Purification and characterization of rat ovarian 20α-hydroxysteroid dehydrogenase

Ken Noda, Kunio Shiota, Michio Takahashi

Research output: Contribution to journalArticle

17 Citations (Scopus)

Abstract

To investigate the regulatory mechanism of 20α-hydroxysteroid dehydrogenase (20α-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 20α-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 μM and 5.16 μM for 20α-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 and HSD2. Thus, it was clarified that two types of 20α-HSD with very similar molecular structures are present in the rat ovary.

Original languageEnglish
Pages (from-to)112-118
Number of pages7
JournalBiochimica et Biophysica Acta (BBA)/Protein Structure and Molecular
Volume1079
Issue number1
DOIs
Publication statusPublished - 1991 Aug 9
Externally publishedYes

Keywords

  • 20α-Hydroxysteroid dehydrogenase
  • Enzyme purification
  • Rat ovary

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Molecular Biology
  • Structural Biology

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