Gian Luigi Sottocasa

Reconstitution in vitro of sulfobromophthalein transport by bilitranslocase

Liposomes containing 150 mM KCl and 0.48 mM sulfobromophthalein have been prepared. The internal pH was set at 6.5, a value at which sulfobromopthalein is colorless. When brought to alkaline pH a certain amount of the dye is deprotonated and can be read spectrophotometrically as external sulfobromophthalein. Upon addition of Triton X-100 the membrane is dissolved and all sulfobromophthalein present in the preparation may be measured. Addition of bilitranslocase to such a preparation of liposomes causes the internal sulfobromophthalein to leave the internal compartment. The rate of this phenomenon may be followed directly and shown to be greatly accelerated by the addition of valinomycin. The latter finding indicates that sulfobromophthalein transport occurs in response to a membrane diffusion potential created by permeabilisation to K+ of liposomes brought about by valinomycin (uniport). The permeability change induced by bilitranslocase is specific and does not reflect an alteration of the normal impermeability of liposomes to small ions such as protons or Ca2+.

Cellular localization of sulfobromophthalein transport activity in rat liver

The movement of sulfobromophthalein is measured in rat liver plasma-membrane vesicles by direct dual-wavelength spectrophotometry. The technique is based on the principle that the dye, when entering a more acidic compartment, changes its absorption in the visible region. From this study it may be concluded that, among the different cellular subfractions, only liver plasma-membrane vesicles can catalyze electrogenic transport of sulfobromophthalein. Plasma membranes from erythrocytes are unable to perform such a function. The movement follows the distribution pattern of (Na+ + K+)-ATPase and it is therefore concluded that this process occurs exclusively at the sinusoidal membrane level. Inhibition studies confirm that the process is catalyzed by bilitranslocase.

Specific sequence-directed anti-bilitranslocase antibodies as a tool to detect potentially bilirubin-binding proteins in different tissues of the rat.

The hypothesis that the uneven distribution of bilirubin in the organism, which occurs in hyperbilirubinemia, could reflect an uneven distribution of bilirubin‐binding proteins was tested by searching for peptides containing the bilirubin‐binding motif identified in bilitranslocase (Battiston et al., 1998). In the rat, positive proteins bands were found to be present only in the liver, gastric mucosa and central nervous system. The electrophoretic mobilities of the positive compounds in the liver and stomach were identical to that of purified bilitranslocase (38 kDa). In the brain, on the contrary, two peptides were found with molecular masses of 79 and 34 kDa, respectively. Their distribution pattern in the central nervous system was different for each of them.

The quinoid structure is the molecular requirement for recognition of phthaleins by the organic anion carrier at the sinusoidal plasma membrane level in the liver

Sulfobromophthalein electrogenic uptake into rat liver plasma membrane vesicles was shown to admit only the quinoid, trivalent anion. The minimum requirement for this electrogenic process has been investigated in rat liver plasma membrane vesicles by using Thymol blue, a pH-indicator phthalein occurring either as a neutral, phenolic molecule or as a quinoid, monovalent anion. It has been found that Thymol blue is taken up electrogenically, in accordance with Michaelis-Menten kinetics. Parallel inhibition experiments have shown that both sulfobromophthalein and Thymol blue electrogenic uptakes are performed by the same carrier. It is, therefore, concluded that the phthalein structure recognized for transport is the quinoid molecule, with the dissociated acidic function on the benzene ring. Moreover, inhibitions by rifamycin-SV and bilirubin suggest that there exists a common uptake system for bilirubin, phthaleins and other anions. Taurocholate, on the contrary, does not appear to be involved in the same process.

Sex differences of nicotinate-induced hyperbilirubinemia in Gilbert's syndrome: Implication of bilitranslocase function

Intravenous administration of nicotinic acid (NA) is followed by an increase in serum unconjugated bilirubin level. This effect is higher in Gilberts syndrome (GS) and this test has been used in the diagnosis of the syndrome. After administration of 5.9 mumol NA/kg body weight, the maximal increment of serum unconjugated bilirubin and the area under the bilirubin concentration time curve (AUC) were significantly higher (P less than 0.01) in GS males than in GS females. The half-life of the first fast slope of plasma disappearance curve of the drug was also significantly prolonged in GS males as compared to GS females (15.91 +/- 1.12 vs 9.13 +/- 1.25 min, mean +/- SEM, P less than 0.005). The maximal bilirubin increment and AUC were linearly correlated (P less than 0.01) with NA plasma half-life. Purified preparations of bilitranslocase, a liver plasma-membrane protein involved in bilirubin and sulfobromophthalein (BSP) transport, specifically bound NA and the drug competitively inhibited BSP uptake in rat liver plasma membrane vesicles (Ki = 50 nM). These data suggest that, in addition to the hemolytic effect of the drug, NA-induced hyperbilirubinaemia could be also due to a competition between the two anions at the sinusoidal plasma membrane level. A possible implication of bilitranslocase in GS is considered.

Measurement of the association of cholephylic organic anions with different binding proteins

The binding of the colored cholephylic anions tetrabromosulfonphthalein (BSP), di-bromosulfonphthalein (DBSP), indocyanine green (ICG) and thymol blue (ThB) to a number of protein preparations including bovine serum albumin, human serum, rat hepatic cytosol and purified rat liver bilitranslocase has been studied by a direct spectrophotometric method. The experimentation provides extinction coefficients, dissociation constants and number of binding sites for the different complexes between dyes and the various proteins. Data obtained by this technique were in excellent agreement with those obtained on the same samples by ultrafiltration. The data presented indicate that the direct spectrophotometry applied to these dyes is simple, rapid and reproducible, making this the approach of choice during the purification of binding proteins when the binding capacity is the only useful criterion to follow the progress of the procedure.

Isolation of bilitranslocase, the anion transporter from liver plasma membrane for bilirubin and other organic anions

Publisher Summary This chapter discusses the isolation of bilitranslocase, the anion transporter from liver plasma membrane for bilirubin and other organic anions. Bilitranslocase––a carrier protein involved in sulfobromophthalein (BSP) and other organic anion transport––has been isolated from rat liver plasma membranes. A more direct demonstration of the origin and involvement of bilitranslocase in the transport function has been obtained from immunochemical studies. The only way to trace bilitranslocase during a purification procedure is to follow the BSP binding capacity at the different purification steps. This can be done either by gel filtration or by membrane ultrafiltration. In both cases, the principle is to separate physically the protein-dye complex and to measure the amount of the dye bound in equilibrium with the free form. This chapter also discusses the estimate of BSP binding to protein by gel filtration on BioGel P-2.

The sulfhydryl groups responsible for bilitranslocase transport activity respond to the interaction of the carrier with bilirubin and functional analogues.

Both inactivation of sulfobromophthalein transport in rat liver plasma membrane vesicles by sulfhydryl group reagents and subsequent reactivation by 2-mercaptoethanol are shown to be modulated by ligands to bilitranslocase. In particular, bilirubin, sulfobromophthalein and Thymol blue behave as negative effectors in the inactivation reaction and as positive effectors in the reactivation reaction. Kinetic data provide further evidence of the existence of two classes of sulfhydryl groups involved in transport activity. The effect brought about by remarkably low concentrations of bilirubin is in line with the physiological function of bilitranslocase as a bilirubin carrier.

Sex steroid modulation of the hepatic uptake of organic anions in rat

To investigate the role of sex steroids in the sex-related difference in the hepatic uptake of organic anions, sulphobromophthalein (bromsulphalein, BSP) transport was measured in hepatocytes isolated from rats either deprived of hormonal influence by castration at prepubertal age or after hormonal substitution. In control animals, the kinetics of BSP uptake showed the presence of two components: one saturable (0-3 microM), with high affinity and low capacity, and the other linear (9-30 microM), probably related to the non-specific component of BSP uptake. Sex difference was detected only in the saturable portion of the uptake process as the apparent Km was significantly lower in females than in males (3.8 +/- 0.7 vs. 6.1 +/- 1.8 microM, mean +/- S.D. of six animals, P less than 0.01). In contrast, no difference was observed in Vmax (2.3 +/- 0.3 vs. 2.2 +/- 0.7 nmol BSP.(mg protein)-1.min-1). Castration was associated with the disappearance of the saturable uptake site and abolished the sex difference. Progesterone treatment of castrated males failed to restore the saturable kinetics of BSP uptake. In contrast, administration of oestradiol to castrated males or testosterone to castrated females did restore the saturable kinetics of the high-affinity BSP uptake. Km and Vmax were comparable to those of adult females and males, respectively, with the exception of testosterone which induced a Vmax value higher than that observed in the other groups of animals. These data suggest that the influence of oestrogen and testosterone is necessary for the expression of the high-affinity, low-capacity carrier-mediated process of hepatic BSP uptake.(ABSTRACT TRUNCATED AT 250 WORDS)

Gastric uptake of nicotinic acid by bilitranslocase

Sabina Passamonti*, Lucia Battiston, Gian Luigi SottocasaBilitranslocase is a 38 kDa membrane protein [1] localised atthe vascular surface of the liver cells. The isolated protein isendowed with transport function of sulphobromophthalein(SBP), as directly demonstrated in reconstituted systems.Thus, its proposed physiological role is to mediate the tra⁄cof SBP and related organic anions across the sinusoidal do-main of the hepatocyte plasma membrane. On account of theknown competition between SBP and bilirubin for the ¢rststep of liver uptake observed in vivo, bilirubin had beenpointed out as the most obvious physiological substrate forbilitranslocase. A strong indication in this sense has come withthe ¢nding, within bilitranslocase primary structure, of thesequence EDSQGQHLSSF [2], which has the peculiarity ofbeing the central portion of a motif highly conserved in K-phycocyanins, biliproteins found in cyanobacteria. Whereasin K-phycocyanins this motif is involved in binding the pros-thetic group phycocyanobilin [3], a compound structurally re-lated to bilirubin, in bilitranslocase it has been shown to act asthe bilirubin-binding site [2]. Indeed, the a⁄nity of the carrierfor the pigment is high enough to ensure bilirubin extractionfrom the blood stream (K