Characterization of electrogenic bromosulfophthalein
transport in carnation petal microsomes and its inhibition
by antibodies against bilitranslocase
Sabina Passamonti
1
, Alessandra Cocolo
1
, Enrico Braidot
2
, Elisa Petrussa
2
, Carlo Peresson
2
,
Nevenka Medic
1
, Francesco Macri
2
and Angelo Vianello
2
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

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 (K
M
¼ 5.3 lm), which is
competitively inhibited by cyanidine 3-glucoside (K
i
¼ 51.6 lm) and mainly
noncompetitively by cyanidin (K
i
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 (K
d
¼ 1.7 nm) in the
carnation carrier. The other antibody identified a high-affinity binding site
for cyanidine 3-glucoside (K
d
¼ 1.7 lm) on the carnation carrier only, and
a high-affinity bilirubin-binding site (K
d
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-

+
-PP
i
ase [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

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-

, 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 K
M
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 (K

)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: A
580)514
of the assay solu-
tion (17.7 l
M 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 A
580)514
(¼ 1.87 nmol BSP).
Bilitranslocase homologue in carnation petals S. Passamonti et al.
3284 FEBS Journal 272 (2005) 3282–3296 ª 2005 FEBS
Inhibition of electrogenic BSP uptake by

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]:
k
A
=k
0
¼ k
2
=k
1
þ K
d
½1 Àðk
A
=k
0
Þ=½Að1Þ
where k
A
and k
0
are the inactivation rate constants
either in the presence or in the absence of various
concentrations of a ligand A, k

ted in rat liver plasma membrane vesicles, both biliru-
bin and biliverdin acted as competitive inhibitors of
BSP uptake (K
i
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 m
M 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 m
M) 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 ¼
V
max
[BSP] ⁄ (K

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 K
d
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 K
M
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 (K

Biliverdin None – Competitive 0.11 ± 0.02
C Interaction of various compounds with site A (K
d
,nM)
Carnation Liver
Bilirubin 1.76 ± 0.03 2.2 ± 0.3
Nicotinic acid 12.7 ± 1.3 11.3 ± 1.3
b
Cyanidin 3-glucoside None None
D Interaction of various compounds with site B (K
d
,nM)
Carnation Liver
Bilirubin None 0.33 ± 0.01
Nicotinic acid None None
Cyanidin 3-glucoside 1.7 ± 0.19 · 10
3
None
a
[30],
b
[39]
Bilitranslocase homologue in carnation petals S. Passamonti et al.
3286 FEBS Journal 272 (2005) 3282–3296 ª 2005 FEBS
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.

0
is the relative uptake rate at the inhibition steady-state, a ¼
1–y
0
, e ¼ 2.7183, t ¼ time and k is the first order inhibition rate con-
stant. The parameters of the three curves were: y
0
¼ 0.70 ± 0.01,
a ¼ 0.30 ± 0.01, k
1
¼ 0.17 ± 0.02 min
)1
(s); y
0
¼ 0.70 ± 0.02, a ¼
0.29 ± 0.02, k
2
¼ 0.08 ± 0.01 min
)1
(n); y
0
¼ 0.71 ± 0.09, a ¼
0.29 ± 0.08, k
3
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

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 l
M BSP) for the deter-
mination of BSP electrogenic uptake activity. Data were fitted to
the equation y ¼ y
0
+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);
k

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

1
, the value of the intercept in the Scrutton
and Utter plot, where k
2
and k
1
are the rate constants of the inhibition of either the bilitranslocase-ligand complex or free bilitranslocase,
respectively; K
d
, dissociation constant of the bilitranslocase–ligand complex.
Relevant experimental conditions
Ab Material
Ligand Parameters
A [A] range (nM) nk
2
⁄ k
1
K
d
(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 · 10
3
)12 · 10
3
7 0.086 ± 0.003 1.73 ± 0.19 · 10
3
Liver Bilirubin 0.25–5 5 0.005 ± 0.008 0.33 ± 0.008
Bilitranslocase homologue in carnation petals S. Passamonti et al.

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 K
M
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

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,

PP
i
-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 PP
i
-
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

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 % 1mgÆmL

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
)1
Æmg 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
)1
Æmg protein
)1
)
ATPase None 241 437 393
400 l
M Na
3

diffusion potential by adding 5 l g 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

examined, the preincubation mixtures included 3 lLofa
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 condi-
tions, all components of the preincubation mixture were dilu-
ted 5.7 · 10
2
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 K
M
and V
max
values were derived with their standard errors. The
competitive and noncompetitive K
i
values were derived from
the equations K
Mi
¼ K
M
(1 + [I] ⁄ K

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 ⁄ P
i
, pH 7.4) for 10 min and then blocked in
100 lL1%(w⁄ v) skimmed milk in NaCl ⁄ P
i
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
)at37°C for
90 min. Control sections were incubated with preimmune
serum. They were then washed three times with 1% (v ⁄ v)
Tween in NaCl ⁄ P
i
and subsequently incubated with a
FITC-conjugated secondary antibody (Sigma-Aldrich; 60 lg
proteinÆmL
)1

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
`

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The following supplementary material is available
online:
Appendix S1. The problem of the primary structure
of bilitranslocase.
Bilitranslocase homologue in carnation petals S. Passamonti et al.
3296 FEBS Journal 272 (2005) 3282–3296 ª 2005 FEBS