Electrogenic Bromosulfalein Transport in Isolated Membrane Vesicles: Implementation in Both Animal and Plant Preparations for the Study of Flavonoid Transporters
Bromosulfalein is an organic anion dye used in the study of a variety of membrane carriers expressed in animal tissues and involved in transport of drugs and metabolites. The spectrophotometric assay of electrogenic bromosulfalein transport in membrane vesicles, isolated from various mammalian organs or tissues, enables to specifically measure the transport activity of bilitranslocase (TCDB 2.A.65.1.1). The latter is a bilirubin- and flavonoid-specific transporter expressed in rat liver, the organ where its function has been best characterized. The spectrophotometric assay of electrogenic bromosulfalein transport requires minimal volumes of membrane vesicles, is completed within 1 min, and, therefore, is a useful tool to screen the transporter spectrum of potential substrates, by testing them as reversible inhibitors of bromosulfalein transport kinetics. Furthermore, the assay enables to study the progress of time-dependent inactivation of bromosulfalein transport, caused by different protein-specific reagents, including specific anti-sequence antibodies. Inactivation can be retarded by the presence of substrates in a concentration-dependent manner, enabling to derive the dissociation constants of the transporter–substrate complex and thus to gain further insight into the transporter structure–function relationship. This assay, implemented in membrane vesicles isolated from plant organs, has paved the way to the discovery of homologues of bilitranslocase in plants.
Key wordsBromosulfalein, membrane transport flavonoids bilitranslocase enzyme kinetics enzyme inactivation rat liver rat kidney carnation petal grape berry
We are grateful for the financial support granted by Regione Friuli Venezia Giulia (L.R. n. 26/2005, Progetto “Miglioramento Delle Produzioni Viti-Vinicole…”) and Fondazione Cassa di Risparmio di Trieste (Rif. 10.1424-2007.0128).
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