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Updated: Jul 29, 2026
11:07
Biochemical Reconstitution of Steroid Receptor•Hsp90 Protein Complexes and Reactivation of Ligand Binding
Published on: September 21, 2011
The 11beta hydroxysteroid dehydrogenase 2 exists as an inactive dimer.
E P Gomez-Sanchez1, V Ganjam, Y J Chen
1Endocrine Section and Research Service, G.V. (Sonny) Montgomery VA Medical Center, 1500 E Woodrow Wilson Dr (151), Jackson, MS 39216, USA. elise.gomezsanchez@med.va.gov
Steroids
|September 29, 2001
Summary
The 11beta-hydroxysteroid dehydrogenase type 2 (11beta-HSD2) enzyme in rat kidneys exists as a dimer, which inactivates it. DTT addition breaks this dimer, revealing active NAD(+)-dependent enzyme kinetics.
Area of Science:
- Biochemistry
- Enzymology
- Molecular Biology
Background:
- 11beta-hydroxysteroid dehydrogenase types 1 and 2 (11beta-HSD1 and 11beta-HSD2) regulate glucocorticoid receptor activity.
- 11beta-HSD2 in the kidney inactivates glucocorticoids, allowing aldosterone access to its receptor.
- Previous studies on rat kidney 11beta-HSD2 kinetics yielded inconsistent results.
Purpose of the Study:
- To investigate the inconsistent kinetic results of 11beta-HSD2 activity in rat kidney.
- To determine the effect of reducing agents on 11beta-HSD2 activity and structure.
Main Methods:
- Western blotting was used to analyze 11beta-HSD2 protein structure with and without beta-mercaptoethanol.
- Enzyme kinetics assays were performed using 3H-corticosterone and varying concentrations of DTT.
- NAD(+)-dependent and NADP(+)-dependent activities were measured.
Main Results:
- Western blots indicated 11beta-HSD2 exists as a 40 kDa monomer and an 80 kDa dimer under non-denaturing conditions.
- DTT addition significantly increased NAD(+)-dependent 11beta-HSD2 activity (approximately 10-fold).
- The K(m) for NAD(+)-dependent activity in the presence of DTT was 15.1 nM, consistent with the cloned enzyme.
Conclusions:
- 11beta-HSD2 in rat kidney microsomes exists predominantly as an inactive dimer.
- Oxidative conditions or protocols may promote 11beta-HSD2 dimerization and inactivation.
- Reducing agents like DTT are crucial for accurately assessing 11beta-HSD2's active kinetic parameters.
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