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-[3H]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-[3H]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-[3H]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.
Lin
M C
MC
Department of Metabolic Diseases, Bristol-Myers Squibb Research Institute, Princeton, New Jersey 08543.
Mullady
E
E
Wilson
F A
FA
D000345:Affinity Labels; D000818:Animals; D001391:Azo Compounds; D001647:Bile Acids and Salts / Q000378:metabolism*; D002352:Carrier Proteins / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism; D004492:Edetic Acid; D006913:Hydroxysteroid Dehydrogenases; D007082:Ileum / Q000166:cytology / Q000378:metabolism*; D008297:Male; D008562:Membrane Glycoproteins; D008861:Microsomes / Q000378:metabolism; D017830:Octoxynol; D011092:Polyethylene Glycols; D051381:Rats; D017207:Rats, Sprague-Dawley; D013997:Time Factors; D045002:3-alpha-Hydroxysteroid Dehydrogenase (B-Specific)
D000345:Affinity Labels; D001391:Azo Compounds; D001647:Bile Acids and Salts; D002352:Carrier Proteins; D004492:Edetic Acid; D006913:Hydroxysteroid Dehydrogenases; D008562:Membrane Glycoproteins; D017830:Octoxynol; D011092:Polyethylene Glycols; C037643:bile acid binding proteins; C037641:(7,7-azo-3,12-dihydroxy-5-cholan-24-oyl)-2-aminoethanesulfonic acid; C010615:Nonidet P-40; C435496:AKR1C2 protein, human; D045002:3-alpha-Hydroxysteroid Dehydrogenase (B-Specific)