Abstract
In human liver, we previously identified one isoform of dihydrodiol dehydrogenase activity that expresses high affinity bile acid binding (HBAB) with minimal 3 alpha-hydroxysteroid dehydrogenase (3 alpha-HSD) activity for bile acids. This protein may assist in the rapid intracellular transport of bile acids from the sinusoidal to the canalicular pole of the cell. We now report the cDNA cloning and bacterial expression of this novel, multifunctional protein. A 1252-base pair HBAB cDNA was cloned from a HepG2 lambda GT11 library using a rat hepatic bile acid binder cDNA probe. Bacterial expressed recombinant HBAB oxidized racemic trans dihydrodiol benzene (0.455 mumol NADPH/mg/min) with minimal 3 alpha-HSD activity for bile acids (< 0.003 mumol NADPH/mg/min). Lithocholic acid and chenodeoxycholic acid dissociation constants as determined by displacement of the fluorescent probe, bis-1-anilino-8 sulfonate, were higher than those previously reported for the native protein (1 microM versus 10 nM). Significant amino acid sequence homology was found with the human chlordecone reductase, bovine prostaglandin F synthetase, and rat hepatic-3 alpha-HSD suggesting, that HBAB is also a member of the recently identified, monomeric oxidoreductase gene family. Future studies will define the physiologic significance of this novel, multifunctional protein in bile acid transport and xenobiotic metabolism.
Highlights
In humanliver, wepreviously identified one isoform tion intobile
high affinity bile acid binding (HBAB) was stored in 50% glycerol a t -20 "C
Bile acid binding studies was purified with the exact same protocol cloning of HBAB was based on obtaining the primary amino except that no Triton X-100 was added after sonification of the cell acid sequence from the protein toverify a clone identified by pellet or used for the initial equilibration of the glutathione affinity screening a human livercDNA libraryusingrh-3a-hydroxysteroid dehydrogenase (3a-HSD)
Summary
Andrew Stolz$gV,Linda Hammondl, Huan Lou$, Hajime Takikawa,lMl ichael Ronk**,and John E.Shively**. Wehave previously established that in ratliver of dihydrodiol dehydrogenase activity that expresses high affinity bile acid binding (HBAB) with minimal 3a-hydroxysteroid dehydrogenase (3a-HSD)activity for bile acids This protein may assist in the rapid intracellular transport of bile acidsfrom the sinusoidal to the canalicular pole of the cell. We have previously purified and characterized a cytosolic36-kDa bile acid-binding protein (HBAB). ’The abbreviations and trivial names used are: rh-3a-HSD, rat hepatic 3a-hydroxysteroid dehydrogenase; HBAB, human bile acid binder; PBS, phosphate-buffered saline; PAGE, polyacrylamide gel electrophoresis; PCR, polymerase chain reaction; chenodeoxycholic acid, 3a,7a-dihydroxyl-5fl-cholaniaccid; lithocholic acid, 3a-hydroxyl-5~-cholanicacid bis-ANS, l-anilinonaphthalene-8-sulfonic acid; trans-dihydrodiol benzene, trans-1,2-dihydroxycyclohexa-3,5diene; l-acenaphthenol, 7 hydroxyacenapthene; BSA: bovine serum albumin; 3a-hydroxysteroid dehydrogenase (EC 1.1.1.50);dihydrodiol dehydrogenase (EC 1.3.120). HBAB was purified to homogeneity by application onto aHi-Trap Blue minicolcDNA Cloning and Expression of HBAB umn (Pharmacia), pre-equilibrated with 5ml of 50 mM Tris-HC1, pH
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