Tài liệu Báo cáo khoa học: Characterization of electrogenic bromosulfophthalein transport in carnation petal microsomes and its inhibition by antibodies against bilitranslocase docx

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
`
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

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:
Website:
(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 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.

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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,
[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 1 shows the continuous
3283


Bilitranslocase homologue in carnation petals

1

5 sec
0.005 A 580-514

2

valinomycin

3

4

1,6
nmoles of BSP disappeared

microsomes

S. Passamonti et al.

1,2

0,8

0,4

0,0
0

1

2

3

4

litre/osmol
Fig. 1. 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 1 lL valinomycin (¼ 5 lg). Vertical bar ¼
0.005 A580)514 (¼ 1.87 nmol BSP).

spectrophotometric recording of a typical transport
assay. Segment 1 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 5 lg valinomycin), as well as of K+ (6.25 ± 0.16 unitsỈmEq)1
K+, with 5 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. 2. 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, 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.

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 binding 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 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.
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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. 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 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 5 lL of cyanidin 3-monoglucoside (21 mM) dissolved in
dimethylsulfoxide (triangles) at room temperature; 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 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 4 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 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 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

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Bilitranslocase homologue in carnation petals

S. Passamonti et al.

Table 1. Kinetic parameters of electrogenic BSP uptake in two materials. Data are collected from experiments shown in Fig. 3 (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 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
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. 4. 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 2.
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
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S. Passamonti et al.

Bilitranslocase homologue in carnation petals

A

relative uptake rate

1,0

0,9

0,8

0,7

0

5

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. 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 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, 2 and 4 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. 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 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 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 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. 4. (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 1 and 2.

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Bilitranslocase homologue in carnation petals

S. Passamonti et al.

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 lgỈmL)1; Ab B, 7 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

5
4
7
5

0.151
0.264
0.086
0.005

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 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
1

2

3

4

4

3

2

1

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 ⁄ 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. 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 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 3 to 6 [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, 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 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 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 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 min 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 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 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 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 (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 3. 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
1 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 2 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 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 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 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 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 1 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 6 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 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-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 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 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-

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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 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, 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 2 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 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.

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 solution (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 chamber at 37 °C for 45 min. Sections were incubated with antibody A as the primary antibody (3.3 lgỈmL)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 lg
proteinỈmL)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 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 1 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 2 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. 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.

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