The sexually differentiated delta 4-3-ketosteroid 5 beta-reductase of rat liver. Purification, characterization, and quantitation.
delta 4-3-Ketosteroid 5 beta-reductase was purified from male rat liver cytosol. The purification scheme consisted of column chromatographies on hydroxylapatite and DEAE-Sepharose, chromatofocusing, and Sephadex G-75 gel filtration followed by sodium dodecyl sulfate-gel electrophoresis. The column chromatography steps gave a 100-fold purification and resulted in a 90% pure preparation as judged by sodium dodecyl sulfate-gel electrophoresis. Kinetic properties with 4-androstene-3,17-dione as substrate were established for the enzyme, and its activity regarding three other delta 4-3-ketosteroids, testosterone, progesterone, and cortisol, was investigated. The relative rates of reduction of these steroids were 1.0, 0.8, 0.7, and 0.62, respectively. The electrophoretically purified 5 beta-reductase, with an Mr of 38,000, was used for immunization of rabbits. The antiserum was shown to be monospecific as judged from immunoblotting of electrophoretically separated rat liver cytosolic proteins. Immunological reactive protein and enzymatic 5 beta-reductase activity co-purified in the chromatographic steps. The sex difference in enzyme activity, 0.26 versus 0.10 nmol of product/mg of proteins/min for males and females, respectively, was shown to be due to a difference in concentration of enzyme protein. The 5 beta-reductase was calculated to constitute 1% of the total cytosolic proteins in male livers, whereas the corresponding figure for female livers was 0.3%.
D000818:Animals; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D002855:Chromatography, Thin Layer; D003600:Cytosol / Q000201:enzymology; D005260:Female; D007163:Immunosorbent Techniques; D007525:Isoelectric Focusing; D008099:Liver / Q000201:enzymology*; D008297:Male; D010088:Oxidoreductases / Q000302:isolation & purification*; D051381:Rats; D011919:Rats, Inbred Strains; D012727:Sex Characteristics
D010088:Oxidoreductases; C021936:3-oxo-5 beta-steroid delta 4-dehydrogenase