Characterization of electrogenic bromosulfophthalein transport in carnation petal microsomes and its inhibition by antibodies against bilitranslocase

Abstract

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 activity was implemented in subcellular membrane fractions, leading to the identification of a bromosulfophthalein carrier (KM = 5.3 µm), which is competitively inhibited by cyanidine 3‐glucoside (Ki = 51.6 µm) and mainly noncompetitively by cyanidin (Ki = 88.3 µm). Two antisequence antibodies against bilitranslocase inhibited this carrier. In analogy to liver bilitranslocase, one antibody identified a bilirubin‐binding site (Kd = 1.7 nm) in the carnation carrier. The other antibody identified a high‐affinity binding site for cyanidine 3‐glucoside (Kd = 1.7 µm) on the carnation carrier only, and a high‐affinity bilirubin‐binding site (Kd = 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.

Evidence for a putative flavonoid translocator similar to mammalian bilitranslocase in grape berries (Vitis vinifera L.) during ripening

“…20 g) were separated electrophoretically in a 12% (w/v) polyacrylamide gel (0.1%, w/v, sodium dodecylsulfate) under reducing conditions and immunoblotting was performed as previously described (Passamonti et al 2005c). The primary antibody, raised against BTL, was used at a concentration of 0.66 g/ml IgG at 4°C overnight and the immune reaction was developed by the activity of the alkaline phosphatase, conjugated to anti-rabbit IgG antibody (1:2,500) produced in goat (Sigma).…”

“…The BTL antibody was obtained from rabbit sera, following immunisation with the peptide EFTYQLTSSPTC and puriWed by immunoaYnity chromatography, as described in Passamonti et al (2005c).…”

“…Such antibodies have indeed been helpful in tracking the expression of a BTL-related protein in the anthocyaninladen epidermal cells of carnation petals (Passamonti et al 2005c). The antibodies enabled to characterize the transport function of this protein, found in both the tonoplast and the plasma membrane fractions isolated from petal homogenates by sub-cellular fractionation (Passamonti et al 2005c).…”

“…Such a protein cross-reacted with antibodies raised against BTL epitopes and was able to mediate an electrogenic uptake of bromosulfalein (BSP). Kinetic studies showed remarkable functional similarities between BTLmediated transport into hepatocytes (Passamonti et al 2002) or gastric epithelial cells (Passamonti et al 2000), and the plant homologue; not surprisingly, the latter has shown a diVerent pattern of aYnity for compounds of animal origin, suggesting its putative function in the membrane transport of plant secondary metabolites (Passamonti et al 2005c). Furthermore, its tissue localization was mainly peripheral as established by immunohistochemical analysis.…”

“…The antibodies enabled to characterize the transport function of this protein, found in both the tonoplast and the plasma membrane fractions isolated from petal homogenates by sub-cellular fractionation (Passamonti et al 2005c). Such a protein cross-reacted with antibodies raised against BTL epitopes and was able to mediate an electrogenic uptake of bromosulfalein (BSP).…”