Skip to main content
NCBI home page
Biochemical Journal logoLink to Biochemical Journal
. 1990 Feb 15;266(1):41–46. doi: 10.1042/bj2660041

Characterization and localization of progesterone 5 alpha-reductase from cell cultures of foxglove (Digitalis lanata EHRH).

S Wendroth 1, H U Seitz 1
PMCID: PMC1131093  PMID: 2106876

Abstract

Progesterone 5 alpha-reductase, which catalyses the reduction of progesterone to 5 alpha-pregnane-3,20-dione, was isolated and characterized from cell cultures of Digitalis lanata (foxglove). Optimum enzyme activity was observed at pH 7.0, and the enzyme had an apparent Km value of 30 microM for its substrate progesterone. The enzyme needs NADPH as reductant, which could not be replaced by NADH. For NADPH, the apparent Km value is 130 microM. The optimum temperature was 40 degrees C; at temperatures below 45 degrees C, the product 5 alpha-pregnane-3,20-dione was reduced by a second reaction to 5 alpha-pregnan-3 beta-ol-20-one. Progesterone 5 alpha-reductase activity was not dependent on bivalent cations. In the presence of EDTA, 0.1 mM-Mn2+ had no influence on enzyme activity, whereas 0.1 mM-Ca2+, -Co2+ and -Zn2+ decreased progesterone 5 alpha-reductase activity. Only 0.1 mM-Mg2+ was slightly stimulatory. EDTA and thiol reagents such as dithiothreitol stimulate progesterone 5 alpha-reductase activity. By means of linear sucrose gradient fractionation of the cellular membranes, progesterone 5 alpha-reductase was found to be located in the endoplasmic reticulum.

Full text

PDF
41

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bach T. J., Rogers D. H., Rudney H. Detergent-solubilization, purification, and characterization of membrane-bound 3-hydroxy-3-methylglutaryl-coenzyme A reductase from radish seedlings. Eur J Biochem. 1986 Jan 2;154(1):103–111. doi: 10.1111/j.1432-1033.1986.tb09364.x. [DOI] [PubMed] [Google Scholar]
  2. Bertics P. J., Karavolas H. J. Partial characterization of the microsomal and solubilized hypothalamic progesterone 5 alpha-reductase. J Steroid Biochem. 1984 Sep;21(3):305–314. doi: 10.1016/0022-4731(84)90284-x. [DOI] [PubMed] [Google Scholar]
  3. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  4. Cheng Y. J., Karavolas H. J. Subcellular distribution and properties of progesterone (delta4-steroid) 5alpha-reductase in rat medial basal hypothalamus. J Biol Chem. 1975 Oct 25;250(20):7997–8003. [PubMed] [Google Scholar]
  5. Frederiksen D. W., Wilson J. D. Partial characterization of the nuclear reduced nicotinamide adenine dinucleotide phosphate: delta 4-3-ketosteroid 5 alpha-oxidoreductase of rat prostate. J Biol Chem. 1971 Apr 25;246(8):2584–2593. [PubMed] [Google Scholar]
  6. Golf S. W., Graef V. Reconstitution of NADPH: 4-ene-3-oxosteroid-5alpha-oxidoreductase from solubilized components of rat liver microsomes. J Steroid Biochem. 1978 Nov;9(11):1087–1092. doi: 10.1016/0022-4731(78)90037-7. [DOI] [PubMed] [Google Scholar]
  7. Graves J. M., Smith W. K. Transformation of pregnenolone and progesterone by cultured plant cells. Nature. 1967 Jun 17;214(5094):1248–1249. doi: 10.1038/2141248a0. [DOI] [PubMed] [Google Scholar]
  8. Hodges T. K., Leonard R. T. Purification of a plasma membrane-bound adenosine triphosphatase from plant roots. Methods Enzymol. 1974;32:392–406. doi: 10.1016/0076-6879(74)32039-3. [DOI] [PubMed] [Google Scholar]
  9. Hudson R. W. Studies of the nuclear 5 alpha-reductase of human hyperplastic prostatic tissue. J Steroid Biochem. 1981 Jun;14(6):579–584. doi: 10.1016/0022-4731(81)90034-0. [DOI] [PubMed] [Google Scholar]
  10. Kondo K., Oba K. Purification and characterization of 3-hydroxy-3-methylglutaryl CoA reductase from potato tubers. J Biochem. 1986 Oct;100(4):967–974. doi: 10.1093/oxfordjournals.jbchem.a121809. [DOI] [PubMed] [Google Scholar]
  11. Krause J. E., Karavolas H. J. Subcellular location of hypothalamic progesterone metabolizing enzymes and evidence for distinct NADH- and NADPH-linked 3 alpha-hydroxysteroid oxidoreductase activities. J Steroid Biochem. 1980 Mar;13(3):271–280. doi: 10.1016/0022-4731(80)90005-9. [DOI] [PubMed] [Google Scholar]
  12. Negm F. B., Marlow G. C. Partial Purification and Characterization of d-Ribose-5-phosphate Reductase from Adonis vernalis L. Leaves. Plant Physiol. 1985 Aug;78(4):758–761. doi: 10.1104/pp.78.4.758. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Robaire B., Ewing L. L., Zirkin B. R., Irby D. C. Steroid delta4-5alpha-reductase and 3alpha-hydroxysteroid dehydrogenase in the rat epididymis. Endocrinology. 1977 Nov;101(5):1379–1390. doi: 10.1210/endo-101-5-1379. [DOI] [PubMed] [Google Scholar]
  14. Schabort J. C., Potgieter D. J. Cucurbitacin B delta 23-reductase from Cucurbita maxima. II. Cofactor requirements, enzyme kinetics, substrate specificity and other characteristics. Biochim Biophys Acta. 1968 Jan 8;151(1):47–53. doi: 10.1016/0005-2744(68)90159-9. [DOI] [PubMed] [Google Scholar]
  15. Scheer H., Robaire B. Solubilization and partial characterization of rat epididymal delta 4-steroid 5 alpha-reductase (cholestenone 5 alpha-reductase). Biochem J. 1983 Apr 1;211(1):65–74. doi: 10.1042/bj2110065. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Vick B. A., Zimmerman D. C. Characterization of 12-oxo-phytodienoic Acid reductase in corn: the jasmonic Acid pathway. Plant Physiol. 1986 Jan;80(1):202–205. doi: 10.1104/pp.80.1.202. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Voigt W., Fernandez E. P., Hsia S. L. Transformation of testosterone into 17 beta-hydroxy-5 alpha-androstan-3-one by microsomal preparations of human skin. J Biol Chem. 1970 Nov 10;245(21):5594–5599. [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES