Purification, characterization and partial peptide microsequencing of progesterone 5 beta-reductase from shoot cultures of Digitalis purpurea.

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  • Author(s): Gärtner DE;Gärtner DE; Keilholz W; Seitz HU
  • Source:
    European journal of biochemistry [Eur J Biochem] 1994 Nov 01; Vol. 225 (3), pp. 1125-32.
  • Publication Type:
    Journal Article; Research Support, Non-U.S. Gov't
  • Language:
    English
  • Additional Information
    • Source:
      Publisher: Blackwell Science Ltd. on behalf of the Federation of European Biochemical Societies Country of Publication: England NLM ID: 0107600 Publication Model: Print Cited Medium: Print ISSN: 0014-2956 (Print) Linking ISSN: 00142956 NLM ISO Abbreviation: Eur J Biochem Subsets: MEDLINE
    • Publication Information:
      Publication: -2004: Oxford, UK : Blackwell Science Ltd. on behalf of the Federation of European Biochemical Societies
      Original Publication: Berlin, New York, Springer.
    • Subject Terms:
      Plants, Medicinal* ; Plants, Toxic*; Digitalis/*enzymology ; Oxidoreductases/*isolation & purification; Amino Acid Sequence ; Cardenolides/metabolism ; Kinetics ; Molecular Sequence Data ; Molecular Weight ; NADP/metabolism ; Oxidoreductases/genetics ; Oxidoreductases/metabolism ; Peptide Fragments/genetics ; Peptide Fragments/isolation & purification ; Steroids ; Substrate Specificity
    • Abstract:
      Progesterone 5 beta-reductase, which catalyzes the reduction of progesterone to 5 beta-pregnane-3,20-dione, was purified 770-fold to homogeneity from the cytosolic fraction of shoot cultures of Digitalis purpurea. This purification involved DEAE-Sephacel, affinity chromatography (Blue-Sepharose CL-6B and adenosine 2',5'-bisphosphate-Sepharose 4B) and elution from a gel matrix after non-dissociating PAGE. The molecular mass determined by SDS/PAGE was 43 kDa and the molecular mass determined by gel-filtration chromatography on calibrated Sephadex G-200 was 280 kDa, thus indicating that the native protein is a polymer consisting of several subunits. The purified enzyme had a Km value of 6 microM for NADPH and 34 microM for progesterone. The enzyme had a strong substrate specificity for progesterone. The relative rates for other steroids such as testosterone, cortisone and cortisol were much lower. The trypsin digestion of the purified progesterone 5 beta-reductase resulted in 100 peptide fragments. The largest fragment after trypsin digestion and sequence analysis consisted of 13 amino acids.
    • Accession Number:
      0 (Cardenolides)
      0 (Peptide Fragments)
      0 (Steroids)
      53-59-8 (NADP)
      EC 1.- (Oxidoreductases)
      EC 1.3.1.- (progesterone 5 beta-reductase)
    • Publication Date:
      Date Created: 19941101 Date Completed: 19941221 Latest Revision: 20190620
    • Publication Date:
      20240829
    • Accession Number:
      10.1111/j.1432-1033.1994.1125b.x
    • Accession Number:
      7957203