Purification and characterization of rat ovarian 20 alpha-hydroxysteroid dehydrogenase.
To investigate the regulatory mechanism of 20 alpha-hydroxysteroid dehydrogenase (20 alpha-HSD) (EC 1.1.1.149) activity in ovarian tissue, the enzyme was purified from ovaries of normal mature female rats. Column chromatography of the cytosolic fraction from ovaries on DEAE-Toyopearl 650M revealed two peaks of the 20 alpha-HSD activity at different ionic strengths. These peaks were designated HSD1 and HSD2, respectively. Each of the active fractions was further purified to homogeneity by dye-affinity chromatography using Matrex Green A and AF Red-Toyopearl. Both the fractions appeared as a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (at Mr = 33,000 under reducing conditions). Under non-reducing conditions, similar values were obtained on gel-exclusion HPLC, indicating that the enzyme fractions were single-stranded, monomeric polypeptides. Homogeneous HSD1 and HSD2 were purified 361-fold and 509-fold, respectively, and differed in their substrate preference. The two enzyme fractions had Km values of 4.75 microM and 5.16 microM for 20 alpha-dihydroprogesterone, respectively, and showed almost the same RF values on reverse-phase HPLC and free-zone capillary electrophoresis. However, amino acid composition was slightly different, i.e. lysin content was higher in HSD1 than HSD2. Thus, it was clarified that two types of 20 alpha-HSD with very similar molecular structures are present in the rat ovary.
Noda
K
K
Department of Veterinary Physiology, University of Tokyo, Japan.
Shiota
K
K
Takahashi
M
M
D015089:20-Hydroxysteroid Dehydrogenases / Q000302:isolation & purification* / Q000378:metabolism; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002853:Chromatography, Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D007527:Isoenzymes / Q000302:isolation & purification* / Q000378:metabolism; D007700:Kinetics; D010053:Ovary / Q000201:enzymology*; D051381:Rats; D011919:Rats, Inbred Strains; D013379:Substrate Specificity
D000596:Amino Acids; D007527:Isoenzymes; D015089:20-Hydroxysteroid Dehydrogenases