3′-Isothiocyanatobenzamido[ 3H]cholate, a new affinity label for hepatocellular membrane proteins responsible for the uptake of both bile acids and phalloidin

Abstract

Substitution of the hydroxyl group on C7 of cholic acid by a benzamido group leads to a derivative with inhibiting quality for the inward transport of both bile acids and phallotoxins by isolated liver cells. The tritiated isothiocyanate derivative was prepared (3′-isothiocyanatobenzamido[ 3H]cholate, [ 3H]IBCA) with a specific activity of 70–80 mCi/mmol. The latter compound was used for affinity labeling of liver plasma membranes in order to detect chemically modified proteins involved in the transport of bile acids. [ 3H]IBCA and the noncovalently binding analogs were recognized by the transport system; they inhibited the uptake of both [ 14C]cholate and of demethyl[ 3H]phalloin in vitro. Isothiocyanatobenzamidocholate (IBCA) was able to protect isolated hepatocytes against phalloidin. In isolated and purified plasma membranes prepared from liver cells [ 3H]IBCA binds to saturable sites in an irreversible manner. Micromolar concentrations of unlabeled IBCA or millimolar concentrations of natural substrates prevented [ 3H]IBCA binding in a concentration dependent manner; some other substrates of the transport system also protected liver membranes against chemical modification. Membranes from AS-3OD hepatoma cells, well known to transport neither bile acids nor phallotoxins, could not be labeled by [ 3H]IBCA. The major targets of labeling in hepatocellular plasma membranes were polypeptides with molecular mass of 67, 60, 54, 50, and 37 kDa as shown by SDS-polyacrylamide gel electrophoresis (10% acrylamide). The 67 kDa protein could be found in the aqueous phase after phase separation in Triton X-114. The 54 kDa and 50 kDa proteins remained in the detergent phase and can therefore be regarded as integral membrane proteins.