Rat hepatic 3 alpha-hydroxysteroid dehydrogenase: expression of cDNA and physiological function in bile acid biosynthetic pathway.
3 alpha-Hydroxysteroid dehydrogenase (3 alpha-HSD) [EC 1.1.1.213]2 plays important multifunctional roles in metabolizing steroid hormones, polycyclic aromatic hydrocarbons, and prostaglandins and also in transforming the steroid nucleus for the biosynthesis of bile acids from cholesterol in liver. To gain insight into the details and physiological functions of 3 alpha-HSD in the bile acid biosynthetic pathway, cDNA clones of 3 alpha-HSD were isolated from rat liver lambda phage cDNA libraries by using specific antibodies to 3 alpha-HSD purified from rat liver. Transfection of the 3 alpha-HSD cDNA in Simian COS7 cells resulted in the expression of an immunoreactive protein to the antibodies against the purified enzyme, and the transfected cells exhibited activities for not only 7 alpha-hydroxy-5 beta-cholestan-3-one, the intermediate of bile acid biosynthesis, but also steroid hormones and 9,10-phenanthrenequinone. Northern blot analysis on poly(A)+ RNA by selective use of different cDNA fragments of the 5'-untranslated region, the coding region, and the 3'-untranslated region as probes revealed three hybridizable bands, 3.6, 2.7, and 2.5 kb, in liver and four bands, 3.6, 2.7, 2.5, and 1.8 kb, in ovary. Of these, the 2.7- and 1.8-kb bands were predominant in liver and ovary, respectively. Northern hybridization analysis also revealed that the coding region of the various sizes of mRNA seemed to be common. Southern blot analysis of genomic DNA by the selective use of the cDNA fragments as probes indicated that the various mRNA species were derived from a single gene, probably due to an alternative splicing mechanism.(ABSTRACT TRUNCATED AT 250 WORDS)
Usui
E
E
Department of Biochemistry, Hiroshima University School of Dentistry.
Okuda
K
K
Kato
Y
Y
Noshiro
M
M
D015096:3-Hydroxysteroid Dehydrogenases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D045002:3-alpha-Hydroxysteroid Dehydrogenase (B-Specific); D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001647:Bile Acids and Salts / Q000096:biosynthesis*; D002460:Cell Line; D002783:Cholestenones / Q000378:metabolism*; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D003907:Dexamethasone / Q000494:pharmacology; D005260:Female; D008099:Liver / Q000201:enzymology; D008297:Male; D008969:Molecular Sequence Data; D010053:Ovary / Q000201:enzymology; D011061:Poly A / Q000737:chemistry; D011441:Propylthiouracil / Q000494:pharmacology; D012313:RNA / Q000737:chemistry; D012333:RNA, Messenger; D051381:Rats; D017208:Rats, Wistar; D012727:Sex Characteristics
D001647:Bile Acids and Salts; D002783:Cholestenones; D018076:DNA, Complementary; D012333:RNA, Messenger; D011061:Poly A; C002656:7 alpha-hydroxy-4-cholesten-3-one; D012313:RNA; D011441:Propylthiouracil; D003907:Dexamethasone; D015096:3-Hydroxysteroid Dehydrogenases; D045002:3-alpha-Hydroxysteroid Dehydrogenase (B-Specific)