphosphatidylcholine (DMPC) vesicles depends on the concentration and structure of the bile salt. The sigmoidal rate increase with cholate saturates at 0.06 mole fraction and changes little at the higher mole fractions. Also, with the rate-lowering bile salts (B), such as taurochenodeoxycholate (TCDOC), the initial sigmoidal rate increase at lower mole fraction is followed by nearly complete reversal to the rate at the pre-activation level at higher mole fractions. The rate-lowering effect of TCDOC is not observed with the (62-66)-loop deleted DeltaPLA2, or with the Naja venom PLA2 that is evolutionarily devoid of the loop. The rate increase is modeled with the assumption that the binding of PLA2 to DMPC interface is cooperatively promoted by bile salt followed by allosteric k(cat)(*)-activation of the bound enzyme by the anionic interface. The rate-lowering effect of bile salts is attributed to the formation of a specific catalytically inert E(*)B complex in the interface, which is noticeably different than the 1:1 EB complex in the aqueous phase. The cholate-activated rate of hydrolysis is lowered by hypolidemic ezetimibe and guggul extract which are not interfacial competitive inhibitors of PLA2. We propose that the biphasic modulation of the pancreatic PLA2 activity by bile salts regulates gastrointestinal fat metabolism and cholesterol homeostasis.     

Relationships between bile salts hydrophilicity and phospholipid composition in bile of various animal species

Bile salts and phospholipids from bile of chicken, dog, sheep, rat, ox, pig, guinea-pig and man were analyzed by high-performance liquid chromatography. Bile salts showed marked differences in their hydrophilic properties, owing to hydroxyl structure and type of conjugation. Phospholipids were generally similar, containing 90-95% of phosphatidylcholine which was made of molecular species containing palmitic acid in the sn-1 position. The comparative analysis of bile salts and phosphatidylcholines profile demonstrated that bile salts hydrophilicity influences the quantity of phosphatidylcholine in bile but not the quality.     

Role of 57-72 loop in the allosteric action of bile salts on pancreatic IB phospholipase A2: Regulation of fat and cholesterol homeostasis

Mono- and biphasic kinetic effects of bile salts on the pancreatic IB phospholipase A2 (PLA2) catalyzed interfacial hydrolysis are characterized. This novel phenomenon is modeled as allosteric action of bile salts with PLA2 at the interface. The results and controls also show that these kinetic effects are not due to surface dilution or solubilization or disruption of the bilayer interface where in the mixed-micelles substrate replenishment becomes the rate-limiting step. The PLA2-catalyzed rate of hydrolysis of zwitterionic dimyristoylphosphatidylcholine (DMPC) vesicles depends on the concentration and structure of the bile salt. The sigmoidal rate increase with cholate saturates at 0.06 mole fraction and changes little at the higher mole fractions. Also, with the rate-lowering bile salts (B), such as taurochenodeoxycholate (TCDOC), the initial sigmoidal rate increase at lower mole fraction is followed by nearly complete reversal to the rate at the pre-activation level at higher mole fractions. The rate-lowering effect of TCDOC is not observed with the (62-66)-loop deleted ΔPLA2, or with the Naja venom PLA2 that is evolutionarily devoid of the loop. The rate increase is modeled with the assumption that the binding of PLA2 to DMPC interface is cooperatively promoted by bile salt followed by allosteric k_cat^?-activation of the bound enzyme by the anionic interface. The rate-lowering effect of bile salts is attributed to the formation of a specific catalytically inert E^?B complex in the interface, which is noticeably different than the 1:1 EB complex in the aqueous phase. The cholate-activated rate of hydrolysis is lowered by hypolidemic ezetimibe and guggul extract which are not interfacial competitive inhibitors of PLA2. We propose that the biphasic modulation of the pancreatic PLA2 activity by bile salts regulates gastrointestinal fat metabolism and cholesterol homeostasis.     

Pyrophosphate is a source of phosphoryl groups for Escherichia coli protein phosphorylation

Abstract Stress tolerance and cross-protection in Enterococcus faecalis ATCC19433 were examined after exposure to bile salts, acid or heat shock. Bile salts and heat adapted cells demonstrated induced homologous tolerance and cross-resistance. No cross-protection of heat adapted cells against acid stress is observed and pretreatment with bile salts even sensitized the cells to this challenge. Whole-cell protein extract analysis revealed that each treatment induced a battery of stress proteins. Some of these polypeptides are induced by more than one treatment. The greatest overlap is observed between bile salts and heat treatments. Eighteen stress proteins, including DnaK and GroEL, are common between these stresses.     

Use of novel cationic bile salts in cholesterol crystallization and solubilization in vitro

Unnatural bile salts have been synthesized with a cationic group at the side chain of natural bile acids. These cationic bile salts aggregate in water and aqueous salt solutions in a manner similar to their natural counterparts. The critical micellar concentrations of the cationic bile salts were measured using a fluorescence method. Cationic bile salts aggregated at a concentration lower than natural deoxycholic acid. Since dihydroxy bile salt micelles are well known for cholesterol dissolution/removal, the dissolution in the cationic micelles has been evaluated. The cationic analogs dissolve approximately 70 mg/dL of cholesterol, which is comparable to taurochenodeoxycholate micelle under identical bile salt concentrations. Cholesterol dissolution in cationic bile salt micelle enhanced upon adding various amounts of PC. Cholesterol crystallization was studied in model bile at various cationic bile salt concentrations. The addition of 5, 15 and 30 mM of the cationic bile salts attenuated the crystallization process, without influencing the crystal observation time or decreasing the final amount of crystals formed. All these effects were comparable to those observed with cholic acid. These findings suggest that cationic bile salts have physico-chemical properties analogous to those of natural anionic bile salts, and thus may have therapeutic potential.     

On the probable involvement of arginine residues in the bile-salt-binding site of human pancreatic carboxylic ester hydrolase

Modification of arginine residues with 2,3-butanedione inhibits the carboxylic-ester hydrolase activity on soluble and emulsified substrates when assayed with bile salts. The alpha-dicarbonyl reagent modifies seven of the nineteen arginine residues present per enzyme molecule. Nevertheless the inactivation with butanedione is greatly diminished when the protein is in the presence of negatively charged micellar bile salt. In these conditions we observe the protection of one arginine residue by sodium taurodeoxycholate and of two arginine residues by sodium cholate. This suggests that the carboxylic-ester hydrolase from human pancreatic juice contains at least two arginine residues essential for the activation by bile salts. All our data confirm the presence of two bile-salt-binding sites on the enzyme in which one arginine per site is involved and plays the general role of an anionic binding site. This study provides evidence that arginine residues may play an essential role in the interaction between bile salts and protein.     

Structural analysis of the high molecular mass aminoacyl-tRNA synthetase complex. Effects of neutral salts and detergents.

The effects of a variety of detergents and neutral salts on the structure of the eukaryotic high molecular mass aminoacyl-tRNA synthetase complex have been directly determined by observing alterations in the composition, sedimentation behavior, and electron microscopic appearance of the rabbit reticulocyte complex. The intact complex is shown to exhibit the enzymatic activities, polypeptide composition, relative stoichiometry, and morphological features that are characteristic of this eukaryotic multienzyme particle. The structure of the high molecular mass aminoacyl-tRNA synthetase complex is seen to be resistant to both ionic and nonionic detergents. However, both 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate and deoxycholate induce formation of large protein aggregates. In contrast, the chaotropic salts LiCl and NaSCN both selectively remove individual polypeptides from the high molecular mass aminoacyl-tRNA synthetase complex and promote formation of specific particulate subcomplexes which have distinct sizes, polypeptide compositions, and structural features. These data support the view that many of the protein interactions within the high molecular mass amino-acyl-tRNA synthetase complex are hydrophobic in nature. This study also provides direct evidence that the complex contains a core of tightly interacting synthetases onto which the remaining polypeptides are arrayed. The structural alterations observed here may account for the ability of these reagents to markedly inhibit several enzymatic activities within the complex.     

Effect of bile anionic polypeptidic fraction on the fate of cholesterol carried by liposomes in the rat

[14C]Cholesterol associated with liposomes with or without anionic polypeptidic fraction was administered intravenously to the rat. The cholesterol originated from liposomes including anionic polypeptidic fraction is secreted in bile much later, is stored in liver in higher quantity, and is metabolized into bile salts in lesser quantity during the 4 hr of experimentation than the cholesterol issued from liposomes exempt of anionic polypeptidic fraction. From these results it can be postulated that the cholesterol associated with liposomes containing anionic polypeptidic fraction might be directed in a particular liver pathway.     

Susceptibility and adaptive response to bile salts in Propionibacterium freudenreichii: physiological and proteomic analysis

Tolerance to digestive stresses is one of the main factors limiting the use of microorganisms as live probiotic agents. Susceptibility to bile salts and tolerance acquisition in the probiotic strain Propionibacterium freudenreichii SI41 were characterized. We showed that pretreatment with a moderate concentration of bile salts (0.2 g/liter) greatly increased its survival during a subsequent lethal challenge (1.0 g/liter, 60 s). Bile salts challenge led to drastic morphological changes, consistent with intracellular material leakage, for nonadapted cells but not for preexposed ones. Moreover, the physiological state of the cells during lethal treatment played an important role in the response to bile salts, as stationary-phase bacteria appeared much less sensitive than exponentially growing cells. Either thermal or detergent pretreatment conferred significantly increased protection toward bile salts challenge. In contrast, some other heterologous pretreatments (hypothermic and hyperosmotic) had no effect on tolerance to bile salts, while acid pretreatment even might have sensitized the cells. Two-dimensional electrophoresis experiments revealed that at least 24 proteins were induced during bile salts adaptation. Identification of these polypeptides suggested that the bile salts stress response involves signal sensing and transduction, a general stress response (also triggered by thermal denaturation, oxidative toxicity, and DNA damage), and an alternative sigma factor. Taken together, our results provide new insights into the tolerance of P. freudenreichii to bile salts, which must be taken into consideration for the use of probiotic strains and the improvement of technological processes.     

Bile salts in submicellar concentrations promote bidirectional cholesterol transfer (exchange) as a function of their hydrophobicity

Cholesterol, despite its poor solubility in aqueous solutions, exchanges efficiently between membranes. Movement of cholesterol between different subcellular membranes in the hepatocyte is necessary for assembly of lipoproteins, biliary cholesterol secretion, and bile acid synthesis. Factors which initiate and facilitate transfer of cholesterol between different membranes in the hepatocyte are incompletely understood. It is known that cholesterol secretion into the bile is linked to bile salt secretion. In the present study, we investigated the effects of bile salts of different physicochemical properties at submicellar concentrations (150- 600 microM) on the transfer of [14C]cholesterol from hepatocytes, or crude hepatocellular membranes (donors), to rat high density lipoproteins (acceptor). Bile salts included taurine conjugates of ursodeoxycholic acid (TUDCA), hyodeoxycholic acid (THDCA), cholic acid (TCA), chenodeoxycholic acid (TCDCA), and deoxycholic acid (TDCA). High density lipoprotein (HDL) was separated from hepatocellular membranes and the transfer of [14C]cholesterol from the membranes to HDL was quantitatively determined. In the absence of HDL, [14C]cholesterol remained confined to the membrane fraction. Following addition of HDL, [4-14C]cholesterol in the HDL fraction increased linearly over time. Addition of hydrophilic bile salts (TUDCA and THDCA) increased transfer of [4-14C]cholesterol to HDL only minimally. By contrast, more hydrophobic bile salts stimulated transfer of labeled cholesterol to HDL, and their potency increased in order of increasing hydrophobicity (TCA less than TCDCA less than TDCA). Both for single bile salts and mixtures of bile salts at a total bile salt concentration of 0.30 mM, the rate of cholesterol transfer exhibited a strong linear correlation with a bile salt monomeric hydrophobicity index (r = 0.95; P less than 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of various denaturing treatments on the structure and activity of a purified CP1 complex

Spinach CP1 complex, purified as previously described [16], was submitted to various dissociating treatments. Chaotropic agents, like urea and thiocyanate salts, remained without effect on the structure and photooxidation of the complex, just SDS at very high concentrations was able to dissociate the chlorophyll from the polypeptides and to abolish the photoreaction. Proteolytic enzymes have no more action on the apparent structure and activity of CP1, but some of them do cleave the large polypeptides (65 kD) into smaller ones, as observed after pigments dissociation. This last result might be an important step in the search for a smaller active P700 protein complex.Abbreviations CP1 pigment-protein complex with the slower mobility in SDS electrophoresis - MW molecular weight - PSI photosystem I - PSII photosystem II - SDS sodium dodecylsulfate     

Susceptibility and Adaptive Response to Bile Salts in Propionibacterium freudenreichii: Physiological and Proteomic Analysis

Tolerance to digestive stresses is one of the main factors limiting the use of microorganisms as live probiotic agents. Susceptibility to bile salts and tolerance acquisition in the probiotic strain Propionibacterium freudenreichii SI41 were characterized. We showed that pretreatment with a moderate concentration of bile salts (0.2 g/liter) greatly increased its survival during a subsequent lethal challenge (1.0 g/liter, 60 s). Bile salts challenge led to drastic morphological changes, consistent with intracellular material leakage, for nonadapted cells but not for preexposed ones. Moreover, the physiological state of the cells during lethal treatment played an important role in the response to bile salts, as stationary-phase bacteria appeared much less sensitive than exponentially growing cells. Either thermal or detergent pretreatment conferred significantly increased protection toward bile salts challenge. In contrast, some other heterologous pretreatments (hypothermic and hyperosmotic) had no effect on tolerance to bile salts, while acid pretreatment even might have sensitized the cells. Two-dimensional electrophoresis experiments revealed that at least 24 proteins were induced during bile salts adaptation. Identification of these polypeptides suggested that the bile salts stress response involves signal sensing and transduction, a general stress response (also triggered by thermal denaturation, oxidative toxicity, and DNA damage), and an alternative sigma factor. Taken together, our results provide new insights into the tolerance of P. freudenreichii to bile salts, which must be taken into consideration for the use of probiotic strains and the improvement of technological processes.     

Identification of different transport systems for bile salts in sinusoidal and canalicular membranes of hepatocytes

The preservation of the functional polarity of hepatocytes in liver snips (1 x 2 x 4 mm) was demonstrated by fluorescent microscopic studies using the sodium salt of (N-[7-(4-nitrobenzo-2-oxa-1,3-diazol)]-3 beta-amino-7 alpha,12 alpha- dihydroxy-5 beta-cholan-24-oyl)-2-aminoethanesulfonic acid. This fluorescent bile salt derivative is not only taken up by hepatocytes of several cell layers at the surface of the snips but also secreted into bile canaliculi. The intact hepatobiliary transport of bile salts by hepatocytes of liver snips demonstrates that they are a useful system for the investigation of those transcellular transport processes which require the integrity of hepatic structure. Photoaffinity labelling of liver snips with the sodium salt of (7,7-azo-3 alpha,12 alpha-dihydroxy-5 beta-[3 beta-3H]cholan- 24-oyl)-2-aminoethanesulfonic acid revealed that the bile-salt-binding membrane polypeptides with apparent Mr values of 54,000 and 48,000 are exclusively located in the sinusoidal membrane, whereas a single bile-salt-binding polypeptide with an apparent Mr of 100,000 is located in the bile-canalicular membrane. Photoaffinity labelling of liver snips at 4 degrees C, when transcellular bile-salt transport is insignificant, resulted in the labelling of the two sinusoidal membrane polypeptides and practically no labelling of the polypeptide with an apparent Mr of 100,000. This latter polypeptide was also not labelled when Ca2 deprivation abolished bile secretion completely. These results indicate that the directed hepatobiliary transport of bile salts in hepatocytes is accomplished by transport systems which are different for sinusoidal uptake and canalicular secretion.     

Comparison of the bile salts and sodium dodecyl sulfate stress responses in Enterococcus faecalis.

The resistance to detergents and detergent-induced tolerance of a gastrointestinal organism, Enterococcus faecalis ATCC 19433, were examined. The most remarkable observation was the rapid response of cells in contact with bile salts and sodium dodecyl sulfate (SDS). The killing by high concentrations of detergents was nearly instantaneous. A 5-s adaptation with moderate sublethal concentrations of bile salts or SDS (0.08 or 0.01%, respectively) was sufficient to induce significant adaptation against homologous lethal conditions (0.3% bile salts or 0.017% SDS). However, resistance to a subsequent lethal challenge progressively increased further to a maximum reached after 30 min of adaptation. Furthermore, extremely strong cross-resistances were observed with bile salts- and SDS-adapted cells. However, no relationship seems to exist between levels of tolerance and de novo-synthesized proteins, since blockage of protein synthesis during adaptation had no effect on induction of resistance to bile salts and SDS. We conclude that this induced tolerance to detergent stress is independent of protein synthesis. Nevertheless, the stress-induced protein patterns of E. faecalis ATCC 19433 showed significant modifications. The rates of synthesis of 45 and 34 proteins were enhanced after treatments with bile salts and SDS, respectively. In spite of the overlap of 12 polypeptides, the protein profiles induced by the two detergents were different, suggesting that these detergents trigger different responses in E. faecalis. Therefore, bile salts cannot be substituted for SDS in biochemical detergent shock experiments with bacteria.     

The superfamily of organic anion transporting polypeptides

Organic anion transporting polypeptides (Oatps/OATPs) form a growing gene superfamily and mediate transport of a wide spectrum of amphipathic organic solutes. Different Oatps/OATPs have partially overlapping and partially distinct substrate preferences for organic solutes such as bile salts, steroid conjugates, thyroid hormones, anionic oligopeptides, drugs, toxins and other xenobiotics. While some Oatps/OATPs are preferentially or even selectively expressed in one tissue such as the liver, others are expressed in multiple organs including the blood-brain barrier (BBB), choroid plexus, lung, heart, intestine, kidney, placenta and testis. This review summarizes the actual state of the rapidly expanding OATP superfamily and covers the structural properties, the genomic classification, the phylogenetic relationships and the functional transport characteristics. In addition, we propose a new species independent and open ended nomenclature and classification system, which is based on divergent evolution and agrees with the guidelines of the Human Genome Nomenclature Committee.