Expression and characterization of a new species of 11 beta-hydroxysteroid dehydrogenase in Xenopus oocytes.
11 beta-Hydroxysteroid dehydrogenase (11-OHSD) plays a critical role in conferring aldosterone specificity to mineralocorticoid target cells. We have recently described a novel isoform of 11-OHSD in the collecting duct (11-OHSD/CD), which differs from the previously characterized isoform (11-OHSD-1) in kinetic parameters, cofactor dependency, and reversibility of the reaction. Unlike 11-OHSD-1, the collecting duct enzyme catalyzes irreversible dehydrogenation of endogenous glucocorticoids, has a very high affinity for its substrate, and is located in mineralocorticoid target cells; it thus appears well suited to "protect" the mineralocorticoid receptors from occupancy by glucocorticoids. As a first step in attempting to isolate the cDNA for the 11-OHSD/CD isoform, we isolated mRNA from immunodissected cortical collecting duct (CCD) cells and characterized the 11-OHSD in oocytes injected with this mRNA. Water-injected oocytes had no measurable 11-OHSD activity. In contrast, oocytes injected with as little as 1 ng CCD mRNA expressed detectable 11-OHSD activity. Expression of 11-OHSD activity was dependent on the amount of mRNA injected and was maximal with 30 ng mRNA. Similar to the findings in CCD cells, the expressed enzyme preferred NAD over NADP (activity was 0.46 +/- 0.04 and 0.011 +/- 0.01 fmol.min-1.oocyte-1 with 0.1 mM NAD and NADP, respectively). The Michaelis constant (Km) for corticosterone was 11.5 +/- 3.7 nM. Similar to the findings in CCD cells, the expressed enzyme worked predominantly in the oxidative direction, as back-conversion of [3H]dehydrocorticosterone to corticosterone was negligible. (ABSTRACT TRUNCATED AT 250 WORDS)
Náray-Fejes-Tóth
A
A
Department of Physiology, Dartmouth Medical School, Lebanon, New Hampshire 03756.
Rusvai
E
E
Denault
D L
DL
St Germain
D L
DL
Fejes-Tóth
G
G
D042842:11-beta-Hydroxysteroid Dehydrogenases; D000818:Animals; D003001:Cloning, Molecular; D003345:Corticosterone / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology; D004789:Enzyme Activation; D006913:Hydroxysteroid Dehydrogenases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D007267:Injections; D007527:Isoenzymes / Q000378:metabolism*; D009243:NAD / Q000494:pharmacology; D009865:Oocytes / Q000201:enzymology; D010084:Oxidation-Reduction; D012333:RNA, Messenger / Q000235:genetics; D014982:Xenopus laevis
D007527:Isoenzymes; D012333:RNA, Messenger; D009243:NAD; D006913:Hydroxysteroid Dehydrogenases; D042842:11-beta-Hydroxysteroid Dehydrogenases; C003552:11-dehydrocorticosterone; D003345:Corticosterone