Identification and Functional Characterization of Bilitranslocase in Sea-Bass (Dicentrarchus labrax) Hepatopancreas

Abstract

The mammalian bilirubin transporter bilitranslocase (BTL, T.C.#2.A.65.1.1) is found in both absorptive (intestine) and excretory epithelia (liver, kidney) and in the vascular endothelium. The aim of this work was to investigate whether a BTL homologue is expressed also in fish hepatopancreas. Immunochemistry based on an antisequence antibody specific for rat liver BTL demonstrated the presence of such homologue in sea-bass (Dicentrarchus labrax) hepatopancreas. Furthermore the transport activity of such a carrier, measured as electrogenic bromosulphophthalein (BSP) uptake, was assayed in sea-bass microsomes, where it was inhibited by the same antibody. Transport activity in fish showed numerous kinetic similarities with rat, such as BSP K m (about 5 lM in both), bilirubin K i (about 0.1 lM), quercetin competitive K i (about 20 lM), and noncompetitive K i (about 85 lM). Biliverdin K i was instead nearly 10-fold higher in fish than in rat (0.97 AE 0.06 lM and 0.11 AE 0.01 lM, respectively). Fish BTL was found to exist in two different allosteric forms with different affinities for the substrate, similarly to rat liver BTL. It was found that sea-bass BTL is very sensitive to inhibition by HgCl 2 , a major water pollutant, making it reasonable to exploit fish BTL activity as an ecotoxicological biosensor.