Expression of 11β-hydroxysteroid dehydrogenase using recombinant vaccinia virus

Anil K. Agarwal, Maria Teresa Tusie-Luna, Carl Monder, Perrin C. White

Research output: Contribution to journalArticle

162 Citations (Scopus)

Abstract

Ligand specificity of the type I steroid receptor is apparently conferred by the activity of 11β-hydroxysteroid dehydrogenase. To determine the kinetic properties of this enzyme, rat liver cDNA was expressed in cultured cells using recombinant vaccinia virus. Although this enzyme catalyzes only dehydrogenation when purified from rat liver, the recombinant enzyme obtained from cell lysates catalyzed both 11β-dehydrogenation of corticosterone to 11-dehydrocorticosterone and the reverse 11-oxoreduction reaction. At pH 8.5, the first order rate constant Kcat/Km for dehydrogenase activity exceeded that for reductase (63 vs. 38 min-1 × 10-4), whereas the rate constants for the two reactions were nearly equal (48 vs. 47 min-1 × 10-4) at pH 7.0. These results are consistent with the previously determined pH optima for these activities in liver microsomes. Removal (with glucose-6-phosphate dehydrogenase) of NADP+ produced by the reductase reaction significantly increased reductase activity. Glycyrrhetinic acid, a known inhibitor of the dehydrogenase reaction, also inhibited the reductase reaction at slightly higher concentrations (50% inhibitory concentration, <5 nu for dehydrogenase, 10-20 nM for reductase). Partial inhibition of glycosylation with A1-tunicamycin decreased dehydrogenase activity 50% without affecting reductase activity. The data demonstrate that a single polypeptide catalyzes both dehydrogenation and reduction, although the presence of additional enzyme forms catalyzing one or the other activity has not been ruled out.

Original languageEnglish (US)
Pages (from-to)1827-1832
Number of pages6
JournalMolecular Endocrinology
Volume4
Issue number12
StatePublished - 1990

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11-beta-Hydroxysteroid Dehydrogenases
Vaccinia virus
Oxidoreductases
Enzymes
Inhibitory Concentration 50
Glycyrrhetinic Acid
Steroid Receptors
Glucosephosphate Dehydrogenase
Liver
Liver Microsomes
Corticosterone
NADP
Glycosylation
Cultured Cells
Complementary DNA

ASJC Scopus subject areas

  • Molecular Biology
  • Endocrinology, Diabetes and Metabolism

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Expression of 11β-hydroxysteroid dehydrogenase using recombinant vaccinia virus. / Agarwal, Anil K.; Tusie-Luna, Maria Teresa; Monder, Carl; White, Perrin C.

In: Molecular Endocrinology, Vol. 4, No. 12, 1990, p. 1827-1832.

Research output: Contribution to journalArticle

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