Purification and characterization of the multiple forms of 3 alpha-hydroxysteroid dehydrogenase in rat liver cytosol.
The presence of multiple forms of 3 alpha-hydroxysteroid dehydrogenase in the cytosol of male rat livers was demonstrated. The enzyme activity was separated into two fractions (F3 and F4) by DEAE-cellulose chromatography, and further fractionation of F3 into four (I-IV) and F4 into three (I-III) fractions was achieved by subsequent CM-Sephadex chromatography. Six forms (F3-II-IV and F4-I-III) were further purified by chromatofocusing and Red-Sepharose 4B chromatography. Two (F4-II and III) of the isolated enzymes were homogeneous, based on polyacrylamide gel electrophoresis. No shift of pI values was observed, when isoelectric focusing was performed with the F4 enzyme species in the presence of NAD(P)+ or NAD(P)H. All six enzyme species migrated closely with each other on dodecyl sulfate-polyacrylamide gel electrophoresis, from which the molecular masses were estimated to be 32 500 Da. Gel filtration gave similar values for the F4 enzyme species, indicating that each enzyme is a monomeric peptide. All enzyme species were able to catalyse the dehydrogenation of 3 alpha-hydroxysteroids (C19 to C26), and not C27 compound having a 1,5-dimethylhexyl side chain. The catalytic properties with steroids were very similar for five of the enzyme species, but F3-IV especially preferred androsterone. When male rat livers were used for isolation, the enzyme activity ratio of F3 to F4 for androsterone was about 1 to 8, whereas the ratio was about 1 to 20 for female rat livers. Considering the biosynthetic pathway of bile acids, the enzymes isolated here might play a specific role in the conversion of a 3 beta-hydroxy group to a 3 alpha-hydroxy group via a 3-oxo group of an intermediate in the synthesis of bile acids.
D015096:3-Hydroxysteroid Dehydrogenases / Q000302:isolation & purification*; D045002:3-alpha-Hydroxysteroid Dehydrogenase (B-Specific); D000818:Animals; D001647:Bile Acids and Salts / Q000494:pharmacology; D003600:Cytosol / Q000201:enzymology; D004591:Electrophoresis, Polyacrylamide Gel; D007527:Isoenzymes / Q000302:isolation & purification*; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008970:Molecular Weight; D051381:Rats; D013379:Substrate Specificity
D001647:Bile Acids and Salts; D007527:Isoenzymes; D015096:3-Hydroxysteroid Dehydrogenases; D045002:3-alpha-Hydroxysteroid Dehydrogenase (B-Specific)