Molecular cloning, tissue distribution, and expression of a 14-kDa bile acid-binding protein from rat ileal cytosol.
A cDNA clone encoding the major intestinal cytosolic 14-kDa bile acid-binding protein (14-kDa I-BABP) was isolated from a rat ileal lambda gt22A library following immunoscreening using a monospecific antiserum raised against a 14-kDa polypeptide found in the rat ileal cytosol. One clone of 516 bp encoded a 128-amino acid protein with a predicted molecular mass of 14,544 Da. The deduced amino acid sequence of 14-kDa I-BABP showed 100% homology to rat intestinal 15-kDa protein (I-15P) and 72% homology to porcine 15-kDa gastrotropin, whereas comparison of I-BABP to rat 14-kDa fatty acid-binding proteins of liver, intestine, and heart revealed homologies of 44%, 25%, and 28%, respectively. Northern blot analysis revealed a single transcript of approximately 0.5 kb in ileum and ovary; however, the abundance of I-BABP mRNA was much greater in ileum than in ovary. No transcript was seen in RNA extracted from stomach, jejunum, colon, liver, adrenal, brain, heart, kidney, or testis. Transfection of the I-BABP cDNA into COS-7 cells resulted in the expression of a 14-kDa protein that was identical to the ileal cytosolic I-BABP as determined by immunoblotting. Photoaffinity labeling of expressed 14-kDa protein was saturable with respect to increasing concentrations of 7,7-azo[3H]taurocholate (Km, 83.3 microM; Vmax, 6.7 pmol/mg per 5 min). Taurocholate inhibited 7,7-azotaurocholate labeling by > 96% with lesser inhibition by taurochenodeoxycholate (83.1%), chenodeoxycholate (74.6%), cholate (50.5%), and progesterone (38.5%), whereas oleic acid and estradiol did not inhibit binding.
Gong
Y Z
YZ
Department of Medicine, Medical University of South Carolina, Charleston 29425.
Everett
E T
ET
Schwartz
D A
DA
Norris
J S
JS
Wilson
F A
FA
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001647:Bile Acids and Salts / Q000378:metabolism*; D015152:Blotting, Northern; D015153:Blotting, Western; D002352:Carrier Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D002460:Cell Line; D003001:Cloning, Molecular; D003600:Cytosol / Q000378:metabolism; D004247:DNA; D006801:Humans; D006913:Hydroxysteroid Dehydrogenases; D007082:Ileum / Q000378:metabolism*; D008562:Membrane Glycoproteins; D008969:Molecular Sequence Data; D009928:Organ Specificity / Q000235:genetics; D051381:Rats; D017207:Rats, Sprague-Dawley; D017386:Sequence Homology, Amino Acid; D014162:Transfection; D045002:3-alpha-Hydroxysteroid Dehydrogenase (B-Specific)
D001647:Bile Acids and Salts; D002352:Carrier Proteins; D004247:DNA; D006913:Hydroxysteroid Dehydrogenases; D008562:Membrane Glycoproteins; C037643:bile acid binding proteins; C435496:AKR1C2 protein, human; D045002:3-alpha-Hydroxysteroid Dehydrogenase (B-Specific)