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-
Trang 1transport in carnation petal microsomes and its inhibition
by antibodies against bilitranslocase
Sabina Passamonti1, Alessandra Cocolo1, Enrico Braidot2, Elisa Petrussa2, Carlo Peresson2,
Nevenka Medic1, Francesco Macri2and 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, 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: passamonti@bbcm.units.it
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 (KM¼ 5.3 lm), which is competitively inhibited by cyanidine 3-glucoside (Ki¼ 51.6 lm) and mainly noncompetitively by cyanidin (Ki¼ 88.3 lm) Two antisequence antibodies against bilitranslocase inhibited this carrier In analogy to liver bilitrans-locase, one antibody identified a bilirubin-binding site (Kd¼ 1.7 nm) in the carnation carrier The other antibody identified a high-affinity binding site for cyanidine 3-glucoside (Kd¼ 1.7 lm) on the carnation carrier only, and
a high-affinity bilirubin-binding site (Kd¼ 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.
Trang 2cytosol [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
tt12gene 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
Trang 3spectrophotometric 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 KMvalue 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 (Ki¼ 51.6 lm) In a similar experiment,
it was found that cyanidin exerted mixed-type inhibi-tion (noncompetitive Ki¼ 88.3 lm, competitive Ki¼ 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
litre/osmol
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 m M 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 l M 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 A580-514
2
3
4 valinomycin
Fig 1 Continuous spectrophotometric recording of BSP uptake in
carnation petal microsomes Segment 1: A 580 )514of 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 A580)514(¼ 1.87 nmol BSP).
Trang 4Inhibition 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
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=k0¼ k2=k1þ 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 (Kd¼ 1.76 nm) can be derived from its slope In a similar experiment, the dissociation constant of the carrier– nicotinic acid complex was obtained (Kd¼ 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 (Ki¼ 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
Vmax[BSP] ⁄ (K M + [BSP]) The parameters found were: Vmax¼
2.77 ± 0.12 (circles) or 2.84 ± 0.11 (triangles) lmol BSPÆmin)1Æ
mg)1protein; KM¼ 5.28 ± 0.92 (circles) or 10.63 ± 1.17 (triangles)
lM BSP The inset displays the double reciprocal plot.
Trang 5contribute to the extracellular domain of the carrier A
rabbit antisequence antibody (referred to as antibody
B) was raised against a peptide corresponding to
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 (KMof elec-trogenic BSP uptake, section A of the table; Kiof cyanidin 3-glucoside, section B), or described in detail in both the experimental procedures (K i of cyanidin, bilirubin, biliverdin, section B) and in Table 2 (K d 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, l M )
B Types and constants of BSP electrogenic uptake inhibition by various compounds
C Interaction of various compounds with site A (Kd, n M )
D Interaction of various compounds with site B (Kd, n M )
a [30], b [39]
Trang 6collected [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 m M 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 l M BSP)
for the determination of BSP electrogenic uptake activity Data were
fitted to the equation y ¼ y 0 + ae –kt , where y is the relative uptake
rate, y 0 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
con-stant The parameters of the three curves were: y0¼ 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, k2¼ 0.08 ± 0.01 min)1 (n); y0¼ 0.71 ± 0.09, a ¼
0.29 ± 0.08, k3¼ 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; r 2 ¼
0.999.
time (min)
0 5 10 15 20 25 30
0,7 0,8 0,9
1,0
A
B
(1-kbr/k0)/[bilirubin] (nM-1)
/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 m M 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 m M 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 0 and k br 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 ¼
k2⁄ k 1 ¼ 0.15 ± 0.005 and slope ¼ K d ¼ 1.76 ± 0.03 n M , r 2 ¼ 0.999 These data are also reported in Tables 1 and 2.
Trang 7blotted, 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 ⁄ PAGE and 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⁄ k 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; Kd, dissociation constant of the bilitranslocase–ligand complex.
Relevant experimental conditions
Trang 8implemented 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 KMwould 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 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.
Trang 9peptides, 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]
Trang 10Experimental 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 nmolÆ min)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).
Fractions
Activity values (nmolÆmin)1Æmg protein)1)