Identification of cytosolic and microsomal bile acid-binding proteins in rat ileal enterocytes.
Studies were performed to determine the subcellular fractions and proteins involved in the intracellular transport of bile acids in rat ileal cells. The photolabile derivative 7,7-azo-taurocholate inhibited the Na(+)-dependent uptake of taurocholate into rat ileal enterocytes reversibly in the dark and irreversibly following photolysis. When photolabeled cells were submitted to subcellular fractionation, greatest radioactivity was found in the soluble protein (SP) fraction with decreasing radioactivity in the brush-border-(BBM), basolateral-(BLM), mitochondria-(MT), microsome-(MC), and Golgi-(GO) enriched fractions. Following trichloroacetic acid precipitation, delipidation, and correction for loss of marker enzyme activity, protein bound radioactivity was in SP greater than BBM greater than MC greater than BLM greater than GO greater than MT. When photolabeled cells were first fractionated and then submitted to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, a 99-kDa polypeptide was associated with BBM, 54- and 59-kDa polypeptides with BLM, 14-, 35-, 43-, 59-, and 68-kDa polypeptides with SP and a 20-kDa polypeptide with MC fractions. Immunoprecipitation with known antisera identified the 68-kDa polypeptide as albumin and the 43-kDa polypeptide as actin. No precipitation on the 14-kDa polypeptide was noted with anti-hepatic and anti-intestinal fatty acid-binding proteins. No precipitation of the 35-kDa polypeptide occurred with antibody to the hepatic cytosolic bile acid-binding protein. These studies reveal a previously unrecognized 20-kDa microsomal, and 14- and 35-kDa cytosolic bile acid-binding polypeptides which may be involved in the transcellular movement of bile acids.
Department of Medicine, Pennsylvania State University College of Medicine, Hershey 17033.
M C
MC
Lin
W
W
Kramer
F A
FA
Wilson
D000818:Animals; D001647:Bile Acids and Salts / Q000378:metabolism*; D002352:Carrier Proteins / Q000302:isolation & purification / Q000378:metabolism* / Q000528:radiation effects; D002458:Cell Fractionation / Q000379:methods; D002499:Centrifugation, Density Gradient; D003600:Cytosol / Q000378:metabolism; D004847:Epithelial Cells; D004848:Epithelium / Q000378:metabolism; D006913:Hydroxysteroid Dehydrogenases; D007082:Ileum / Q000166:cytology / Q000378:metabolism*; D007700:Kinetics; D008562:Membrane Glycoproteins; D008861:Microsomes / Q000378:metabolism*; D008970:Molecular Weight; D009130:Muscle, Smooth / Q000166:cytology / Q000378:metabolism; D051381:Rats; D013656:Taurocholic Acid / Q000378:metabolism; D014466:Ultraviolet Rays; D045002:3-alpha-Hydroxysteroid Dehydrogenase (B-Specific)
D001647:Bile Acids and Salts; D002352:Carrier Proteins; D006913:Hydroxysteroid Dehydrogenases; D008562:Membrane Glycoproteins; D013656:Taurocholic Acid; C037643:bile acid binding proteins; C435496:AKR1C2 protein, human; D045002:3-alpha-Hydroxysteroid Dehydrogenase (B-Specific)