Molecular cloning and expression of rat liver 3 alpha-hydroxysteroid dehydrogenase.
Complementary DNA clones encoding 3 alpha-hydroxysteroid dehydrogenase (3 alpha HSD) were isolated from a rat liver cDNA lambda gt11 expression library using monoclonal antibodies as probes. The sizes of the cDNA inserts ranged from 1.3-2.3 kilobases. Sequence analysis indicated that variation in the DNA size was due to heterogeneity in the length of 3' noncoding sequences. A full-length cDNA clone of 1286 basepairs contained an open reading frame encoding a protein of 322 amino acids with an estimated mol wt of 37 kDa. When expressed in E. coli, the encoded protein migrated to the same position on sodium dodecyl sulfate-polyacrylamide gels as the enzyme purified from rat liver cytosols. The protein expressed in bacteria was highly active in androsterone reduction in the presence of NAD as cofactor, and this activity was inhibited by indomethacin, a potent inhibitor of 3 alpha HSD. The predicted amino acid sequence of 3 alpha HSD was related to sequences of several other enzymes, including bovine prostaglandin F synthase, human chlordecone reductase, human aldose reductase, human aldehyde reductase, and frog lens epsilon-crystalline, suggesting that these proteins belong to the same gene family.
Cheng
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KC
Department of Pediatrics Cornell University Medical College, New York, New York 10021.
White
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PC
Qin
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KN
D015096:3-Hydroxysteroid Dehydrogenases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods; D003600:Cytosol / Q000201:enzymology; D004247:DNA / Q000235:genetics / Q000302:isolation & purification; D004926:Escherichia coli / Q000235:genetics; D015723:Gene Library; D014644:Genetic Variation; D005822:Genetic Vectors; D006801:Humans; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009693:Nucleic Acid Hybridization; D015345:Oligonucleotide Probes; D016366:Open Reading Frames; D051381:Rats; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D012689:Sequence Homology, Nucleic Acid
D015345:Oligonucleotide Probes; D011994:Recombinant Proteins; D004247:DNA; D015096:3-Hydroxysteroid Dehydrogenases