Expression of 11β-hydroxysteroid dehydrogenase using recombinant vaccinia virus

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

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164 Scopus citations


Ligand specificity of the type I steroid receptor is apparently conferred by the activity of 11β-hydroxy-steroid 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-oxored-uction 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 micro-somes. 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 nM for dehydrogenase, IQ-20 nM for reductase). Partial inhibition of glycosyla-tion with Ai-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
Issue number12
StatePublished - Dec 1990

ASJC Scopus subject areas

  • Molecular Biology
  • Endocrinology


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