Mechanisms of hepatic uptake of organic anions

The focus of this article is mainly on bilirubin and related organic anions, and the processes involved in their passage from plasma into the hepatocyte across the sinusoidal plasma-membrane. Bile acids will be considered only in relation to transport of the former

Bilitranslocase is the protein responsible for the electrogenic movement of sulfobromo-phthalein in plasma membrane vesiscles from rat liver: immuno chemical evidence using mono and poly clonal antibodies

Monoclonal antibodies raised against bilitranslocase, may display either inhibitory or enhancing activity on the electrogenic transport of sulfobromophthalein, evoked in rat liver plasma-membrane vesicles by the addition of valinomycin in the presence of K+. In both cases, the target protein is identified with a 37 kDa band in SDS-mercaptoethanol gel electrophoresis of solubilized membranes. The electrophoretically homogeneous protein isolated by ion-exchange chromatography, corresponds in all respects to the 37 kDa protein band of bilitranslocase, obtained in the past by different techniques. Using this protein as antigen, a polyclonal monospecific antibody preparation has been obtained. As expected, the antibody preparation inhibits the electrogenic movement of sulfobromophthalein in plasma membrane vesicles from rat liver. It is concluded that the 37 kDa protein of bilitranslocase is at least a necessary component of the transport system involved in the sulfobromophthalein movement in plasma membrane

Localization and function of bilitranslocase in the basolateral plasma membrane of the proximal renal tubule in the rat.

Bilirubin and phthalein dyes are taken up by the liver via a carrier-mediated mechanism operated at least in part by bilitranslocase (BTL). Because they also undergo renal transport, the presence and function of BTL was investigated in rat renal tubular plasma membrane vesicles. Transport of sulfobromophthalein (BSP) was enriched in basolateral domain of plasma membrane and followed the distribution pattern of Na+-K+-ATPase but not of \u3b3-glutamyltransferase. BSP uptake was inhibited by addition of monospecific antibodies raised against hepatic BTL. As in liver vesicles, BSP transport was electrogenic, being greatly accelerated by addition of valinomycin in presence of an inwardly directed K+ gradient. Apparent K(m) of BSP transport was 17 \ub1 2 \u3bcM (n = 3 expts), one order of magnitude higher than that measured in liver; however, V(max) was similar to that described in liver vesicles (429 \ub1 18 nmol BSP\ub7mg protein-1\ub7min-1, n = 3 expts). Competitive inhibition was observed with both unconjugated bilirubin (K(i), 2.9 \ub1 0.2 \u3bcM) and rifamycin SV (K(i), 76 \ub1 10 \u3bcM), known competitors for hepatic BTL-mediated transport of BSP. Immunoblotting studies with anti-BTL monospecific antibodies revealed presence of a single positive band only in basolateral-enriched membrane fraction; its apparent molecular mass was 37 kDa, virtually identical to that of hepatic protein. Immunohistochemistry confined presence of BTL to renal proximal tubules (RPT). We conclude that BTL is present in basolateral plasma membrane of RPT cells. Lower affinity of renal, compared with hepatic protein, for substrates might explain the marginal role of kidney in plasma clearance of bilirubin and cholephilic dyes

Isolation of a sulfobromophthalein binding protein from hepatocyte plasma membrane

This paper deals with the isolation and partial characterization of a protein capable of high affinity sulfobromophthalein-binding from liver plasma membrane. The purification involves acetone powder of a crude preparation of rat liver plasma membrane, salt extraction and purification through two chromatographic steps. Based on sulfobromophthalein binding, the process gives a yield of approximately 40%, with a purification of about 300 times with respect to the starting homogenate. The best preparation can bind more than 100 nmol sulfobromophthalein/mg protein. The protein behaves as a single species in dodecyl sulphate polyacrylamide gel electrophoresis, with an apparent molecular weight of 1.7 \ub7 105. The molecule does not contain sugars. The dissociation constant of the protein \ub7 sulfobromophthalein complex has been found to be 4 \ub7 10 126 M, a value in agreement with that of high affinity binding sites described on isolated liver plasma membrane