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

Fingerprint

20-Hydroxysteroid Dehydrogenases
Purification
Rats
Ovary
Enzymes
20-alpha-Dihydroprogesterone
High Pressure Liquid Chromatography
Affinity chromatography
Capillary electrophoresis
Column chromatography
Capillary Electrophoresis
Ionic strength
Molecular Structure
Electrophoresis
Affinity Chromatography
Sodium Dodecyl Sulfate
Osmolar Concentration
Molecular structure
Chromatography
Polyacrylamide Gel Electrophoresis

Keywords

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

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Molecular Biology
  • Structural Biology

Cite this

Purification and characterization of rat ovarian 20α-hydroxysteroid dehydrogenase. / Noda, Ken; Shiota, Kunio; Takahashi, Michio.

In: Biochimica et Biophysica Acta (BBA)/Protein Structure and Molecular, Vol. 1079, No. 1, 09.08.1991, p. 112-118.

Research output: Contribution to journalArticle

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