Expression of 11 beta-hydroxysteroid dehydrogenase using recombinant vaccinia virus.
Ligand specificity of the type I steroid receptor is apparently conferred by the activity of 11 beta-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 11 beta-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 x 10(-4], whereas the rate constants for the two reactions were nearly equal (48 vs. 47 min-1 x 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, less than 5 nM 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.
Agarwal
A K
AK
Division of Pediatric Endocrinology, Cornell University Medical College, New York, New York 10021.
Tusie-Luna
M T
MT
Monder
C
C
White
P C
PC
D042842:11-beta-Hydroxysteroid Dehydrogenases; D000818:Animals; D002460:Cell Line; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D004274:DNA, Recombinant; D015870:Gene Expression; D006034:Glycyrrhetinic Acid / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D006913:Hydroxysteroid Dehydrogenases / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D007163:Immunosorbent Techniques; D007700:Kinetics; D009249:NADP / Q000494:pharmacology; D051381:Rats; D014415:Tunicamycin / Q000494:pharmacology; D014616:Vaccinia virus / Q000235:genetics*
D004274:DNA, Recombinant; D014415:Tunicamycin; D009249:NADP; D004247:DNA; D006913:Hydroxysteroid Dehydrogenases; D042842:11-beta-Hydroxysteroid Dehydrogenases; D006034:Glycyrrhetinic Acid