Circular dichroism analysis of the secondary structure of Z protein and its complexes with bilirubin and other organic anions.

Circular dichroism (CD) methods were employed to study the conformation of Z protein and characterize its complexes with bilirubin and other organic anions. Z protein-bilirubin complexes exhibited a spectrum with overlapping ellipticity bands of opposite sign in the bilirubin absorption region. These results were compared with those obtained with ligandin, the other major organic anion binding protein of liver. Secondary structural differences between the two proteins were easily demonstrated since ligandin is predominantly an alpha-helical protein and Z features mainly beta-structure. Furthermore, the optical activity pattern generated by bilirubin binding to Z was virtually a mirror image of that of the ligandin bilirubin system. CD experiments were designed to study the direct transfer of bilirubin between Z protein and ligandin, and it was shown that both proteins have almost equal affinities for bilirubin. The bilirubin on Z was readily displaced by oleic acid and displaced to a lesser extent by sulfobromophthalein,     

Hepatic organic anion transport kinetics and bile flow during short-term total parenteral nutrition in the rabbit

Plasma disappearance of sulfobromophthalein (BSP) after an intravenous bolus (5 mg/kg) was determined in six lab chow-fed (LCF) rabbits and in six rabbits maintained on total parenteral nutrition (TPN) for 5 days. A common bile duct cannula enabled measurements of bile flow and biliary BSP excretion. Compartmental analysis of the biexponential plasma disappearance curve yielded three fractional transfer rates, plasma to liver (hepatic uptake), liver to plasma (reflux), and liver to bile (canalicular excretion). The transfer rates for hepatic uptake were 0.253 +/- 0.061/min for LCF and 0.147 +/- 0.040/min for TPN (P less than 0.01) and for the canalicular excretion of BSP were 0.038 +/- 0.019/min for LCF and 0.019 +/- 0.002/min for TPN (P less than 0.05). Model-computed rates for BSP excretion in bile over 60 min were lower with TPN (61%) than with LCF (80%); the measured excretory rates were 53% for TPN rabbits and 75% of injected dose for LCF animals. Basal biliary flow was reduced by 50% in the TPN group. With a two-compartmental model, assuming two pools and three transfer rates, we have demonstrated for the first time significant decreases in hepatic uptake and canalicular excretion of the organic anion BSP during TPN. A decrease in hepatic blood flow due to the enteral fast of TPN could have contributed in part to the decreased hepatic uptake. But, because the second exponent of the biexponential curve is independent of hepatic blood flow, the decrease in liver to bile transfer rate is a true approximation of a diminished canalicular excretory capacity during TPN. It is concluded that the movement of organic anions along the hepatic BSP/bilirubin transport system is impaired early during TPN.     

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

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

Effect of free fatty acid anion on the in vitro binding of 1-anilino-8-naphthalene sulfonate to cytoplasmic proteins from rat liver.

The influence of oleate ion, a free fatty acid anion, on the binding characteristics of 1-anilino-8-naphthalene sulfonate (ANS) with the cytoplasmic proteins (Y and Z) from rat liver has been examined using fluorescence spectroscopy. ANS binds strongly with both ligandin (Y) and Z protein at a single binding site with dissociation constants of 0.6 and 1.4 micron respectively. Increasing concentrations of oleate ion decreased the ANS binding with either protein by competing with the ANS binding site. Relative binding constant of oleate ion for the hepatic ligandin or Z protein was about 2 micron as determined from the competitive inhibition of ANS binding. These results suggest that variations in the hepatic cytoplasmic free fatty acid concentration may be important in regulating the capacity of Y and Z proteins to transport other organic anions.     

Multiplicity of Hepatic Excretory Mechanisms for Organic Anions

Previous studies based upon competition between different organic anions for biliary excretion in vivo have suggested that all organic anions share a common hepatic secretory mechanism. Corriedale sheep with an inherited defect in organic anion excretion by the liver were used to study this problem directly without the need for competition studies, the results of which are difficult to analyze. Maximal biliary excretion of sulfobromphthalein (BSP) in mutant Corriedale sheep was less than 7% of that observed in normal sheep whereas maximal biliary excretion of taurocholate, the major organic anion in sheep bile, was not different in mutant and normal sheep. Taurocholate infusion enhanced maximal hepatic excretion of BSP in normal but not in mutant sheep. These studies of an inheritable disorder which appears to be identical to the Dubin-Johnson syndrome in man, demonstrate that taurocholate excretion requires at least one step in biliary excretion which is not required by other organic anions such as bile pigment, porphyrins, drugs, and dyes.     

Imide Products from Photo-oxidation of Bilirubin and Mesobilirubin

IN 1958 Cremer et al.¹ observed that the concentration of bilirubin (la) in the plasma could be reduced by exposing newborn infants to fluorescent light. Since that time phototherapy has come into wide use to lower elevated bilirubin levels associated with neonatal jaundice (hyperbilirubin-aemia)^{2, 3}. This condition in the newborn has been associated with retarded motor development, irreversible brain damage or even death. Phototherapy lowers bilirubin levels (by conversion of this lipid-soluble pigment to water-soluble products)⁴ and therefore presumably helps to prevent brain damage. But there are two possible dangers in this treatment: light may have other deleterious effects on the newborn and the photo-products of bilirubin may themselves be toxic. At present neither the structures of the bilirubin photo-products nor their toxicities have been established. Although the photo-destruction of bilirubin has been studied in vivo and in vitro by Ostrow^5–7, Schmid^{4, 7} and others^{8, 9}, these authors investigated principally the visible-ultraviolet spectral changes during the course of bilirubin photo-oxidation and recorded paper chromatographic separations of the photo-products for comparison with the bile or urine of the congenitally jaundiced Gunn rat. More recently McDonagh has shown that singlet oxygen is involved in the self-sensitized photo-oxidation of bilirubin¹⁰. In view of the paucity of structural.information on the bilirubin photo-products, we wish to report preliminary findings on the in vitro photo-oxidation products from bilirubin IXa (1a), mesobilirubin IXa (1b) and 5′-oxo-3′,4,4′-triethyl-3,5-dimethyl-l′,5′-dihydro-(2.2′)-dipyrrylmethene (2). We synthesized and carried out our initial studies on 2, which serves as a simplified model for rings I and II of 1a and 1b because it lacks the vinyl group of 1a and the propionic acid β-substituent of 1a and 1b. The visible-ultraviolet spectrum of 2 is quite similar to that of either 1a or 1b^11–13.     

On the mechanism of bilitranslocase transport inactivation by phenylmethylsulphonyl fluoride

Bilitranslocase is a plasma membrane carrier involved in the uptake of bilirubin and other organic anions from the blood into the liver cell. In the membrane, the carrier occurs as two interchangeable metastable forms, with high and low affinity for the substrates, respectively. The latter form can be specifically produced by either cysteine- or arginine modification. In liver plasma membrane vesicles, the serine-specific reagent phenylmethylsulphonyl fluoride is a partial inhibitor of bilitranslocase-mediated BSP transport rate. In this work, phenylmethylsulphonyl fluoride is shown to reduce the carrier maximal transport rate, without affecting its affinity for that substrate. In addition, it is found that the chemical modification caused by this reagent neither influences the equilibrium between the high- and the low-affinity forms nor prevents their free interconversion. From the effects of combined derivatizations of cysteine(s), arginine(s) and serine(s), it is concluded that the functionally relevant aminoacid residues lie in a close spatial arrangement. Also, in this study, the PMSF-modified serine(s) is shown to be involved in bilirubin binding by bilitranslocase.     

On the mechanism of bilitranslocase transport inactivation by phenylmethylsulphonyl fluoride.

Bilitranslocase is a plasma membrane carrier involved in the uptake of bilirubin and other organic anions from the blood into the liver cell. In the membrane, the carrier occurs as two interchangeable metastable forms, with high and low affinity for the substrates, respectively. The latter form can be specifically produced by either cysteine- or arginine modification. In liver plasma membrane vesicles, the serine-specific reagent phenylmethylsulphonyl fluoride is a partial inhibitor of bilitranslocase-mediated BSP transport rate. In this work, phenylmethyl-sulphonyl fluoride is shown to reduce the carrier maximal transport rate, without affecting its affinity for that substrate. In addition, it is found that the chemical modification caused by this reagent neither influences the equilibrium between the high- and the low-affinity forms nor prevents their free interconversion. From the effects of combined derivatizations of cysteine(s), arginine(s) and serine(s), it is concluded that the functionally relevant aminoacid residues lie in a close spatial arrangement. Also, in this study, the PMSF-modified serine(s) is shown to be involved in bilirubin binding by bilitranslocase.     

Z protein: Isolation and characterization of multiple forms in rat liver cytosol

The Z protein fraction of rat liver cytosol contains one or more proteins which have been associated with organic anion transport, fatty acid metabolism, and aminoazodye binding. To study the possible identity of these proteins and investigate their function, Z was purified using ammonium sulfate fractionation, gel filtration, and preparative isoelectric focusing. Three protein fractions were obtained (pI 5.2, 6.0, 7.3) which reacted specifically with anti-Z IgG. These three fractions were homogenous as determined by several electrophoretic systems. Monospecific antibody prepared against two of the proteins cross-reacted specifically with all three. Each fraction bound BSP with different affinity; acidic Z bound the least BSP. The molecular weight of each fraction was 12,500 as determined by SDS-gel electrophoresis. Amino acid analyses of the three Z protein bands were virtually identical. Heterogeneity in Z probably results from interaction of the protein with ampholytes or exogenous ligands.     

Bilirubin binding to hepatic Y and Z protein (ligandin): tissue bilirubin concentrations in phenobarbital treated Gunn rats (38466).

NaP treatment significantly reduced plasma bilirubin levels in the (jj) Gunn rat. It is suggested that NaP significantly increased extravascular bilirubin binding in the liver by induction of the synthesis of Y anion binding protein. The changes in bilirubin concentration of blood and other tissues subsequent to NaP treatment were consistent with the establishment of a new tissue bilirubin equilibrium.     

Characterization of the Mechanisms Involved in the Increased Renal Elimination of Bromosulfophthalein During Cholestasis: Involvement of Oatp1

The kidneys and liver are the major routes for organic anion elimination. We have recently shown that acute obstructive jaundice is associated with increased systemic and renal elimination of two organic anions, p-aminohippurate and furosemide, principally excreted through urine. This study examined probable adaptive mechanisms involved in renal elimination of bromosulfophthalein (BSP), a prototypical organic anion principally excreted in bile, in rats with acute obstructive jaundice. Male Wistar rats underwent bile duct ligation (BDL rats). Pair-fed sham-operated rats served as controls. BSP renal clearance was performed by conventional techniques. Renal organic anion-transporting polypeptide 1 (Oatp1) expression was evaluated by immunoblotting and IHC. Excreted, filtered, and secreted loads of BSP were all higher in BDL rats compared with Sham rats. The higher BSP filtered load resulted from the increase in plasma BSP concentration in BDL rats, because glomerular filtration rate showed no difference with the Sham group. The increase in the secreted load might be explained by the higher expression of Oatp1 observed in apical membranes from kidneys of BDL animals. This likely adaptation to hepatic injury, specifically in biliary components elimination, might explain, at least in part, the huge increase in BSP renal excretion observed in this experimental model. (J Histochem Cytochem 57:449–456, 2009)     

Hepatic uptake of bilirubin diglucuronide: analysis by using sinusoidal plasma membrane vesicles

In order to characterize the mechanism for bilirubin transport in the liver, the uptake of bilirubin diglucuronide (BDG) into purified sinusoidal plasma membrane vesicles was investigated. BDG uptake was saturable, and was inhibited by sulfobromophthalein and unconjugated bilirubin, but was not affected by sodium taurocholate. BDG uptake was sodium-independent and was stimulated by intravesicular bilirubin or BDG (trans-stimulation). BDG transport showed strong potential sensitivity; vesicle inside-negative membrane potential created by different anion gradients inhibited BDG uptake whereas vesicle inside-positive membrane potential generated by potassium gradients and valinomycin markedly stimulated BDG transport. These data suggest that BDG, sulfobromophthalein, and probably unconjugated bilirubin share a common transporter in liver cells which is sodium independent, membrane-potential-dependent and capable of exchange. The direction of transport in vivo may be governed by the intracellular concentration of BDG and of other yet unidentified organic anions sharing this transporter.     

Overexpression of Constitutively Active OsRab11 in Plants Enhances Tolerance to High Salinity Levels

Rab proteins are key regulators of intracellular trafficking between specific compartments in a cell. Among them, Rab11, a widely conserved sub-group, mainly regulates plasma membrane (PM) trafficking. Previously, we reported that Oryza sativa Rab11 (OsRab11) plays an important role in the intracellular trafficking from the trans-Golginetwork (TGN) to the plasma membrane (PM) and prevacuolar compartments (PVCs), and in the plant’s response to high salt stress. In this study, when the constitutively active mutant of OsRab11, (CA OsRab11(Q73L)) was co-transformed with Arabidopsis Ca²⁺-ATPase8-GFP (ACA8-GFP) or sporamin-GFP (Spo-GFP) into Arabidopsis protoplasts, the PM or vacuolar trafficking proportion of the reporter proteins was highly increased. Transgenic Arabidopsis plants overexpressing (OE) CA OsRab11(Q73L) exhibited enhanced tolerance to high salt stress and exogenous abscisic acid (ABA) compared to Col plants. Moreover, certain stress-responsive genes were expressed under high salt stress and ABA treatment in OEOsRab11(Q73L) plants. Thus, these results suggest that the active conformation of OsRab11 may be required to modulate plant responses to salt and ABA via the regulation of the expression of stress-responsive genes.     

Isolation of an organic anion binding protein from rat liver plasma membrane fractions by affinity chromatography

As part of a study of hepatic organic anion transport, solubilized liver plasma membrane proteins were subjected to affinity chromatography on bilirubin- and sulfobromophthalein-labeled agarose columns. Both columns retained a Sudan Black and PAS negative protein of molecular weight 60,000 daltons, which cochromatographed with [³⁵S]sulfobromophthalein on Sephadex G-75, and reversibly bound [³⁵S]sulfobromophthalein in vitro with high affinity (K_a ? 10⁷ M^?1) and a valence of 2. Erythrocyte ghost membranes did not contain this protein. Sulfobromophthalein-agarose retained two additional smaller proteins which did not cochromatograph with [³⁵S]sulfobromophthalein. Their significance is unclear. This study supports the hypothesis that liver cell plasma membranes participate in the hepatic transport of organic anions.     

Uptake of bilirubin into HepG2 cells assayed by thermal lens spectroscopy. Function of bilitranslocase

Bilitranslocase is a carrier protein localized at the basolateral domain of the hepatocyte plasma membrane. It transports various organic anions, including bromosulfophthalein and anthocyanins. Functional studies in subcellular fractions enriched in plasma membrane revealed a high-affinity binding site for bilirubin, associated with bilitranslocase. The aim of this work was to test whether the liver uptake of bilirubin depends on the activity of bilitranslocase. To this purpose, an assay of bilirubin uptake into HepG2 cell cultures was set up. The transport assay medium contained bilirubin at a concentration of approximately 50 nm in the absence of albumin. To analyse the relative changes in bilirubin concentration in the medium throughout the uptake experiment, a highly sensitive thermal lens spectrometry method was used. The mechanism of bilirubin uptake into HepG2 cells was investigated by using inhibitors such as anti-sequence bilitranslocase antibodies, the protein-modifying reagent phenylmethanesulfonyl fluoride and diverse organic anions, including nicotinic acid, taurocholate and digoxin. To validate the assay further, both bromosulfophthalein and indocyanine green uptake in HepG2 cells was also characterized. The results obtained show that bilitranslocase is a carrier with specificity for both bilirubin and bromosulfophthalein, but not for indocyanine green.     

Effects of chaotropic anions on bicuculline inhibition of high affinity γ-aminobutyric acid binding in bovine retina and rat brain membranes

Chaotropic anions (ions that favour transfer of apolar groups to water) increased bicuculline inhibition of ³H-γ-aminobutyric acid binding to bovine retinal membranes as previously reported for rat forebrain membranes. The increased bicuculline inhibition was reversible when the chaotropic anion was removed thus ruling out the possibility that ‘endogenous regulators’ were being removed by the chaotropic anions. Another possible explanation for the enhanced bicuculline inhibition is an increase in the solubility of bicuculline, an organic compound that is sparingly soluble in water; however, when bicuculline-methiodide, a more water soluble form of bicuculline was used, no difference was noted in this enhancement.Although the chaotropic anions temporarily increase the bicuculline inhibition of γ-aminobutyric acid binding, they do not increase ³H-γ-aminobutyric acid receptor binding as previously suggested. Thiocyanate or perchlorate have no effect on ³H-γ-aminobutyric acid receptor binding to rat forebrain or cerebellar membranes. Although thiocyanate slightly inhibits γ-aminobutyric acid receptor binding to bovine retinal membranes, perchlorate has no effect. The previous observation that sodium perchlorate enhanced γ-aminobutyric acid binding in bovine retina was due to the enhancement of sodium-dependent binding to a nipecotic acid-sensitive binding site (perhaps an uptake site).The mechanism of action of chaotropic anions in vitro on γ-aminobutyric acid binding may be through an alteration in the interaction of the receptor with the antagonists, bicuculline or bicuculline-methiodide, but is not through the removal of a component that blocks the binding site or regulates the receptor's properties, as evidenced by the reversibility of the chaotropic anion effect and the lack of effect on agonist binding.     

Functional characterization of plasma membrane-localized organic acid transporter (CsALMT1) involved in aluminum tolerance in <Emphasis Type="Italic">Camelina sativa</Emphasis> L.

Aluminum (Al) toxicity is a major limiting factor that inhibits root elongation and decreases crop production in acidic soils. The symptoms of inhibited root growth include a reduced uptake of nutrients because the roots become stubby and brittle. The release of organic anions from roots can protect a plant from Al toxicity. The mechanism relies on the efflux of organic anions, such as malate or citrate, which protect roots by chelating the Al³⁺. In this study, homologs of TaALMT1, a Camelina gene that encodes an aluminum-activated malate transporter, were investigated. The expression of this gene was induced by Al in the root, but not in the shoots. Using green fluorescent protein (GFP) fusion constructs and Western-blot analysis, we observed that CsALMT1 was localized in the plasma membrane. Also, to determine the degree to which Al tolerance was affected by malate secretion in Camelina root, we generated CsALMT1 overexpressing plants. CsALMT1 overexpressing transgenic plants showed a higher root elongation rate than the wild-type plant. Damaged cell staining analysis by hematoxylin under 25 µM Al treatment for 2, 4, and 6 h showed a pattern of less damage in CsALMT1 transgenic plants than in wild-type plant, especially in the root elongation zone. Furthermore, the rate of increase of secretion of organic acid in overexpressed plants after Al treatment was higher than that in the wild-type plant. In addition, in the Al-specific dye morin staining on root protoplast under 50 µM Al treatment, less Al accumulation was observed in the CsALMT1 transgenic plants than in the wild-type plant. The Al contents in the roots of the transgenic plants were at a lower level than those in the wild-type plant. These results show that the overexpression of CsALMT1 improves Al tolerance by increasing the release of malate from the root to the soil and, thereby, detoxifies the Al³⁺.     

Conjugative chromosomal gene transfer in <Emphasis Type="Italic">Bacillus subtilis</Emphasis>

Conjugative transfer of 20-kb chromosomal fragment carrying genes encoding tetracycline (tet ^r ) and lincomycin (lin ^r ) resistance in the soil strain Bacillus subtilis 19 is described. Transfer was preceded by this fragment insertion into the large conjugative p19cat plasmid producing a hybrid plasmid. Insertion frequency was 10^?4?10^?5. Then genes tet ^r and lin ^r were transferred to the recipient strains. The transfer of chromosomal genes inserted into the plasmid and plasmid gene cat occurred sequentially and resembled sexduction, which represents chromosomal gene transfer by F′ and R′ plasmids during conjugation in Escherichia coli and other gram negative bacteria.     

Channel-like slippage modes in the human anion/proton exchanger ClC-4

The ClC family encompasses two classes of proteins with distinct transport functions: anion channels and transporters. ClC-type transporters usually mediate secondary active anion–proton exchange. However, under certain conditions they assume slippage mode behavior in which proton and anion transport are uncoupled, resulting in passive anion fluxes without associated proton movements. Here, we use patch clamp and intracellular pH recordings on transfected mammalian cells to characterize exchanger and slippage modes of human ClC-4, a member of the ClC transporter branch. We found that the two transport modes differ in transport mechanisms and transport rates. Nonstationary noise analysis revealed a unitary transport rate of 5 × 10⁵ s⁻¹ at +150 mV for the slippage mode, indicating that ClC-4 functions as channel in this mode. In the exchanger mode, unitary transport rates were 10-fold lower. Both ClC-4 transport modes exhibit voltage-dependent gating, indicating that there are active and non-active states for the exchanger as well as for the slippage mode. ClC-4 can assume both transport modes under all tested conditions, with exchanger/channel ratios determined by the external anion. We propose that binding of transported anions to non-active states causes transition from slippage into exchanger mode. Binding and unbinding of anions is very rapid, and slower transitions of liganded and non-liganded states into active conformations result in a stable distribution between the two transport modes. The proposed mechanism results in anion-dependent conversion of ClC-type exchanger into an anion channel with typical attributes of ClC anion channels.