TIMED PHOTOAFFINITY-LABELING AND CHARACTERIZATION OF BILE-ACID BINDING AND TRANSPORT PROTEINS IN RAT ILEUM

Rat ileal enterocytes were radiolabeled by flash photolysis with a photolabile derivative of taurocholate (7,7-azo-[H-3]TC) and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Maximal labeling of the bile acid binding proteins (BABPs) was achieved between 15 and 90 s. When enterocytes were pulsed with 7,7-azo-[H-3]TC for 2 min, and then 0.5 mM TC was added to chase the radiolabel, the radioactivity in the BABPs was displaced by 50% after 2 min. The 99-kDa brush-border membrane (BBM) protein had the highest initial labeling rate, followed by 43-kDa actin, 35- and 14-kDa cytosolic proteins, 54-kDa basolateral membrane (BLM) protein, 59-kDa BLM-associated protein, and 20-kDa microsomal protein. When a mixed microsomal and cytosolic fraction was photolabeled with 7,7-azo-[H-3]TC and then separated, the 20-kDa microsomal protein was labeled. However, if the microsomal fraction alone was photolabeled, the 20-kDa protein was not labeled, suggesting this protein required a cytosolic cofactor for labeling. Using Triton X-114 phase separation and EDTA extraction, the BABPs were separated into amphiphilic integral membrane proteins (99- and 54-kDa proteins) and hydrophilic proteins (14-,35-,43-, and 59-kDa proteins). From these data, a model is proposed for transcellular bile acid transport in rat ileal enterocytes.