characterization of electrogenic bromosulfophthalein transport in carnation petal microsomes and its...

Characterization of electrogenic bromosulfophthaleintransport in carnation petal microsomes and its inhibitionby antibodies against bilitranslocaseSabina Passamonti1, Alessandra Cocolo1, Enrico Braidot2, Elisa Petrussa2, Carlo Peresson2,Nevenka Medic1, Francesco Macri2 and Angelo Vianello2

1 Dipartimento di Biochimica Biofisica e Chimica delle Macromolecole, Universita di Trieste, Italy

2 Dipartimento di Biologia ed Economia Agro-Industriale, Sezione di Biologia Vegetale, Universita di Udine, Italy

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 col-

oration, 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 anti-

tumour 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

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: [email protected]

Website: http://www.bbcm.units.it

(Received 7 March 2005, revised 15 April

2005, accepted 5 May 2005)

doi:10.1111/j.1742-4658.2005.04751.x

Bilitranslocase is a rat liver plasma membrane carrier, displaying a high-

affinity 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 activ-

ity 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 bilitrans-

locase, 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 bilitranslo-

case in both plasma membrane and tonoplast fractions, isolated from car-

nation 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.

Abbreviations

BSP, bromosulfophthalein; FITC, fluorescein isothiocyanate; PVPP, polyvinylpoly pyrrolidone.

3282 FEBS Journal 272 (2005) 3282–3296 ª 2005 FEBS

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 shiki-

mate 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 mecha-

nisms 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 sec-

ond model postulates the existence of carriers exploit-

ing 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 trans-

porters [20]. The vacuolar transport of anthocyanins

is, however, a complex event, requiring not only mem-

brane 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 flavonoid-

binding proteins rather than as enzymes, because no

conjugate species is formed in vitro [23]. Besides that,

vesicle trafficking also participates in delivering antho-

cyanins 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], locali-

zed at the sinusoidal domain of liver cells [28] and

in the epithelium of the gastric mucosa [29]. The

activity of bilitranslocase, assayed as bromosulfophta-

lein (BSP) uptake in rat liver plasma membrane

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 antho-

cyanins led us to consider the hypothesis that a sim-

ilar carrier protein could be present in the vacuolar

membrane of plant cells. To this purpose, we investi-

gated the presence of bilitranslocase in carnation

petals and found a BSP uptake, inhibited by anti-

bodies 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 epi-

dermal 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 suit-

able 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 par-

ticular, BSP is first allowed to diffuse from the external

medium (pH 8.0) into the intravesicular compart-

ment(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, valino-

mycin-dependent BSP uptake into rat liver plasma

membrane vesicles is a marker activity of the sinusoi-

dal 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 ani-

ons [34], including anthocyanins [30]. Moreover, BSP

uptake is ascribed to purified bilitranslocase [27,35]

and, indeed, a single carrier accounts for it, as indica-

ted 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, micro-

somes prepared thereof were assayed for valinomycin-

induced BSP uptake. Figure 1 shows the continuous

S. Passamonti et al. Bilitranslocase homologue in carnation petals

FEBS Journal 272 (2005) 3282–3296 ª 2005 FEBS 3283

spectrophotometric recording of a typical transport

assay. Segment 1 of the trace records the BSP absorb-

ance 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 vali-

nomycin concentrations. Uptake of 29.5 lm BSP was

found to linearly depend on the addition of protein

(2.6 ± 0.04 lmolÆmin)1Æmg protein)1, with 5 lg vali-

nomycin), as well as of K+ (6.25 ± 0.16 unitsÆmEq)1

K+, with 5 lg valinomycin) and valinomycin (0.51 ±

0.03 unitsÆlg)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 rela-

ted to the vesicular volume. In order to test this pos-

sibility, the assay medium was supplemented with

increasing sucrose concentrations, to provoke an

osmotic shrinking of the vesicles. Figure 2 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 bind-

ing of BSP to vesicles occurs.

The dependence of BSP uptake rate on the substrate

concentration is shown in Fig. 3. 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 inhibi-

tion (noncompetitive Ki ¼ 88.3 lm, competitive Ki ¼136.1 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.

litre/osmol

0 1 2 3 4

nmol

es o

f BS

P d

isap

pear

ed

0,0

0,4

0,8

1,2

1,6

Fig. 2. The dependence of valinomycin-induced disappearance of

BSP on the osmolarity of the extra-vesicular medium in the pres-

ence 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 (¼ 295.6

mosmolÆL)1) potassium phosphate (pH 8.0), containing 29 lM BSP

and increasing concentrations of sucrose. After attainment of the

steady state, 1 lL (¼ 5 lg) valinomycin was added. Data (n ¼ 3)

are means ± SEM and were fitted to a straight line by linear

regression.

microsomes

1

5 sec

0.005 A 580-514

2

3

4

valinomycin

Fig. 1. Continuous spectrophotometric recording of BSP uptake in

carnation petal microsomes. Segment 1: A580)514 of the assay solu-

tion (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 1 lL valinomycin (¼ 5 lg). Vertical bar ¼0.005 A580)514 (¼ 1.87 nmol BSP).

Bilitranslocase homologue in carnation petals S. Passamonti et al.


Inhibition of electrogenic BSP uptake by

antisequence anti-bilitranslocase

The primary structure of bilitranslocase includes a seg-

ment (residues 58–99) that is 58% homologous to a

highly conserved segment (residues 6–45) in a-phycocy-anins, where it is in close contact with the biline pros-

thetic group [38]. An antisequence antibody, targeting

the sequence 65–75 (EDSQGQHLSSF) of bilitrans-

locase, has been shown to react with purified bilitrans-

locase, 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 carna-

tion petal microsomes is supported by a protein related

to bilitranslocase, microsomes were preincubated with

antibody A and then assayed for BSP uptake activity.

Figure 4 shows the time-dependence of activity inhi-

bition at three different IgG concentrations. Neither

bovine IgG nor IgG purified from the rabbit preim-

mune serum (both in the range 1–10 lgÆmL)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 inhi-

bition by antibody A, an effect depending on the for-

mation of a complex between the carrier and the

ligands [39]. The occurrence of this effect was also

investigated in carnation petal microsomes by preincu-

bating 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]:

kA=k0 ¼ k2=k1 þ Kd½1� ðkA=k0Þ�=½A� ð1Þ

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, respect-

ively. Kd is the dissociation constant of the apparent

bilitranslocase–ligand complex. Figure 5B shows the

Scrutton and Utter plot; the value of the dissocia-

tion constant of the carrier–bilirubin complex (Kd ¼1.76 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 bili-

rubin and nicotinic acid are listed in Table 2.

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 tes-

ted in rat liver plasma membrane vesicles, both biliru-

bin 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

Fig. 3. 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 des-

cribed in Experimental procedures. Three microlitres of micro-

somes [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 5 lL of cyanidin 3-monoglucoside (21 mM) dissolved in

dimethylsulfoxide (triangles) at room temperature; after attain-

ment of the steady state, 1 lL (¼ 5 lg) valinomycin was added.

Data (n ¼ 3) are means ± SEM and were fitted to v ¼Vmax[BSP] ⁄ (KM + [BSP]). The parameters found were: Vmax ¼2.77 ± 0.12 (circles) or 2.84 ± 0.11 (triangles) lmol BSPÆmin)1Æ

mg)1 protein; KM ¼ 5.28 ± 0.92 (circles) or 10.63 ± 1.17 (triangles)

lM BSP. The inset displays the double reciprocal plot.



contribute to the extracellular domain of the carrier. A

rabbit antisequence antibody (referred to as antibody

B) was raised against a peptide corresponding to seg-

ment 235–246, to assess the possible role of this seg-

ment 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. 4. Unlike in carnation petal

microsomes, bilirubin delayed the progress of the

activity inhibition in rat liver plasma membrane vesi-

cles and the data fitted the Scrutton and Utter equa-

tion. The parameters obtained are listed in Table 2.

The dissociation constant of the bilitranslocase–biliru-

bin 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 biliru-

bin concentrations (i.e. when the carrier occurs as a

complex with the pigment) antibody B could not inhi-

bit the carrier activity. This might result from either a

perfect shield of site B afforded by bilirubin, or, other-

wise, 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 micro-

somes, 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 iden-

tical KM values of BSP uptake were found (5.4 ± 0.5

and 5.3 ± 0.7 lm, respectively). The plasma mem-

brane fraction was purified by two-phase partitioning.

Under these conditions it is well established that a

homogeneous population of right-side-out vesicles is

Table 1. Kinetic parameters of electrogenic BSP uptake in two materials. Data are collected from experiments shown in Fig. 3 (KM of elec-

trogenic 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 2 (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 Liver

5.28 ± 0.9 5.32± 0.63a

B Types and constants of BSP electrogenic uptake inhibition by various compounds

Carnation Liver

Types Constant (Ki, lM) Types Constant (Ki, lM)

Cyanidin 3-glucoside Competitive 51.6 ± 5.7 Competitive 5.8 ± 0.4a

Cyanidin Noncompetitive 88.3 ± 4.5

Competitive 136.1 ± 15.9 Competitive 17.5 ± 1.7a

Bilirubin None – Competitive 0.11 ± 0.01

Biliverdin None – Competitive 0.11 ± 0.02

C Interaction of various compounds with site A (Kd, nM)

Carnation Liver

Bilirubin 1.76 ± 0.03 2.2 ± 0.3

Nicotinic acid 12.7 ± 1.3 11.3 ± 1.3b

Cyanidin 3-glucoside None None

D Interaction of various compounds with site B (Kd, nM)

Carnation Liver

Bilirubin None 0.33 ± 0.01

Nicotinic acid None None

Cyanidin 3-glucoside 1.7 ± 0.19 · 103 None

a [30], b [39]



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 carna-

tion petals were separated by SDS ⁄PAGE and immuno-

Fig. 4. 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 proteinÆmL)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, 2 and 4 lg IgGÆmL)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 ¼1–y0, e ¼ 2.7183, t ¼ time and k is the first order inhibition rate con-

stant. 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 ¼0.29 ± 0.02, k2 ¼ 0.08 ± 0.01 min)1 (n); y0 ¼ 0.71 ± 0.09, a ¼0.29 ± 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 ¼0.999.

time (min)

0 5 10 15 20 25 30

rela

tive

upta

ke r

ate

0,7

0,8

0,9

1,0

A

B

(1-kbr/k0)/[bilirubin] (nM-1)

0,0 0,1 0,2 0,3

k br/

k 0

0,0

0,2

0,4

0,6

0,8

Fig. 5. (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 proteinÆmL)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 IgGÆmL)1) and 0 (d), 1

(e), 2.5 (,), 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 deter-

mination of BSP electrogenic uptake activity. Data were fitted to

the equation y ¼ y0 + ae–kt, and the individual inhibition rate con-

stants were obtained as detailed in the legend to Fig. 4. (B) Scrut-

ton 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, respect-

ively. Data were fitted to a straight line by linear regression and the

following parameters were obtained: intercept at the y axis ¼k2 ⁄ 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 1 and 2.



blotted, in order to detect their reactivity with both the

antibodies A and B. Figure 6 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 4

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 asso-

ciated 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

the tonoplast is almost in contact with the plasma

membrane. Interestingly, if observed with little magni-

fication, these are the only cells containing a large

vacuole stored with red pigments, presumably antho-

cyanins (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 elec-

trogenic 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

1 2 3 4

B

4 3 2 1

45

31

38.4

31

38.4

45

Fig. 6. 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 ⁄ PAGEand blotted. The blot was developed with either antibody A (A) or antibody B (B), as detailed in the Experimental procedures.

Table 2. 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, 4 lgÆmL)1; Ab B, 7 lgÆmL)1), in either carnation microsomes (2.6 mg

proteinÆmL)1) or rat liver plasma membrane vesicles (2.76 mg proteinÆmL)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

Ab Material

Ligand Parameters

A [A] range (nM) n k2 ⁄ k1 Kd (nM)

A Carnation Bilirubin 1–20 5 0.151 ± 0.005 1.76 ± 0.03

Nicotinic acid 5–120 4 0.264 ± 0.031 12.73 ± 1.27

B Carnation Cyanidin 3-glucoside 1.5 · 103)12 · 103 7 0.086 ± 0.003 1.73 ± 0.19 · 103

Liver Bilirubin 0.25–5 5 0.005 ± 0.008 0.33 ± 0.008



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 osmo-

larity (Fig. 2); it was inferred that BSP disappeared

because of its uptake into an osmotically active com-

partment. Interestingly, the regression line fitting the

experimental data intersected the ordinate at its origin,

consistently with the obvious prediction that BSP dis-

appearance will never occur in a virtual vesicular com-

partment. 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 bilitran-

slocase. The two carriers appear to share the following

functional features: (a) identical KM values of BSP

uptake (Table 1, section A); (b) inhibition of electro-

genic 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

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 pre-

sumably 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 chlo-

rophyll 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 differ-

ence 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-glu-

coside in carnation but not in the liver. Obviously,

these divergent functions have to be supported by par-

tially 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

Fig. 7. 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 ¼100 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.



peptides, corresponding to two segments of the

primary structure of bilitranslocase. The ability of anti-

body 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 structur-

ally 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 mem-

brane 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 pri-

mary structure of bilitranslocase or, otherwise, with

two distinct conformational epitopes on the same car-

rier. Nevertheless, both antibodies appear to be useful

tools for the identification and functional characteriza-

tion of the membrane transport of BSP and are cur-

rently 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 mem-

brane 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 fea-

tures in common with phthaleins. They undergo

pH-dependent tautomerism [44], although at pH ran-

ges 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 pre-

dict 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 car-

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-depend-

ent 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 pre-

parations 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 mem-

brane level, ATP-dependent pumps build up an electri-

cal 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 PPi-

dependent 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 3 to 6 [45]. Therefore, the bio-

energetic 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 me-

tabolites at the level of cell wall in maize cells, engi-

neered 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 differ-

ence of BSP concentration, achieved by intracellular

binding to glutathione transferase (EC 2.5.1.18), subse-

quent conjugation with one or two glutathione moiet-

ies [51,52] and primary active transport into the bile

canaliculus [53].



Experimental procedures

Plant material

Red carnation flowers (Dianthus caryophyllus L) were pur-

chased 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 (Ika-

Werk, Sweden) blender in 220 mL 0.25 m sucrose, 20 mm

Hepes ⁄Tris pH 7.6, 5 mm EDTA, 1 mm DTE, 1 mm

phenlymethylsulfonyl fluoride, 0.6% (w ⁄ v) polyvinylpoly

pyrrolidone and 0.3% (w ⁄ v) BSA at 4 �C. The homogenate

was filtered through eight layers of gauze and centrifuged

at 2800 g for 5 min in a Sorvall RC-5B centrifuge (SS-34

rotor). The supernatant was re-centrifuged at 13 000 g for

12 min. The new supernatant was re-filtered through two

layers of gauze and ultracentrifuged at 100 000 g for

36 min 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 microsom-

al 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 mgÆmL)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 min 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 � 1 mgÆmL)1.

The vanadate-sensitive ATPase activity, a marker of the

plasma membrane, was found to be 331 and 30 nmolÆmin)1Æmg)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 des-

cribed 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 min in a

Beckman L7-55 centrifuge (SW 28 rotor). A sharp band of

membranes was collected at the interface, diluted about 20-

fold in 20 mm Tris ⁄HCl pH 7.5, 0.25 m sucrose and ultra-

centrifuged at 120 000 g for 70 min 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 � 1 mgÆmL)1.

Marker enzyme assays

The level of purification of tonoplast and plasma mem-

brane vesicles was evaluated by measuring some marker

enzymes [54], whose activities are reported in Table 3.

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 (endo-

plasmic reticulum marker). As shown in Table 3, both the

plasmalemma and tonoplast fractions were slightly contam-

inated 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 Ame-

slvoort et al. [56], using three rat livers (Rattus norvegicus,

Table 3. Markers of enzyme activities in plasma membrane and tonoplast fractions purified from carnation petals. Activity values are

expressed as nmolÆmin)1Æmg protein)1. All activities were performed in the presence of 0.05% (w ⁄ v) Brij 58 in order to determine total activ-

ity (naked and latent).

Enzyme Additions

Fractions

Microsome Plasma membrane Tonoplast

Activity values (nmolÆmin)1Æmg protein)1)

ATPase None 241 437 393

400 lM Na3VO4 181 106 335

100 nM bafilomycin 187 412 13

1 lgÆmL)1 oligomycin 163 418 325

Cytochrome c reductase – 158 15 32

Latent IDPase – 125 14 12



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 spectrophoto-

metrically 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 2 mL assay med-

ium (0.1 m potassium phosphate, pH 8.0), with different

BSP concentrations (in the range 3.5–45 lm) at room tem-

perature. 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 valinomycin-

induced K+ diffusion potential by adding 5 lg valinomycin

(Fluka) in 1 lL methanol. Such K+ diffusion drove the

substrate into the vesicles [28]. The slope of the linear phase

of this absorbance drop, lasting about 1 s, is referred to as

electrogenic BSP uptake and is related to bilitranslocase

transport activity [57]. The pH in the assay medium was con-

stant 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, dis-solved in dimethylsulfoxide) were added to the medium 5 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 condi-

tions 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 inter-

fered 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

of the primary structure of bilitranslocase. The peptide was

conjugated to maleimide-activated keyhole limpet haemo-

cyanin 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 immedi-

ately neutralized with 1 m Tris. The IgG concentration in

the fractions was assayed by the method of Bradford, using

bovine IgG (Sigma) as standard. Fractions were supplemen-

ted with 1.5 mgÆmL)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 6 lL antibody A or antibody B at the concentra-

tions indicated in the figure legends. Controls were carried

out by using equivalent amounts of IgG, purified from pre-

immune rabbit sera. When the effect of various ligands was

examined, the preincubation mixtures included 3 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-lLaliquots 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 condi-

tions, all components of the preincubation mixture were dilu-

ted 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 1 ⁄VmaxI ¼ 1 ⁄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 characteri-

zation 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-



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 anti-

sequence anti-bilitranslocase Igs were used at a concentra-

tion of 1.5–3 lg IgGÆmL)1 at 4 �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 1 : 5000 dilution, fol-

lowed by the addition of the chemiluminescent substrate

ECL (Amersham Biosciences). Negative controls were

obtained by using preimmune sera instead of the primary

antibodies.

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 4 h. During the procedure, the tissues were

infiltrated under vacuum four times for 10 min at intervals of

1 h. After each vacuum infiltration, the fixing solution was

renewed. Fixed samples were kept at 4 �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 solu-

tion (NaCl ⁄Pi, pH 7.4) for 10 min and then blocked in

100 lL 1% (w ⁄ v) skimmed milk in NaCl ⁄Pi in a moist cham-

ber at 37 �C for 45 min. Sections were incubated with anti-

body A as the primary antibody (3.3 lgÆmL)1) at 37 �C for

90 min. 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 lgproteinÆmL)1 were used, according to the manufacturer’s

instructions). After incubation at 37 �C for 1 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 2 h at room tempera-

ture; they were then washed several times in the same buf-

fer and twice in deionized water, dehydrated in ethanol and

embedded in LR White M acrylic resin (Sigma). Immuno-

labelling of ultra-thin sections (120 nm, supported on

300-mesh nickel grids) was carried out by grids flotation

technique at room temperature for 1 h on drops of block-

ing buffer [1% (w ⁄ v) BSA (Sigma), 20% (v ⁄ v) normal goat

serum in 0.1 m Tris-buffered saline pH 7.4], and then incu-

bated for 2 h in Tris-buffered saline pH 7.4 containing the

primary anti-bilitranslocase Ig [3 lgÆmL)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 2 h in the same

incubation medium, except that the pH was 8.4, containing

the gold-conjugated 20-nm goat anti-rabbit secondary anti-

body (Britsh BioCell, Cardiff, UK, diluted 1 : 100 as the

primary one). Finally, the sections were counterstained with

uranyl acetate (2% w ⁄ v) for 3 min and with a lead citrate

solution (0.25% w ⁄ v) for 2 min. They were observed with

Philips EM 208 electron microscope at 80 Kv accelerating

voltages. The primary antibody was omitted from the

controls.

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 (Sand-

nes, 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 Anto-

nella Bandiera (University of Trieste) for useful discus-

sions, 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 affin-

ity 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 Uni-

versities of Trieste and Udine (Fondi 60%), the Regi-

one Friuli Venezia Giulia (L.R. 3 ⁄ 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.

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Supplementary material

The following supplementary material is available

online:

Appendix S1. The problem of the primary structure

of bilitranslocase.



characterization of electrogenic bromosulfophthalein transport in carnation petal microsomes and its...

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