Oestradiol-17 alpha dehydrogenase from chicken liver.
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Journal: 1984/November - Biochemical Journal
ISSN: 0264-6021
PUBMED: 6593066
Abstract:
The NADP+-linked oestradiol-17 alpha dehydrogenase (EC 1.1.1.148) present in cell-free extracts of chicken liver was investigated with the aim of separating it from a closely related oestradiol-17 beta dehydrogenase (EC 1.1.1.62) found in the same subcellular fraction. However, its chromatographic behaviour on CM-cellulose and DEAE-cellulose was almost identical with that previously reported for the latter enzyme, including resolution into two peaks on the anion-exchanger. Both peaks contained oestradiol-17 alpha dehydrogenase and oestradiol-17 beta dehydrogenase activity. Further attempts to separate the putative enzymes by dye-ligand chromatography with the use of the dyes Procion Yellow, Reactive Red and Cibachron Blue linked to Sepharose were unsuccessful, and they behaved identically on affinity columns of adenosine 2',5'-bisphosphate-agarose and 17 beta-oestradiol 3-hemisuccinate bound to Sepharose. A previous report of partial separation on Sephadex G-200 was not confirmed. Slab gel electrophoresis of enzyme preparations after affinity chromatography on adenosine 2',5'-bisphosphate-agarose revealed multiple bands in systems containing sodium dodecyl sulphate, whereas analysis by rod gel electrophoresis gave two major and one minor bands that stained coincidently for oestradiol-17 alpha dehydrogenase, oestradiol-17 beta dehydrogenase, epitestosterone dehydrogenase and testosterone dehydrogenase activities. Isoelectric focusing gave four enzymically active peaks that each oxidized oestradiol-17 alpha and -17 beta. Apparent Km values for the two forms of oestradiol-17 alpha dehydrogenase obtained by DEAE-cellulose chromatography were 17 and 23 microM for oestradiol-17 alpha, and 8.7 and 11.0 microM for NADP+. Limited kinetic studies with oestradiol-17 alpha and -17 beta with the use of the mixed-substrate method showed that the total velocity was equal to the sum of the separate velocities. The active-site inhibitor-alkylating agent 17 beta-(1-oxoprop-2-ynyl)androst-4-en-3-one did not cause time- or temperature-dependent inhibition, in contrast with the reported case of the oestradiol-17 beta dehydrogenase and 20 alpha-hydroxysteroid dehydrogenase activities of the human placental oestradiol dehydrogenase. NADP+ appeared to afford some protection against inhibition. Investigation of substrate specificity with a limited range of steroids suggests that the enzyme(s) from chicken liver differs substantially from the oestradiol-17 beta dehydrogenase from human placenta, and although the evidence is not conclusive it suggests the existence of one enzyme.
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Biochem J 222(3): 761-768
PMID: 6593066

Oestradiol-17 alpha dehydrogenase from chicken liver.

Abstract

The NADP+-linked oestradiol-17 alpha dehydrogenase (EC 1.1.1.148) present in cell-free extracts of chicken liver was investigated with the aim of separating it from a closely related oestradiol-17 beta dehydrogenase (EC 1.1.1.62) found in the same subcellular fraction. However, its chromatographic behaviour on CM-cellulose and DEAE-cellulose was almost identical with that previously reported for the latter enzyme, including resolution into two peaks on the anion-exchanger. Both peaks contained oestradiol-17 alpha dehydrogenase and oestradiol-17 beta dehydrogenase activity. Further attempts to separate the putative enzymes by dye-ligand chromatography with the use of the dyes Procion Yellow, Reactive Red and Cibachron Blue linked to Sepharose were unsuccessful, and they behaved identically on affinity columns of adenosine 2',5'-bisphosphate-agarose and 17 beta-oestradiol 3-hemisuccinate bound to Sepharose. A previous report of partial separation on Sephadex G-200 was not confirmed. Slab gel electrophoresis of enzyme preparations after affinity chromatography on adenosine 2',5'-bisphosphate-agarose revealed multiple bands in systems containing sodium dodecyl sulphate, whereas analysis by rod gel electrophoresis gave two major and one minor bands that stained coincidently for oestradiol-17 alpha dehydrogenase, oestradiol-17 beta dehydrogenase, epitestosterone dehydrogenase and testosterone dehydrogenase activities. Isoelectric focusing gave four enzymically active peaks that each oxidized oestradiol-17 alpha and -17 beta. Apparent Km values for the two forms of oestradiol-17 alpha dehydrogenase obtained by DEAE-cellulose chromatography were 17 and 23 microM for oestradiol-17 alpha, and 8.7 and 11.0 microM for NADP+. Limited kinetic studies with oestradiol-17 alpha and -17 beta with the use of the mixed-substrate method showed that the total velocity was equal to the sum of the separate velocities. The active-site inhibitor-alkylating agent 17 beta-(1-oxoprop-2-ynyl)androst-4-en-3-one did not cause time- or temperature-dependent inhibition, in contrast with the reported case of the oestradiol-17 beta dehydrogenase and 20 alpha-hydroxysteroid dehydrogenase activities of the human placental oestradiol dehydrogenase. NADP+ appeared to afford some protection against inhibition. Investigation of substrate specificity with a limited range of steroids suggests that the enzyme(s) from chicken liver differs substantially from the oestradiol-17 beta dehydrogenase from human placenta, and although the evidence is not conclusive it suggests the existence of one enzyme.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Betz G. Reaction mechanism of 17-beta-estradiol dehydrogenase determined by equilibrium rate exchange. J Biol Chem. 1971 Apr 10;246(7):2063–2068. [PubMed] [Google Scholar]
  • BROWN JB. A chemical method for the determination of oestriol, oestrone and oestradiol in human urine. Biochem J. 1955 Jun;60(2):185–193.[PMC free article] [PubMed] [Google Scholar]
  • Cuatrecasas P. Protein purification by affinity chromatography. Derivatizations of agarose and polyacrylamide beads. J Biol Chem. 1970 Jun;245(12):3059–3065. [PubMed] [Google Scholar]
  • Descomps B, de Paulet AC. Etude du site actif de al 17-beta-hydroxystéroïde: NAD (P) oxydoréductase soluble du placenta humain. Bull Soc Chim Biol (Paris) 1969;51(12):1591–1611. [PubMed] [Google Scholar]
  • Engel LL, Groman EV. Human placental 17beta-estradiol dehydrogenase: characterization and structural studies. Recent Prog Horm Res. 1974;30(0):139–169. [PubMed] [Google Scholar]
  • Hasnain S, Williamson DG. The separation and partial purification of the soluble 17 alpha- and 17 beta-hydroxysteroid dehydrogenases of rabbit liver. Can J Biochem. 1974 Feb;52(2):120–125. [PubMed] [Google Scholar]
  • Hasnain S, Williamson DG. Purification of multiple forms of the soluble 17alpha-hydroxy steroid dehydrogenase or rabbit liver. Biochem J. 1975 Jun;147(3):457–461.[PMC free article] [PubMed] [Google Scholar]
  • Hasnain S, Williamson DG. Properties of the multiple forms of the soluble 17alpha-hydroxy steroid dehydrogenase of rabbit liver. Biochem J. 1977 Feb 1;161(2):279–283.[PMC free article] [PubMed] [Google Scholar]
  • Henderson LL, Warren JC. Purification and characterization of epimeric estradiol dehydrogenases (17 alpha and 17 beta) from equine placenta. Biochemistry. 1984 Jan 31;23(3):486–491. [PubMed] [Google Scholar]
  • Houslay MD, Garrett NJ, Tipton KF. Mixed substrate experiments with human brain monoamine oxidase. Biochem Pharmacol. 1974 Jul 15;23(14):1937–1944. [PubMed] [Google Scholar]
  • Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. [PubMed] [Google Scholar]
  • LANGER LJ, ALEXANDER JA, ENGEL LL. Human placental estradiol-17 beta dehydrogenase. II. Kinetics and substrate specificities. J Biol Chem. 1959 Oct;234:2609–2614. [PubMed] [Google Scholar]
  • Lau PC, Layne DS, Williamson DG. A 3(17) alpha-hydroxysteroid dehydrogenase of female rabbit kidney cytosol. Purification and characterization of multiple forms of the enzyme. J Biol Chem. 1982 Aug 25;257(16):9444–9449. [PubMed] [Google Scholar]
  • Lau PC, Layne DS, Williamson DG. Comparison of the multiple forms of the soluble 3(17) alpha-hydroxysteroid dehydrogenases of female rabbit kidney and liver. J Biol Chem. 1982 Aug 25;257(16):9450–9456. [PubMed] [Google Scholar]
  • Renwick AG, Engel LL. The partial purification of 17 alpha- and 17 beta-estradiol dehydrogenase activities from chicken liver. Biochim Biophys Acta. 1967;146(2):336–348. [PubMed] [Google Scholar]
  • Renwick AG, Chambers SM, Willcox P. "Affinity" chromatography of steroid-transforming enzymes with a non-steroidal ligand. Biochem J. 1979 Feb 1;177(2):401–408.[PMC free article] [PubMed] [Google Scholar]
  • Renwick AG, Soon CY, Chambers SM, Brown CR. Estradiol-17 beta dehydrogenase from chicken liver. J Biol Chem. 1981 Feb 25;256(4):1881–1887. [PubMed] [Google Scholar]
  • Tobias B, Covey DF, Strickler RC. Inactivation of human placental 17 beta-estradiol dehydrogenase and 20 alpha-hydroxysteroid dehydrogenase with active site-directed 17 beta-propynyl-substituted progestin analogs. J Biol Chem. 1982 Mar 25;257(6):2783–2786. [PubMed] [Google Scholar]
  • Travis J, Bowen J, Tewksbury D, Johnson D, Pannell R. Isolation of albumin from whole human plasma and fractionation of albumin-depleted plasma. Biochem J. 1976 Aug 1;157(2):301–306.[PMC free article] [PubMed] [Google Scholar]
  • VELLE W, ENGEL LL. ENZYMES FROM BOVINE PLACENTA AND SEMINAL VESICLES THAT OXIDIZE D(-)-1,2-PROPANEDIOL AND OTHER POLYOLS: THEIR POSSIBLE RELATION TO FRUCTOSE FORMATION. Endocrinology. 1964 Mar;74:429–439. [PubMed] [Google Scholar]
Copyright notice
Abstract
The NADP+-linked oestradiol-17 alpha dehydrogenase (EC 1.1.1.148) present in cell-free extracts of chicken liver was investigated with the aim of separating it from a closely related oestradiol-17 beta dehydrogenase (EC 1.1.1.62) found in the same subcellular fraction. However, its chromatographic behaviour on CM-cellulose and DEAE-cellulose was almost identical with that previously reported for the latter enzyme, including resolution into two peaks on the anion-exchanger. Both peaks contained oestradiol-17 alpha dehydrogenase and oestradiol-17 beta dehydrogenase activity. Further attempts to separate the putative enzymes by dye-ligand chromatography with the use of the dyes Procion Yellow, Reactive Red and Cibachron Blue linked to Sepharose were unsuccessful, and they behaved identically on affinity columns of adenosine 2',5'-bisphosphate-agarose and 17 beta-oestradiol 3-hemisuccinate bound to Sepharose. A previous report of partial separation on Sephadex G-200 was not confirmed. Slab gel electrophoresis of enzyme preparations after affinity chromatography on adenosine 2',5'-bisphosphate-agarose revealed multiple bands in systems containing sodium dodecyl sulphate, whereas analysis by rod gel electrophoresis gave two major and one minor bands that stained coincidently for oestradiol-17 alpha dehydrogenase, oestradiol-17 beta dehydrogenase, epitestosterone dehydrogenase and testosterone dehydrogenase activities. Isoelectric focusing gave four enzymically active peaks that each oxidized oestradiol-17 alpha and -17 beta. Apparent Km values for the two forms of oestradiol-17 alpha dehydrogenase obtained by DEAE-cellulose chromatography were 17 and 23 microM for oestradiol-17 alpha, and 8.7 and 11.0 microM for NADP+. Limited kinetic studies with oestradiol-17 alpha and -17 beta with the use of the mixed-substrate method showed that the total velocity was equal to the sum of the separate velocities. The active-site inhibitor-alkylating agent 17 beta-(1-oxoprop-2-ynyl)androst-4-en-3-one did not cause time- or temperature-dependent inhibition, in contrast with the reported case of the oestradiol-17 beta dehydrogenase and 20 alpha-hydroxysteroid dehydrogenase activities of the human placental oestradiol dehydrogenase. NADP+ appeared to afford some protection against inhibition. Investigation of substrate specificity with a limited range of steroids suggests that the enzyme(s) from chicken liver differs substantially from the oestradiol-17 beta dehydrogenase from human placenta, and although the evidence is not conclusive it suggests the existence of one enzyme.
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