Electrogenic Bromosulfalein Transport in Isolated Membrane Vesicles:Implementation in Both Animal and Plant Preparations for the Studyof Flavonoid Transporters

Bromosulfalein is an organic anion dye used in the study of a variety of membrane carriers expressed inanimal tissues and involved in transport of drugs and metabolites. The spectrophotometric assay of electrogenicbromosulfalein transport in membrane vesicles, isolated from various mammalian organs or tissues, enablesto specifically measure the transport activity of bilitranslocase (TCDB 2.A.65.1.1). The latter is a bilirubin- andflavonoid-specific transporter expressed in rat liver, the organ where its function has been better characterized. Thespectrophotometric 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 tostudy the progress of time-dependent inactivation of bromosulfalein transport, caused by different protein-specificreagents, including specific anti-sequence antibodies. Inactivation can be retarded by the presence of substratesin a concentration-dependent manner, enabling to derive the dissociation constants of the transporter–substratecomplex and thus to gain further insight into the transporter structure–function relationship. This assay, implementedin membrane vesicles isolated from plant organs, has paved the way to the discovery of homologues of bilitranslocasein plants.