title: 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.
mesh_terms: D000345:Affinity Labels / Q000138:chemical synthesis*; D000818:Animals; D001647:Bile Acids and Salts / Q000378:metabolism*; D002352:Carrier Proteins / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D019826:Cholic Acid; D002793:Cholic Acids / Q000138:chemical synthesis* / Q000378:metabolism / Q000494:pharmacology; D006913:Hydroxysteroid Dehydrogenases; D017879:Isothiocyanates; D007700:Kinetics; D008099:Liver / Q000378:metabolism*; D008562:Membrane Glycoproteins; D008970:Molecular Weight; D009842:Oligopeptides / Q000378:metabolism*; D010590:Phalloidine / Q000378:metabolism*; D051381:Rats; D011956:Receptors, Cell Surface / Q000302:isolation & purification / Q000378:metabolism*; D013861:Thiocyanates / Q000138:chemical synthesis* / Q000494:pharmacology; D014316:Tritium; D045002:3-alpha-Hydroxysteroid Dehydrogenase (B-Specific)
chemical_list: D000345:Affinity Labels; D001647:Bile Acids and Salts; D002352:Carrier Proteins; D019826:Cholic Acid; D002793:Cholic Acids; D006913:Hydroxysteroid Dehydrogenases; D017879:Isothiocyanates; D008562:Membrane Glycoproteins; D009842:Oligopeptides; D010590:Phalloidine; D011956:Receptors, Cell Surface; D013861:Thiocyanates; D014316:Tritium; C037643:bile acid binding proteins; C041686:3'-isothiocyanatobenzamidecholic acid; C435496:AKR1C2 protein, human; D045002:3-alpha-Hydroxysteroid Dehydrogenase (B-Specific)