Isolation and partial characterization of a full-length cDNA clone for 3 alpha-hydroxysteroid dehydrogenase: a potential target enzyme for nonsteroidal anti-inflammatory drugs.
Homogeneous 3 alpha-hydroxysteroid dehydrogenase (3 alpha-HSD; EC 1.1.1.50) of rat liver cytosol is a monomeric (Mr 34000) NAD(P)+ dependent oxidoreductase which displays 9-, 11- & 15-hydroxyprostaglandin dehydrogenase activity. The enzyme is potently inhibited by nonsteroidal anti-inflammatory drugs (NSAIDs), suggesting that 3 alpha-HSD may be a target enzyme for NSAIDs. A monospecific, polyclonal anti-sera raised against the purified enzyme was used to screen a lambda gt11 expression library and oligonucleotide probes complementary to the 5' and 3' ends of immunopositive clones were used to isolate a 2.1 kb full-length cDNA. Digestion of the full-length cDNA with Eco RI generated two fragments of 1.1 and 1.0 kb in length. Both fragments were subcloned into pGEM3 and partially sequenced. The 1.1 kb fragment contains the C-terminus of 3 alpha-HSD which was confirmed by an in-frame stop codon and comparison of the predicted amino acid sequence to peptide sequence obtained from two endo lys-C peptides of 3 alpha-HSD. The 1.0 kb fragment is 5' to the 1.1 kb fragment and is sufficient in length to contain the remainder of the entire open reading frame for 3 alpha-HSD. Dideoxysequencing reveals significant sequence homology with bovine lung prostaglandin PGF2 alpha synthase. These findings support the role 3 alpha-HSD in inflammation and suggest that hydroxysteroid dehydrogenases, hydroxyprostaglandin dehydrogenases and prostaglandin F2 alpha synthase may be members of a common gene family.
Pawlowski
J
J
Department of Pharmacology, University of Pennsylvania School of Medicine, Philadelphia 19104-6084.
Huizinga
M
M
Penning
T M
TM
D015096:3-Hydroxysteroid Dehydrogenases / Q000235:genetics*; D045002:3-alpha-Hydroxysteroid Dehydrogenase (B-Specific); D000595:Amino Acid Sequence; D000818:Animals; D000894:Anti-Inflammatory Agents, Non-Steroidal / Q000494:pharmacology*; D003001:Cloning, Molecular; D004247:DNA / Q000032:analysis / Q000302:isolation & purification*; D008297:Male; D008969:Molecular Sequence Data; D011817:Rabbits; D051381:Rats; D011919:Rats, Inbred Strains; D014961:X-Ray Diffraction
D000894:Anti-Inflammatory Agents, Non-Steroidal; D004247:DNA; D015096:3-Hydroxysteroid Dehydrogenases; D045002:3-alpha-Hydroxysteroid Dehydrogenase (B-Specific)