BILITRANSLOCASE AND SULFOBROMOPHTHALEIN BILIRUBIN-BINDING PROTEIN AREBOTH INVOLVED IN THE HEPATIC-UPTAKE OF ORGANIC-ANIONS

The hepatic uptake of cholephilic organic anions is a carrier-mediated process. Three distinct proteins [bilitranslocase (BTL), sulfobromoph thalein (BSP)/bilirubin-binding protein (BBBP), and organic anion-bind ing protein] have been isolated from the basolateral plasma-membrane d omain of the hepatocyte. To investigate the relative role of the first two of them in accounting for the hepatic uptake of organic anions, w e measured the initial rates of uptake of S-35-labeled BSP into rat li ver plasma-membrane vesicles. Because transport by BTL is electrogenic but transport by BBBP is electro-neutral, studies were done either wi th or without a positive-inside membrane potential produced by adding valinomycin in the presence of an inwardly directed K+ gradient (outsi de K+ > inside K+). Both electrogenic and electroneutral transport sys tems followed saturation kinetics. Electroneutral uptake showed an app arent K(m) of 20 +/- 3 muM (mean +/- SD) and a V(max) of 1.0 +/- 0.13 nmol.(mg of prot)-1.15 sec-1, whereas the electrogenic portion of BSP uptake exhibited a K(m) of 5.2 +/- 0.8 muM and a V(max) of 1.1 +/- 0.1 nmol.(mg of prot)-1.15 sec-1. In this case, an overshoot was observed 15 sec after valinomycin addition. Electroneutral BSP uptake was inhi bited by incubation with anti-BBBP antibody, whereas anti-BTL antibody did not show any inhibitory effect. Conversely, the electrogenic upta ke was inhibited by anti-BTL antibody at a BSP concentration of 5 muM; no inhibition was seen either at 20 muM BSP or upon addition of anti- BBBP antibody. From these data we conclude that the hepatic uptake of organic ions occurs via two immunologically distinct carrier proteins (BTL and BBBP) operating in parallel. BTL is a higher affinity electro genic transporting system of organic ions, whereas BBBP is a lower aff inity electroneutral transporter.