Activity of 11 beta-hydroxysteroid dehydrogenase in toad bladder: effects of 11-dehydrocorticosterone.
11 beta-Hydroxysteroid dehydrogenase (11 beta-OHSD) transforms circulating glucocorticoids to their "biologically inert" 11-dehydro derivatives. Isoforms of 11 beta-OHSD with different cofactor requirements and biochemical properties [Michaelis constant (Km) and maximal velocity (Vmax)] exist in the kidney. Since epithelial cells derived from the toad bladder also contain this enzyme, we wished to further characterize its properties in prepared cell homogenates. 11 beta-OHSD from toad bladder demonstrated a clear preference for NAD+ over NADP+ as a cofactor similar to that observed in renal cortical collecting duct (CCD) cells. Furthermore, 11 beta-OHSD had a rapid onset of action. The apparent Km for corticosterone was 16.3 x 10(-8) M, a value comparable to that observed for enzyme from CCD, and a Vmax of 4.8 x 10(-12) mol.mg protein-1.min-1. The end product, 11-dehydrocorticosterone (compound A), influenced enzyme activity; it increased 11 beta-OHSD activity at corticosterone concentrations below the apparent Km for the enzyme and inhibited 11 beta-OHSD activity at corticosterone concentrations above the Km for the enzyme. The inhibitory effects of compound A appeared noncompetitive with an apparent equilibrium constant (Ki) of 2.8 x 10(-7) M. Consistent with its inhibitory action on 11 beta-OHSD, compound A (10(-6) M) enhanced the short-circuit current response to corticosterone (10(-7) M) in the intact toad bladder (experimental 2.03 +/- 0.33 vs. control 1.40 +/- 0.17 times above baseline; n = 7, P < 0.01). Thus 11 beta-OHSD in toad bladder resembles the isoform found in CCD, and compound A may be biologically important as a regulator of 11 beta-OHSD.
Division of Pediatric Nephrology, Rhode Island Hospital, Providence.
A S
AS
Brem
K L
KL
Matheson
S
S
Latif
D J
DJ
Morris
D042842:11-beta-Hydroxysteroid Dehydrogenases; D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D002024:Bufo marinus; D003345:Corticosterone / Q000031:analogs & derivatives* / Q000494:pharmacology; D004594:Electrophysiology; D006913:Hydroxysteroid Dehydrogenases / Q000378:metabolism*; D066298:In Vitro Techniques; D007700:Kinetics; D009243:NAD / Q000494:pharmacology; D009249:NADP / Q000494:pharmacology; D012964:Sodium / Q000378:metabolism; D001743:Urinary Bladder / Q000201:enzymology* / Q000502:physiology
D009243:NAD; D009249:NADP; D012964:Sodium; D006913:Hydroxysteroid Dehydrogenases; D042842:11-beta-Hydroxysteroid Dehydrogenases; C003552:11-dehydrocorticosterone; D003345:Corticosterone