Characterization of electrogenic bromosulfophthalein transport in carnation petal microsomes and its inhibition by antibodies against bilitranslocase Sabina Passamonti1, Alessandra Cocolo1, Enrico Braidot2, Elisa Petrussa2, Carlo Peresson2, Nevenka Medic1, Francesco Macri2 and Angelo Vianello2 ` Dipartimento di Biochimica Biofisica e Chimica delle Macromolecole, Universita di Trieste, Italy ` Dipartimento di Biologia ed Economia Agro-Industriale, Sezione di Biologia Vegetale, Universita di Udine, Italy Keywords anthocyanin; bilitranslocase; bromosulfophthalein; liver, plant Correspondence S Passamonti, Dipartimento di Biochimica Biofisica e Chimica delle Macromolecole, ` Universita di Trieste, via L Giorgeri 1, I-34127 Trieste, Italy Fax: +39 40 558 3691 Tel: +39 40 558 3681 E-mail: passamonti@bbcm.units.it Website: http://www.bbcm.units.it (Received March 2005, revised 15 April 2005, accepted May 2005) doi:10.1111/j.1742-4658.2005.04751.x Bilitranslocase is a rat liver plasma membrane carrier, displaying a highaffinity binding site for bilirubin It is competitively inhibited by grape anthocyanins, including aglycones and their mono- and di-glycosylated derivatives In plant cells, anthocyanins are synthesized in the cytoplasm and then translocated into the central vacuole, by mechanisms yet to be fully characterized The aim of this work was to determine whether a homologue of rat liver bilitranslocase is expressed in carnation petals, where it might play a role in the membrane transport of anthocyanins The bromosulfophthalein-based assay of rat liver bilitranslocase transport activity was implemented in subcellular membrane fractions, leading to the identification of a bromosulfophthalein carrier (KM ¼ 5.3 lm), which is competitively inhibited by cyanidine 3-glucoside (Ki ¼ 51.6 lm) and mainly noncompetitively by cyanidin (Ki ¼ 88.3 lm) Two antisequence antibodies against bilitranslocase inhibited this carrier In analogy to liver bilitranslocase, one antibody identified a bilirubin-binding site (Kd ¼ 1.7 nm) in the carnation carrier The other antibody identified a high-affinity binding site for cyanidine 3-glucoside (Kd ¼ 1.7 lm) on the carnation carrier only, and a high-affinity bilirubin-binding site (Kd ¼ 0.33 nm) on the liver carrier only Immunoblots showed a putative homologue of rat liver bilitranslocase in both plasma membrane and tonoplast fractions, isolated from carnation petals Furthermore, only epidermal cells were immunolabelled in petal sections examined by microscopy In conclusion, carnation petals express a homologue of rat liver bilitranslocase, with a putative function in the membrane transport of secondary metabolites Anthocyanins are red to purple pigments belonging to the vast family of plant secondary metabolites, which accumulate in the central vacuole of plant cells Those pigments belong to the family of flavonoids and occur mainly as glycosides, playing several roles related to ecological aspects of plant life, e.g petal and leaf coloration, UV-B protection, antimicrobial activity and plant–animal interactions [1] In addition, they are endowed with diverse medicinal properties, including antioxidant, anti-inflammatory, estrogenic and antitumour activities [2] The biosynthesis of anthocyanins occurs in the cytoplasm, where many of the enzymes involved have been detected [3,4] It is thought that most of them get assembled as a membrane-associated, multienzyme complex, in contact with multiple proteins in the Abbreviations BSP, bromosulfophthalein; FITC, fluorescein isothiocyanate; PVPP, polyvinylpoly pyrrolidone 3282 FEBS Journal 272 (2005) 3282–3296 ª 2005 FEBS S Passamonti et al cytosol [5–7] Flavonoids originate from the central phenylpropanoid and the acetate-malonate pathways Therefore, all flavonoids may be considered as derived from phenylalanine, synthesized by the shikimate pathway, whereas malonyl-CoA originates from the reaction catalysed by acetyl-CoA carboxylase The phenylpropanoid biosynthesis is highly regulated both at the gene and the protein level [8] Based on these properties, genetic manipulations have been carried out in order to improve the defence response of plants [9] Being synthesized in the cytoplasm, anthocyanins have to be transported into the vacuole The mechanisms of transport through the tonoplast are not fully understood yet At least three carrier-mediated models have been proposed The first involves an H+-driven antiport [10], whose activity depends on the proton electrochemical potential generated both by the H+-ATPase and H+-PPiase [11] By analogy, this model may also include the protein encoded by the tt12 gene in Arabidopsis thaliana [12], a member of the multidrug and toxic compound extrusion family that functions as a Na+ ⁄ multidrug antiporter [13] The second model postulates the existence of carriers exploiting either structural modifications of anthocyanins occurring in the cytosol [14] or conformational changes of anthocyanins, occurring in the vacuolar lumen, possibly depending on their protonation [15] The third model is an ATP-energised mechanism catalysed by ATP-binding cassette transporters They are insensitive to protonophores, strongly inhibited by vanadate and also utilized for the translocation of xenobiotics [16– 18] and anthocyanins [19] It has been proposed that naturally occurring glycosylated secondary metabolites enter the vacuole by an H+-driven antiport, whereas glycosylated xenobiotics are transferred by ABC transporters [20] The vacuolar transport of anthocyanins is, however, a complex event, requiring not only membrane transporters but also the presence of glutathione transferases (EC 2.5.1.18), such as BZ2 in maize and AN9 in petunia [21], or TT19 in A thaliana [22] These glutathione transferases appear to act as flavonoidbinding proteins rather than as enzymes, because no conjugate species is formed in vitro [23] Besides that, vesicle trafficking also participates in delivering anthocyanins and other secondary metabolites to subcellular compartments [24] Bilitranslocase (TC 2.A.65.1.1, http://tcdb.ucsd.edu/tcdb/background.php [25]) is a plasma membrane organic anion carrier [26,27], localized at the sinusoidal domain of liver cells [28] and in the epithelium of the gastric mucosa [29] The activity of bilitranslocase, assayed as bromosulfophtalein (BSP) uptake in rat liver plasma membrane FEBS Journal 272 (2005) 3282–3296 ª 2005 FEBS Bilitranslocase homologue in carnation petals vesicles, is competitively inhibited by a number of anthocyanins, including mono- and di-glycosylated derivatives, suggesting that this carrier could be involved in anthocyanin uptake from the blood into the liver [30], as well as from the gastric lumen into the blood [31] The ability of bilitranslocase to interact with anthocyanins led us to consider the hypothesis that a similar carrier protein could be present in the vacuolar membrane of plant cells To this purpose, we investigated the presence of bilitranslocase in carnation petals and found a BSP uptake, inhibited by antibodies against bilitranslocase, in microsomal, plasma membrane and tonoplast vesicle fractions In addition we showed that a protein cross-reacted with these antibodies in both isolated membranes and fixed epidermal cells Carnation petals were chosen because they have a relatively simple anatomical structure, with a single layer of epidermal cells, featured by a large vacuole containing anthocyanins On the other hand, carnation petals have already provided a suitable material for studying alterations of membrane structure and activity associated to plant senescence [32,33] Results Bilitranslocase transport activity is assayed in rat liver subcellular fractions by a spectrophotometric method, exploiting the pH-indicator properties of BSP In particular, BSP is first allowed to diffuse from the external medium (pH 8.0) into the intravesicular compartment(s) (pH 7.4) up to its electro-chemical equilibrium The subsequent addition of valinomycin generates an inwardly directed potassium diffusion potential, which further drives BSP into vesicles Electrogenic, valinomycin-dependent BSP uptake into rat liver plasma membrane vesicles is a marker activity of the sinusoidal domain of the hepatic plasma membrane [28] BSP uptake is carrier-mediated, as it displays both substrate saturation and inhibition by a number of organic anions [34], including anthocyanins [30] Moreover, BSP uptake is ascribed to purified bilitranslocase [27,35] and, indeed, a single carrier accounts for it, as indicated by kinetic analysis [36] Kinetics of electrogenic BSP uptake in carnation petal microsomes To determine whether bilitranslocase-specific transport activity does occur also in carnation petals, microsomes prepared thereof were assayed for valinomycininduced BSP uptake Figure shows the continuous 3283 Bilitranslocase homologue in carnation petals sec 0.005 A 580-514 valinomycin 1,6 nmoles of BSP disappeared microsomes S Passamonti et al 1,2 0,8 0,4 0,0 litre/osmol Fig Continuous spectrophotometric recording of BSP uptake in carnation petal microsomes Segment 1: A580)514 of the assay solution (17.7 lM BSP in 0.1 M potassium phosphate, pH 8.0); Segment 2: deflection caused by the addition of 7.5 lL (9.75 lg protein) microsomes; Segment 3: steady state; Segment 4: deflection caused by the addition of lL valinomycin (¼ lg) Vertical bar ¼ 0.005 A580)514 (¼ 1.87 nmol BSP) spectrophotometric recording of a typical transport assay Segment of the trace records the BSP absorbance in the assay medium Addition of microsomes causes a decrease in the signal (segment 2) After the signal has levelled off (segment 3), valinomycin is added and a second deflection follows (segment 4) In rat liver plasma membrane vesicles, the latter has been shown to be due to the entry of BSP into vesicles and has been referred to as electrogenic BSP uptake [28] Preliminary tests were carried out to examine the dependence of the rate of electrogenic BSP uptake in carnation petal microsomes on protein, K+ and valinomycin concentrations Uptake of 29.5 lm BSP was found to linearly depend on the addition of protein (2.6 ± 0.04 lmolỈmin)1Ỉmg protein)1, with lg valinomycin), as well as of K+ (6.25 ± 0.16 unitsỈmEq)1 K+, with lg valinomycin) and valinomycin (0.51 ± 0.03 unitsỈlg)1 valinomycin, with 0.3780 mEq K+ in the assay) If the disappearance of BSP from the assay medium represents an uptake into the vesicular compartment, it is expected that the former parameter be directly related to the vesicular volume In order to test this possibility, the assay medium was supplemented with increasing sucrose concentrations, to provoke an 3284 Fig The dependence of valinomycin-induced disappearance of BSP on the osmolarity of the extra-vesicular medium in the presence of carnation petal microsomes The assay was carried out as described in Experimental procedures Three microlitres of microsomes [3.3 lg protein in 0.25 M sucrose, 0.1% (w ⁄ v) BSA, 20 mM Tris ⁄ HCl pH 7.5] were added to 2.0 mL 0.1 M (ẳ 295.6 mosmolặL)1) potassium phosphate (pH 8.0), containing 29 lM BSP and increasing concentrations of sucrose After attainment of the steady state, lL (¼ lg) valinomycin was added Data (n ¼ 3) are means ± SEM and were fitted to a straight line by linear regression osmotic shrinking of the vesicles Figure shows the extent of valinomycin-dependent BSP disappearance as a function of the litre ⁄ osmol ratio BSP disappearance approaches the zero at infinite solute concentration in the medium, when the apparent internal volume of vesicles is null Thus, it can be deduced that no binding of BSP to vesicles occurs The dependence of BSP uptake rate on the substrate concentration is shown in Fig The data could fit the Michaelis–Menten equation The KM value derived was 5.3 lm, i.e the same as that found in plasma membrane vesicles from both rat liver [36] and rat gastric mucosa [37] As shown in the same figure, this activity was competitively inhibited by cyanidin 3-glucoside (Ki ¼ 51.6 lm) In a similar experiment, it was found that cyanidin exerted mixed-type inhibition (noncompetitive Ki ¼ 88.3 lm, competitive Ki ¼ 136.1 lm) These data (collected in Table 1, sections A and B) point to the conclusion that the electrogenic BSP uptake activity in carnation petal microsomes is a carrier-mediated process FEBS Journal 272 (2005) 3282–3296 ª 2005 FEBS S Passamonti et al Bilitranslocase homologue in carnation petals mune serum (both in the range 1–10 lgỈmL)1) affected the transport activity (data not shown) In rat liver plasma membrane vesicles, both bilirubin and nicotinic acid reduce the rate of BSP uptake inhibition by antibody A, an effect depending on the formation of a complex between the carrier and the ligands [39] The occurrence of this effect was also investigated in carnation petal microsomes by preincubating them with antibody A in the presence of increasing concentrations of bilirubin BSP uptake was assayed to track the progress of the antibody-induced inhibition Figure 5A shows that increasing bilirubin concentrations more and more retarded the progress of activity inhibition The inhibition rate constants can be related to bilirubin concentration by the Scrutton and Utter equation [40]: Fig The dependence of the valinomycin-induced BSP uptake rate into carnation petal microsomal vesicles on [BSP] and the effect of cyanidin 3-glucoside The assay was carried out as described in Experimental procedures Three microlitres of microsomes [9.75 lg protein in 0.25 M sucrose, 0.1% BSA (w ⁄ v) and 20 mM Tris ⁄ HCl pH 7.5] were added to 2.0 mL 0.1 M potassium phosphate (pH 8.0), containing increasing [BSP], without (circles) or with lL of cyanidin 3-monoglucoside (21 mM) dissolved in dimethylsulfoxide (triangles) at room temperature; after attainment of the steady state, lL (¼ lg) valinomycin was added Data (n ¼ 3) are means ± SEM and were fitted to v ¼ Vmax[BSP] ⁄ (KM + [BSP]) The parameters found were: Vmax ¼ 2.77 ± 0.12 (circles) or 2.84 0.11 (triangles) lmol BSPặmin)1ặ mg)1 protein; KM ẳ 5.28 ± 0.92 (circles) or 10.63 ± 1.17 (triangles) lM BSP The inset displays the double reciprocal plot Inhibition of electrogenic BSP uptake by antisequence anti-bilitranslocase The primary structure of bilitranslocase includes a segment (residues 58–99) that is 58% homologous to a highly conserved segment (residues 6–45) in a-phycocyanins, where it is in close contact with the biline prosthetic group [38] An antisequence antibody, targeting the sequence 65–75 (EDSQGQHLSSF) of bilitranslocase, has been shown to react with purified bilitranslocase, with a 38-kDa protein in rat liver plasma membrane vesicles, and to inhibit electrogenic BSP uptake by rat liver plasma membrane vesicles [39] For clarity, this antibody will be referred to as antibody A and the sequence 65–75 in bilitranslocase as site A To test whether electrogenic BSP uptake in carnation petal microsomes is supported by a protein related to bilitranslocase, microsomes were preincubated with antibody A and then assayed for BSP uptake activity Figure shows the time-dependence of activity inhibition at three different IgG concentrations Neither bovine IgG nor IgG purified from the rabbit preim- FEBS Journal 272 (2005) 32823296 ê 2005 FEBS kA =k0 ẳ k2 =k1 ỵ Kd ẵ1 kA =k0 ị=ẵA 1ị where kA and k0 are the inactivation rate constants either in the presence or in the absence of various concentrations of a ligand A, k2 and k1 are the rate constants of the inhibition of the bilitranslocase– bilirubin complex and of free bilitranslocase, respectively Kd is the dissociation constant of the apparent bilitranslocase–ligand complex Figure 5B shows the Scrutton and Utter plot; the value of the dissociation constant of the carrier–bilirubin complex (Kd ¼ 1.76 nm) can be derived from its slope In a similar experiment, the dissociation constant of the carrier– nicotinic acid complex was obtained (Kd ¼ 12.7 nm) Further details about the parameters of the Scrutton and Utter equation applied to data obtained with bilirubin and nicotinic acid are listed in Table As shown in Table 1, section C, these data are quite similar to those found in rat liver plasma membrane vesicles [39] and suggest again that the carnation petal carrier is indeed functionally related to the liver one The possibility arises that it could also be a bilirubin carrier In that case, it is expected that bilirubin could engage with the bilitranslocase transport pore, thus inhibiting BSP electrogenic uptake Indeed, when tested in rat liver plasma membrane vesicles, both bilirubin and biliverdin acted as competitive inhibitors of BSP uptake (Ki ¼ 113.3 nm and 111.8 nm, respectively; see Table 1, section B) However, in carnation petal microsomes, none of these effects could be observed According to a tentative model of bilitranslocase topology in the membrane (D Juretic & A Lucin, University of Split, Croatia, personal communication), the segment 235–246 of the bilitranslocase amino acid sequence (for clarity, referred to as site B) is relatively close to the segment 65–75 (site A), and both sites 3285 Bilitranslocase homologue in carnation petals S Passamonti et al Table Kinetic parameters of electrogenic BSP uptake in two materials Data are collected from experiments shown in Fig (KM of electrogenic BSP uptake, section A of the table; Ki of cyanidin 3-glucoside, section B), or described in detail in both the experimental procedures (Ki of cyanidin, bilirubin, biliverdin, section B) and in Table (Kd of the complexes of bilitranslocase with bilirubin, nicotinic acid and cyanidin 3-glucoside, sections C and D) A Michaelis–Menten constants of BSP electrogenic uptake (KM, lM) Carnation 5.28 ± 0.9 B Liver 5.32 ± 0.63a Types and constants of BSP electrogenic uptake inhibition by various compounds Carnation Liver Types Constant (Ki, lM) Types 51.6 ± 5.7 88.3 ± 4.5 136.1 ± 15.9 – – Competitive 5.8 ± 0.4a Bilirubin Biliverdin Competitive Noncompetitive Competitive None None Competitive Competitive Competitive 17.5 ± 1.7a 0.11 ± 0.01 0.11 ± 0.02 C Interaction of various compounds with site A (Kd, nM) Cyanidin 3-glucoside Cyanidin Carnation Liver Bilirubin Nicotinic acid Cyanidin 3-glucoside 1.76 ± 0.03 12.7 ± 1.3 None 2.2 ± 0.3 11.3 ± 1.3b None D Constant (Ki, lM) Interaction of various compounds with site B (Kd, nM) Carnation Bilirubin Nicotinic acid Cyanidin 3-glucoside a [30], b Liver None None 1.7 ± 0.19 · 103 0.33 ± 0.01 None None [39] contribute to the extracellular domain of the carrier A rabbit antisequence antibody (referred to as antibody B) was raised against a peptide corresponding to segment 235–246, to assess the possible role of this segment in the electrogenic BSP uptake in both rat liver plasma membrane vesicles and in carnation petal microsomes In both materials, antibody B inhibited the BSP uptake activity at rates depending on IgG concentration The data (not shown) were thus similar to those shown in Fig Unlike in carnation petal microsomes, bilirubin delayed the progress of the activity inhibition in rat liver plasma membrane vesicles and the data fitted the Scrutton and Utter equation The parameters obtained are listed in Table The dissociation constant of the bilitranslocase–bilirubin complex was found to be 0.33 nm (Table 1, section D) In contrast to what found with antibody A, in this case the straight line of the plot intersected the origin of the axes (Table 2) This means that at infinite bilirubin concentrations (i.e when the carrier occurs as a 3286 complex with the pigment) antibody B could not inhibit the carrier activity This might result from either a perfect shield of site B afforded by bilirubin, or, otherwise, by an alternative conformation of the bilirubin– bilitranslocase complex, totally missed by antibody B Cyanidin 3-glucoside was found to delay the kinetics of antibody B inhibition in carnation petal microsomes, but not in rat liver plasma membrane vesicles (data not shown) The Scrutton and Utter plot allowed calculation of a Kd value of 1.73 lm for the complex of the carrier with this anthocyanin (Table 1, section D and Table 2) Electrogenic BSP uptake was also checked in both tonoplast and plasma membrane fractions, purified from microsomes In both preparations, virtually identical KM values of BSP uptake were found (5.4 ± 0.5 and 5.3 ± 0.7 lm, respectively) The plasma membrane fraction was purified by two-phase partitioning Under these conditions it is well established that a homogeneous population of right-side-out vesicles is FEBS Journal 272 (2005) 3282–3296 ª 2005 FEBS S Passamonti et al Bilitranslocase homologue in carnation petals A relative uptake rate 1,0 0,9 0,8 0,7 10 15 20 25 30 time (min) collected [41] However, orientation is also known to randomly revert by freezing and thawing the vesicle suspension Because as many as three cycles of freezing and thawing did not decrease the specific activity of BSP electrogenic uptake, it is suggested that BSP movement may occur in both directions Finally, it was found that the electrogenic BSP uptake in both rat liver plasma membrane vesicles and in carnation petal microsomes was insensitive to reduced glutathione and was not stimulated by ATP (data not shown) Immunoblots of carnation petal membrane fractions Membrane proteins from subcellular fractions of carnation petals were separated by SDS ⁄ PAGE and immuno- FEBS Journal 272 (2005) 3282–3296 ª 2005 FEBS B 0,8 0,6 kbr/k0 Fig Inhibition of electrogenic BSP uptake into carnation petal microsomes by an antibody (antibody A) directed against the sequence EDSQGQHLSSF (site A) The effect of [IgG] Experimental conditions: microsomes [2.6 mg proteinỈmL)1 in 0.25 M sucrose, 0.1% (w ⁄ v) BSA and 20 mM Tris ⁄ HCl pH 7.5] were preincubated with antibody A (1, and lg IgGỈmL)1; h, n and s, respectively) at 37 °C Aliquots (3.5 lL ¼ 9.1 lg proteins) were withdrawn at the times indicated and added to 2.0 mL assay medium (29.5 lM BSP) for the determination of BSP electrogenic uptake activity Data were fitted to the equation y ¼ y0 + ae–kt, where y is the relative uptake rate, y0 is the relative uptake rate at the inhibition steady-state, a ¼ 1–y0, e ¼ 2.7183, t ¼ time and k is the first order inhibition rate constant The parameters of the three curves were: y0 ¼ 0.70 ± 0.01, a ¼ 0.30 ± 0.01, k1 ¼ 0.17 ± 0.02 min)1 (s); y0 ¼ 0.70 ± 0.02, a ¼ 0.29 ± 0.02, k2 ¼ 0.08 ± 0.01 min)1 (n); y0 ¼ 0.71 ± 0.09, a ¼ 0.29 ± 0.08, k3 ¼ 0.05 ± 0.02 min)1 (h) The inset shows the relationship between k and [IgG] Data were fitted to a straight line by linear regression The parameters were: intercept at the y axis ¼ 0.003 ± 0.004; slope ¼ 0.042 ± 0.001 min)1lg)1ml; r2 ¼ 0.999 0,4 0,2 0,0 0,0 0,1 0,2 0,3 -1 (1-kbr/k0)/[bilirubin] (nM ) Fig (A) Time course of inhibition of electrogenic BSP uptake into carnation petal microsomes by antibody A The effect of [bilirubin] Experimental conditions: microsomes [2.6 mg proteinỈmL)1 in 0.25 M sucrose, 0.1% (w ⁄ v) BSA and 20 mM Tris ⁄ HCl pH 7.5] were preincubated at 37 °C with antibody A (4 lg IgGỈmL)1) and (d), (e), 2.5 (,), (n), 10 (s) and 20 (h) nM bilirubin dissolved in 0.25 M sucrose, 10 mM Hepes pH 7.4 ⁄ dimethylsulfoxide (9 : 1, v ⁄ v; dimethylsulfoxide in the suspension ¼ 1%, v ⁄ v) Aliquots (3.5 lL ¼ 9.1 lg proteins) were withdrawn at the times indicated and added to 2.0 mL assay medium (29.5 lM BSP) for the determination of BSP electrogenic uptake activity Data were fitted to the equation y ¼ y0 + ae–kt, and the individual inhibition rate constants were obtained as detailed in the legend to Fig (B) Scrutton and Utter plot Inactivation rate constants were related to [bilirubin], according to the Scrutton and Utter equation (see text); k0 and kbr are the inactivation rate constants in either the absence or in the presence of various concentrations of bilirubin, respectively Data were fitted to a straight line by linear regression and the following parameters were obtained: intercept at the y axis ¼ k2 ⁄ k1 ¼ 0.15 ± 0.005 and slope ¼ Kd ¼ 1.76 ± 0.03 nM, r2 ¼ 0.999 These data are also reported in Tables and 3287 Bilitranslocase homologue in carnation petals S Passamonti et al Table Parameters of the Scrutton and Utter equation applied to data obtained under various conditions Inhibition of electrogenic BSP uptake activity by two antisequence anti-bilitranslocase Igs (Ab) (Ab A, lgỈmL)1; Ab B, lgỈmL)1), in either carnation microsomes (2.6 mg proteinỈmL)1) or rat liver plasma membrane vesicles (2.76 mg proteinỈmL)1), was carried out as detailed in the text and in Fig 5A or with minor modifications The rate constants of inhibition in either the absence (k0) or the presence (kA) of a series of ligand (A) concentrations are related to [A] by Eqn (1), as detailed in the text and in Fig 5B n, Number of [A] tested; k2 ⁄ k1, the value of the intercept in the Scrutton and Utter plot, where k2 and k1 are the rate constants of the inhibition of either the bilitranslocase-ligand complex or free bilitranslocase, respectively; Kd, dissociation constant of the bilitranslocase–ligand complex Relevant experimental conditions Ligand Parameters Ab Material A [A] range (nM) n k2 ⁄ k1 A Carnation B Carnation Liver Bilirubin Nicotinic acid Cyanidin 3-glucoside Bilirubin 1–20 5–120 1.5 · 103)12 · 103 0.25–5 0.151 0.264 0.086 0.005 blotted, in order to detect their reactivity with both the antibodies A and B Figure shows the immunoblot developed with either antibody A (Fig 6A) or antibody B (Fig 6B) Lanes 1–3 were loaded with microsomal (lane 1), plasma membrane (lane 2) and tonoplast (lane 3) vesicles obtained from carnation petals, while lane was loaded with rat liver plasma membrane vesicles In all samples, antibodies A and B both revealed a protein band of % 38 kDa (arrow) Immunolabelling of carnation petals In order to visualize the immuno-complexes in intact petals, the latter were fixed and cut into sections, which were incubated with antibody A As shown in Fig 7A, an anti-rabbit secondary antibody conjugated with the fluorophore fluorescein isothiocyanate (FITC) revealed that the primary immunocomplexes are associated with the plasma membrane of epidermal cells At this magnification, the vacuolar membrane and the plasma membrane could not be resolved, because the vacuole takes a large part of the lumen of the cell and Kd (nM) ± ± ± ± 0.005 0.031 0.003 0.008 1.76 12.73 1.73 0.33 ± ± ± ± 0.03 1.27 0.19 · 103 0.008 the tonoplast is almost in contact with the plasma membrane Interestingly, if observed with little magnification, these are the only cells containing a large vacuole stored with red pigments, presumably anthocyanins (Fig 7B) A section of a carnation petal was fixed, incubated with antibody A and immunostained with colloidal gold-conjugated secondary antibodies (Fig 7C) Under these conditions, the relevant antigen was again found to be in contact with the cell wall Taken collectively, these observations are consistent with the subcellular distribution of both the BSP electrogenic transport activity and the immuno-reactivity toward the anti-bilitranslocase Igs Discussion Electrogenic BSP uptake into carnation petal and rat liver membrane vesicles: two subtly different carriers In this work, the assay of electrogenic BSP uptake into rat liver plasma membrane vesicles has been A B 45 45 38.4 38.4 31 31 4 Fig Identification of membrane proteins reacting with two antisequence anti-bilitranslocase Igs Subcellular fractions from carnation petals (microsomes, lane 1; plasma membranes, lane 2; tonoplast, lane 3) and rat liver plasma membranes (lane 4) were separated by SDS ⁄ PAGE and blotted The blot was developed with either antibody A (A) or antibody B (B), as detailed in the Experimental procedures 3288 FEBS Journal 272 (2005) 3282–3296 ª 2005 FEBS S Passamonti et al Bilitranslocase homologue in carnation petals Fig Immunolabelling of carnation petals (A) Transverse section of fixed carnation petal, incubated with antibody A as primary antibody and, subsequently, with a FITC-conjugated secondary antibody, as described in Experimental procedures The immunocomplexes were detected by epifluorescence microscopy Scale bar ¼ 100 lm (B) Micrograph of a carnation petal section under visible light Scale bar ¼ 100 lm (C) Ultra-thin section of fixed carnation petal, incubated with antibody A as primary antibody and, subsequently, with a colloidal-gold conjugated secondary antibody, as described in Experimental procedures Scale bar ¼ 100 nm implemented in analogous preparations obtained from carnation petals, yielding an identical phenomenology (Fig 1) The valinomycin-dependent disappearance of BSP from the extra-vesicular compartment was found to decrease linearly as a function of the medium osmolarity (Fig 2); it was inferred that BSP disappeared because of its uptake into an osmotically active compartment Interestingly, the regression line fitting the experimental data intersected the ordinate at its origin, consistently with the obvious prediction that BSP disappearance will never occur in a virtual vesicular compartment Thus, valinomycin-dependent disappearance of BSP reflects exclusively an electrogenic transport into vesicles, whose kinetics obeys the Michaelis– Menten law (Fig 3) The further results collected show that the transport activity identified in carnation petal microsomes is functionally related to rat liver bilitranslocase The two carriers appear to share the following functional features: (a) identical KM values of BSP uptake (Table 1, section A); (b) inhibition of electrogenic BSP uptake by anthocyanins (Table 1, section B); (c) inhibition by two antisequence, anti-bilitranslocase Igs; (d) very close Kd values of the complexes with bilirubin and nicotinic acid (Table 1, section C) However the two carriers are not identical at all, in view of a number of functional differences Considering both cyanidin 3-glucoside and its aglycone (Table 1, section B), there are differences in both the type and the magnitude of the inhibition constants in the two cases As a competitive inhibitor, cyanidin 3-glucoside is nearly 10 times more effective in the liver than in carnation petals Similarly cyanidin, a relatively good competitive inhibitor in liver, is a poor, mixed-type inhibitor in carnation petals These data show that the affinity for anthocyanins of the plant carrier is lower than that of the liver carrier Perhaps, this could be the result of the different, evolutionary pressures acting in the plant and the animal kingdoms The liver carrier FEBS Journal 272 (2005) 3282–3296 ª 2005 FEBS has presumably evolved to facilitate the uptake of the low concentrations of anthocyanins found in plasma after ingestion of red fruits and their derivatives [42] The plant carrier, on the contrary, is exposed to presumably higher local concentrations of those secondary metabolites, and a higher KM would enable the carrier to respond to oscillating substrate concentrations with significant changes in activity Moreover, anthocyanin glycosylation appears to be critical in regulating their interaction with the BSP carriers in both materials This is in keeping with the view that, in plants, conjugation of secondary metabolites and xenobiotics promotes their recognition by vacuolar membrane carriers [20] Another notable difference between the two carriers is given by the evidence that bilirubin and biliverdin inhibit only the hepatic carrier (Table 1, section B) The effect on the plant carrier of other tetrapyrroles, in particular those derived from phytochrome or chlorophyll breakdown, is still to be investigated The data obtained by testing the effect of antibody B on the BSP transport activities in the two materials further support the evidence of the functional difference of the two carriers In fact, the site targeted by that antibody is involved in high-affinity bilirubin binding only in the liver, but not in carnation (Table 1, section D) Conversely, antibody B identifies a site involved in the high-affinity binding of cyanidin 3-glucoside in carnation but not in the liver Obviously, these divergent functions have to be supported by partially different structures The structural difference is probably as subtle as the functional one, because the electrophoretic mobility exhibited by the carnation petal and the rat liver carriers is the same The antisequence anti-bilitranslocase Igs The antibodies (A and B) used to obtain the above summarized results were raised against two different 3289 Bilitranslocase homologue in carnation petals peptides, corresponding to two segments of the primary structure of bilitranslocase The ability of antibody A to inhibit the electrogenic BSP carrier in rat liver has already been demonstrated [39] and, as shown in this work, this antibody also reacts with a structurally similar protein of carnation petals Unfortunately, a database search for the corresponding gene in rat and plant genomes has been unsuccessful so far In principle, such absence in silico does not preclude its existence in nature As a matter of fact, this carrier has been isolated [26] and utilized for the reconstitution of the electrogenic BSP transport in two different membrane models [27,43] In our opinion, the question about the primary structure of bilitranslocase needs to be approached experimentally At this stage, we cannot decide whether the biological effects of both antibodies have to be ascribed to their interaction with the primary structure of bilitranslocase or, otherwise, with two distinct conformational epitopes on the same carrier Nevertheless, both antibodies appear to be useful tools for the identification and functional characterization of the membrane transport of BSP and are currently used in our laboratories to isolate this protein from plants by immunoaffinity chromatography Bioenergetics of BSP uptake and physiological implications in plants and the liver The electrogenic uptake of BSP in subcellular membrane fractions from carnation petals, described in this work, is apparently a newly described mechanism of membrane transport in plant cells Its key feature is to recognize de-protonated, quinoid and planar phthalein structures [28,34] This peculiar molecular recognition, not involving the protonated and phenolic tautomers, is at the basis of the sequestration of phthaleins into vesicles Such property accounts for the remarkable sensitivity of the transport assay Anthocyanins display a number of structural features in common with phthaleins They undergo pH-dependent tautomerism [44], although at pH ranges far lower than BSP and thymol blue That makes them unsuitable substrates under the conditions of the BSP uptake assay Nonetheless, it is reasonable to predict that anthocyanin interactions with bilitranslocase are analogous to that of phthaleins, i.e as anionic, quinoid species Hence they could be driven into the vacuole by the H+ electrochemical potential In the vacuole, the prevailing species would be the flavylium cation Although it still displays the overall planar geometry required by bilitranslocase substrates, unlike BSP, the absence of either negative charges or quinoid moieties could make anthocyanins unfit for this car3290 S Passamonti et al rier In conclusion, the pH conditions occurring in the vacuole could also favour the trapping of anthocyanin tautomer(s) The relationship between the electrogenic BSP uptake activity and that of H+ gradient-dependent transporters in the vacuolar membrane is still to be clarified That could be possibly elucidated by using vacuolar vesicles energized by either ATP- or PPi-dependent H+ translocation Because BSP uptake is found in highly purified preparations of both tonoplast and plasma membranes, a dual localization of the same carrier can be envisaged This view is also supported by both immunoblot (Fig 6) and immunohistochemical data (Fig 7) The localization of the electrogenic BSP carrier on the carnation petal plasma membrane is apparently intriguing, as it could promote an efflux of metabolites into the cell wall, favoured by the plasma membrane potential Indeed, the latter appears to be opposite to that occurring in the tonoplast At the plasma membrane level, ATP-dependent pumps build up an electrical potential (DY) of 120–160 mV (negative inside) and a DpH of 1.5–2 units (cell wall pH % 5.5; cytoplasmic pH % 7) Similarly, at the tonoplast level ATP- or PPidependent proton pumps generate an electrochemical proton gradient with a DY of 30 mV (positive inside) and a DpH of some units, depending on the lumenal pH, which ranges from to [45] Therefore, the bioenergetic conditions on the plasma membrane seem to favour an export of anthocyanins by the electrogenic BSP carrier The physiological significance of this export may be related to the role performed by the cell wall against pathogens This function appears to be particularly interesting if the electrogenic BSP carrier of plant cells could also transport other flavonoids In this context, the identification of these secondary metabolites at the level of cell wall in maize cells, engineered to express P transcriptional activators, strongly supports this hypothesis [46] The bioenergetics of bilitranslocase-dependent BSP uptake in the liver is quite different When BSP is administered into the blood as a clinical test of liver function, it is rapidly and efficiently cleared by the liver [47,48] The slight pH difference between the liver cell (pH 7.07) and the plasma (pH 7.40) [49] acts as a positive driving force although it is outbalanced by the electrical membrane potential, negative inside, as directly shown in isolated rat hepatocytes [50] In the liver, a major driving force is the large difference of BSP concentration, achieved by intracellular binding to glutathione transferase (EC 2.5.1.18), subsequent conjugation with one or two glutathione moieties [51,52] and primary active transport into the bile canaliculus [53] FEBS Journal 272 (2005) 3282–3296 ª 2005 FEBS S Passamonti et al Bilitranslocase homologue in carnation petals Experimental procedures Plant material Red carnation flowers (Dianthus caryophyllus L) were purchased at a local market Isolation of subcellular fractions from carnation petals Microsomes About 40 g of petals claw-deprived were cut into small pieces and then homogenized by an Ultra-turrax (IkaWerk, Sweden) blender in 220 mL 0.25 m sucrose, 20 mm Hepes ⁄ Tris pH 7.6, mm EDTA, mm DTE, mm phenlymethylsulfonyl fluoride, 0.6% (w ⁄ v) polyvinylpoly pyrrolidone and 0.3% (w ⁄ v) BSA at °C The homogenate was filtered through eight layers of gauze and centrifuged at 2800 g for in a Sorvall RC-5B centrifuge (SS-34 rotor) The supernatant was re-centrifuged at 13 000 g for 12 The new supernatant was re-filtered through two layers of gauze and ultracentrifuged at 100 000 g for 36 in a Beckman L7-55 centrifuge (Ty 70ti rotor) The pellet was resuspended in 0.25 m sucrose, 20 mm Tris ⁄ HCl pH 7.5 and ultracentrifuged again as above The microsomal membrane fraction was resuspended in 0.25 m sucrose, 0.1% (w ⁄ v) fatty acid free BSA, 20 mm Tris ⁄ HCl pH 7.5 at a final protein concentration of 3–5 mgỈmL)1 Plasma membrane vesicles Plasma membrane vesicles were isolated from microsomes, using a modified aqueous polymer two-phase partitioning system [54] [6.5% (w ⁄ v) Dextran T-500 and 6.5% (w ⁄ v) PEG 3350] The upper phase was diluted in 0.25 m sucrose, 20 mm Tris ⁄ HCl pH 7.5, and ultracentrifuged at 120 000 g for 70 in a Beckman L7-55 centrifuge (Ty 70ti rotor) The plasma membrane fraction was resuspended in 0.25 m sucrose, 0.1% (w ⁄ v) fatty acid free BSA and 20 mm Tris ⁄ HCl pH 7.5 at a final protein content of % mgỈmL)1 The vanadate-sensitive ATPase activity, a marker of the plasma membrane, was found to be 331 and 30 nmolỈ min)1Ỉmg)1 protein in the presence and in the absence of 0.05% (w ⁄ v) Brij 58, respectively This shows that about 90% of plasma membrane vesicles are right-side-out Tonoplast vesicles Tonoplast vesicles were isolated from microsomes as described by Koren’kov et al [55] Membranes were layered over 22 mL 6% (w ⁄ v) Dextran T-500 step gradient, and purified by centrifugation at 40 000 g for 130 in a Beckman L7-55 centrifuge (SW 28 rotor) A sharp band of membranes was collected at the interface, diluted about 20fold in 20 mm Tris ⁄ HCl pH 7.5, 0.25 m sucrose and ultracentrifuged at 120 000 g for 70 in a Beckman L7-55 centrifuge (Ty 70ti rotor) The tonoplast vesicle fraction was re-suspended in 0.25 m sucrose, 0.1% (w ⁄ v) fatty acid free BSA and 20 mm Tris ⁄ HCl pH 7.5 at a final protein concentration of % mgỈmL)1 Marker enzyme assays The level of purification of tonoplast and plasma membrane vesicles was evaluated by measuring some marker enzymes [54], whose activities are reported in Table These included vanadate-sensitive ATPase (plasmalemma marker), bafilomycin-sensitive ATPase (tonoplast marker), oligomycin-sensitive ATPase (mitochondria marker), latent IDPase (Golgi marker) and cytochrome c reductase (endoplasmic reticulum marker) As shown in Table 3, both the plasmalemma and tonoplast fractions were slightly contaminated by endoplasmic reticulum or Golgi membranes and negligibly contaminated by mitochondria Rat liver plasma membrane vesicles The preparation was carried out as described by van Ameslvoort et al [56], using three rat livers (Rattus norvegicus, Table Markers of enzyme activities in plasma membrane and tonoplast fractions purified from carnation petals Activity values are expressed as nmolỈmin)1Ỉmg protein)1 All activities were performed in the presence of 0.05% (w ⁄ v) Brij 58 in order to determine total activity (naked and latent) Fractions Microsome Plasma membrane )1 Tonoplast )1 Enzyme Additions Activity values (nmolỈmin Ỉmg protein ) ATPase None 400 lM Na3VO4 100 nM bafilomycin lgỈmL)1 oligomycin – – 241 181 187 163 158 125 Cytochrome c reductase Latent IDPase FEBS Journal 272 (2005) 3282–3296 ª 2005 FEBS 437 106 412 418 15 14 393 335 13 325 32 12 3291 Bilitranslocase homologue in carnation petals Wistar Hannover strain) Throughout this work a single vesicle pool (resuspended in 0.25 m sucrose, 10 mm Hepes ⁄ NaOH pH 7.4 and stored in aliquots under liquid nitrogen) was used Its qualities were assessed and found to be consistent with those previously described [28,30] Bilitranslocase transport activity assay Bilitranslocase transport activity was assayed spectrophotometrically as previously described in detail [28,57] Briefly, 3– 10 lL (% 10 lg protein) of the various membrane fractions were added to a stirred cuvette containing mL assay medium (0.1 m potassium phosphate, pH 8.0), with different BSP concentrations (in the range 3.5–45 lm) at room temperature This addition caused an instantaneous decrease in absorbance (recorded at the wavelength pair 580–514 nm) (Fig 1) After the attainment of a steady-state (4 s), a second decrease in absorption was brought about by valinomycininduced K+ diffusion potential by adding lg valinomycin (Fluka) in lL methanol Such K+ diffusion drove the substrate into the vesicles [28] The slope of the linear phase of this absorbance drop, lasting about s, is referred to as electrogenic BSP uptake and is related to bilitranslocase transport activity [57] The pH in the assay medium was constant throughout the duration of the test, as previously shown with an analogous preparation from rat liver [28] Effect of various inhibitors on the electrogenic BSP uptake kinetics For transport inhibition assays, the inhibitors (2–6 lL, dissolved in dimethylsulfoxide) were added to the medium s before the addition of the vesicles The inhibitors were: 52.4 lm cyanidin 3-glucoside; 24.6 and 41 lm cyanidin; 100 nm bilirubin and 100 nm biliverdin Under the conditions of the assay, bilirubin is freely soluble in the buffer [58] The presence of these inhibitors in the assay medium may interfere with absorbance at 580–514 nm (in particular for anthocyanins) However, systematic control experiments in the absence of BSP indicated that the optical signal remained constant on addition of valinomycin to the vesicle suspension, thus confirming that the inhibitors never interfered with the assay Antibody production Antibody A was raised in one rabbit (Oryctolagus cuniculus, white New Zealand strain), immunized with a multiantigen peptide-based system as described in [39], using the peptide EDSQGQHLSSF, corresponding to the segment 65–75 of the primary structure of bilitranslocase Sera were purified by affinity chromatography as described previously [39] Antibody B was obtained by injecting the peptide EFTYQLTSSPTC, corresponding to the segment 235–246 3292 S Passamonti et al of the primary structure of bilitranslocase The peptide was conjugated to maleimide-activated keyhole limpet haemocyanin and injected into a rabbit; sera were purified by affinity chromatography Both conjugation of the peptide to haemocyanin and affinity purification of the antibodies were carried out by using the EZTM Antibody Production and Purification Kit, Sulfhydryl reactive (Pierce, Rockford, IL, USA, catalogue number 77614) and following the instructions provided therein Specific IgG were eluted from the columns with 0.1 m glycine ⁄ HCl (pH 2.5) and immediately neutralized with m Tris The IgG concentration in the fractions was assayed by the method of Bradford, using bovine IgG (Sigma) as standard Fractions were supplemented with 1.5 mgỈmL)1 BSA and stored at )20 °C Electrogenic BSP uptake inhibition by antibodies The kinetics of bilitranslocase transport activity inhibition by antibodies were examined by preincubating 24 lL rat liver plasma membrane vesicles or carnation petal microsomes at 37 °C with lL antibody A or antibody B at the concentrations indicated in the figure legends Controls were carried out by using equivalent amounts of IgG, purified from preimmune rabbit sera When the effect of various ligands was examined, the preincubation mixtures included lL of a given ligand at various concentrations, prepared in 0.25 m sucrose, 10 mm Hepes-NaOH pH 7.4 ⁄ dimethylsulfoxide (9 : 1, v ⁄ v) immediately before the experiment Eight 3.5-lL aliquots of the preincubation mixture were withdrawn during a 20-min span and added to the transport medium for the assay of bilitranslocase transport activity Under these conditions, all components of the preincubation mixture were diluted 5.7 · 102 times, so that they did not interfere with the activity of bilitranslocase It was thus legitimate to apply the Scrutton and Utter equation [40] to the inhibition data Data analyses Data were analysed by means of sigmaplot 2001 (SPSS Science Software Gmbh, Erkrath, Germany) Data for the characterization of the kinetics of electrogenic BSP uptake fitted the Michaelis–Menten equation and the apparent KM and Vmax values were derived with their standard errors The competitive and noncompetitive Ki values were derived from the equations KMi ¼ KM (1 + [I] ⁄ Ki) and ⁄ VmaxI ¼ ⁄ Vmax (1 + [I] ⁄ Ki), respectively, where i stands for inhibitor The data fitted the single exponential decay equation, as specified in the figure legends, thus enabling the characterization of the kinetics of electrogenic BSP uptake inhibition Immunoblot Membrane proteins (% 20 lg) were separated by SDS ⁄ PAGE in a 12% polyacrylamide gel under reducing condi- FEBS Journal 272 (2005) 3282–3296 ª 2005 FEBS S Passamonti et al tions and immunoblotting was performed according to standard techniques [59], with minor modifications: the transfer buffer was composed of 48 mm Tris, 39 mm glycine and 20% (w ⁄ v) methanol (pH 9.2) The two primary antisequence anti-bilitranslocase Igs were used at a concentration of 1.5–3 lg IgGỈmL)1 at °C overnight The immune reaction was detected by means of a goat anti-rabbit IgG, conjugated to horseradish peroxidase (KPL, Inc., Gaithersburg, MD, USA), used at : 5000 dilution, followed by the addition of the chemiluminescent substrate ECL (Amersham Biosciences) Negative controls were obtained by using preimmune sera instead of the primary antibodies Bilitranslocase homologue in carnation petals primary anti-bilitranslocase Ig [3 lgỈmL)1, in 1% (w ⁄ v) BSA, 1% (v ⁄ v) normal goat serum, 4% (v ⁄ v) fetal bovine serum and 0.1% (v ⁄ v) Tween 20 (Merck)] After several washes in Tris-buffered saline to remove the antibody in excess, the sections were incubated for h in the same incubation medium, except that the pH was 8.4, containing the gold-conjugated 20-nm goat anti-rabbit secondary antibody (Britsh BioCell, Cardiff, UK, diluted : 100 as the primary one) Finally, the sections were counterstained with uranyl acetate (2% w ⁄ v) for and with a lead citrate solution (0.25% w ⁄ v) for They were observed with Philips EM 208 electron microscope at 80 Kv accelerating voltages The primary antibody was omitted from the controls Epifluorescence microscopy analysis Carnation petals were cut into small pieces and incubated with freshly made fixing solution (50% ethanol, 35% water, 10% formaldehyde, 5% acetic acid, v ⁄ v ⁄ v ⁄ v) at room temperature for h During the procedure, the tissues were infiltrated under vacuum four times for 10 at intervals of h After each vacuum infiltration, the fixing solution was renewed Fixed samples were kept at °C overnight Then, the samples were washed twice with 63% (v ⁄ v) ethanol and 10–15-lm sections were obtained by cryomicrotomy Sections were incubated in phosphate-buffered saline solution (NaCl ⁄ Pi, pH 7.4) for 10 and then blocked in 100 lL 1% (w ⁄ v) skimmed milk in NaCl ⁄ Pi in a moist chamber at 37 °C for 45 Sections were incubated with antibody A as the primary antibody (3.3 lgỈmL)1) at 37 °C for 90 Control sections were incubated with preimmune serum They were then washed three times with 1% (v ⁄ v) Tween in NaCl ⁄ Pi and subsequently incubated with a FITC-conjugated secondary antibody (Sigma-Aldrich; 60 lg proteinỈmL)1 were used, according to the manufacturer’s instructions) After incubation at 37 °C for h, sections were washed three times with 1% (v ⁄ v) Tween in NaCl ⁄ Pi and finally analysed by a Leitz Fluovert microscope under UV light Transmission electron microscopy analysis A postembedding technique was implemented Small pieces of carnation petals were fixed with a mixture of 4% (v ⁄ v) paraformaldehyde and 0.2% (v ⁄ v) glutaraldehyde in 0.1 m sodium phosphate buffer (pH 6.8) for h at room temperature; they were then washed several times in the same buffer and twice in deionized water, dehydrated in ethanol and embedded in LR White M acrylic resin (Sigma) Immunolabelling of ultra-thin sections (120 nm, supported on 300-mesh nickel grids) was carried out by grids flotation technique at room temperature for h on drops of blocking buffer [1% (w ⁄ v) BSA (Sigma), 20% (v ⁄ v) normal goat serum in 0.1 m Tris-buffered saline pH 7.4], and then incubated for h in Tris-buffered saline pH 7.4 containing the FEBS Journal 272 (2005) 3282–3296 ª 2005 FEBS Protein determination The protein content was measured by the Bradford method with the Bio-Rad protein assay, using crystalline BSA as a standard Reagents Anthocyanins were from Polyphenols Laboratories (Sandnes, Norway), biliverdin from Frontier Scientific Europe Ltd (Carnforth, UK) All other chemicals were purchased from Sigma-Aldrich and Carlo Erba (Milan, Italy), and were of the highest available grade Acknowledgements Thanks are due to Prof G.L Sottocasa and Dr Antonella Bandiera (University of Trieste) for useful discussions, to Dr Marco Stebel (Animal Facility Manager, C.S.P.A – University of Trieste) for the immunization and bleeding of rabbits; to Silvia Zezlina for the affinity purification of antibody A from rabbit sera; to Dr Paolo Ermacora and Prof Giorgio Honsell (University of Udine) and Mr Fulvio Micali (University of Trieste) for the histology work Financial support by the Universities of Trieste and Udine (Fondi 60%), the Regione Friuli Venezia Giulia (L.R ⁄ 98, art.16, fondo ` anno 2002), the Ministero dell’Istruzione, Universita e Ricerca (PRIN projects 2002055532 and 2004070118) and the Progetto D4 (European Social Fund, Regione Friuli Venezia Giulia and Italian Ministry of Welfare) are acknowledged References Winkel-Shirley B (2002) Biosynthesis of flavonoids and effects of stress Curr Opin Plant Biol 5, 218–223 Middleton E Jr, Kandaswami C & Theoharides TC (2000) The effects of plant flavonoids on mammalian 3293 Bilitranslocase homologue in carnation petals 10 11 12 13 14 15 cells: implications for inflammation, heart disease, and cancer Pharmacol Rev 52, 673–751 Hrazdina G & Jensen RA (1992) Spatial organization of enzymes in plant metabolic pathways Annu Rev Plant Physiol Plant Mol Biol 43, 241–267 Fujiwara H, Tanaka Y, Yonekura-Sakakibara K, Fukuchi-Mizutani M, Nakao M, Fukui Y, Yamaguchi M, Ashikari T & Kusumi T (1998) 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Sodium-dependent alanine transport in plasmamembrane vesicles from rat liver Biochem J 174, 1083–1086 57 Passamonti S & Sottocasa GL (2002) Bilitranslocase: structural and functional aspects of an organic anion carrier In Recent Research Developments in Biochemistry (Pandalai GS, ed), pp 371–391 Research Signpost, Kerala, India, Kerala 58 Brodersen R (1979) Bilirubin Solubility and interaction with albumin and phospholipid J Biol Chem 254, 2364– 2369 3296 S Passamonti et al 59 Sambrook J, Fritsch EF & Magnatis T (1989) Molecular Cloning: a Laboratory Manual, 2nd edn Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY Supplementary material The following supplementary material is available online: Appendix S1 The problem of the primary structure of bilitranslocase FEBS Journal 272 (2005) 3282–3296 ª 2005 FEBS ... mgỈmL)1 BSA and stored at )20 °C Electrogenic BSP uptake inhibition by antibodies The kinetics of bilitranslocase transport activity inhibition by antibodies were examined by preincubating 24 lL... addition of protein (2.6 ± 0.04 lmolỈmin)1Ỉmg protein)1, with lg valinomycin), as well as of K+ (6.25 ± 0.16 unitsỈmEq)1 K+, with lg valinomycin) and valinomycin (0.51 ± 0.03 unitsỈlg)1 valinomycin,... also investigated in carnation petal microsomes by preincubating them with antibody A in the presence of increasing concentrations of bilirubin BSP uptake was assayed to track the progress of the