Cloning, expression, and tissue distribution of the rat nicotinamide adenine dinucleotide-dependent 11 beta-hydroxysteroid dehydrogenase.
A pcDNAI adult rat kidney complementary DNA (cDNA) library was screened using a sheep 11-hydroxysteroid dehydrogenase 2 (11 beta HSD-2) probe, and the isolated clones were sequenced. The 5'-end of the cDNA was determined by 5'-rapid amplification of cDNA ends. The rat 11 beta HSD-2 cDNA is 1864 base pair (bp) long. It consists of a 5'-untranslated region of 126 bp, an open reading frame of 1203 bp, and a 3'-untranslated region of 535 bp. The predicted protein contains 400 amino acid residues, with a calculated mol wt of 43,700. The rat 11 beta HSD-2 protein sequence is 85% homologous to human 11 beta HSD-2 and 76% to sheep 11 beta HSD-2. Expression of 11 beta HSD-2 messenger RNA by Northern blot and reverse transcription-polymerase chain reaction was high in kidney, distal colon, and adrenal and lower in the lung, hypothalamus, hippocampus, and midbrain. The rat 11 beta HSD-2 was transiently transfected into modified Chinese hamster ovary cells. Cells transfected with the 11 beta HSD-2 cDNA converted corticosterone into 11-dehydrocorticosterone. Conversion of corticosterone to 11-dehydrocorticosterone was NAD+ dependent and had a Km of 10.1 +/- 2.1 nM. In conclusion, we have cloned a rat NAD(+)-dependent 11 beta HSD with tissue distribution and kinetic characteristics suggesting that it could play a significant role in mineralocorticoid receptor selectivity.
Zhou
M Y
MY
Department of Internal Medicine, University of Missouri, Harry S. Truman Memorial Veterans Hospital, Columbia 65201, USA.
Gomez-Sanchez
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EP
Cox
D L
DL
Cosby
D
D
Gomez-Sanchez
C E
CE
D042842:11-beta-Hydroxysteroid Dehydrogenases; D000311:Adrenal Glands / Q000737:chemistry / Q000201:enzymology*; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D001921:Brain / Q000201:enzymology; D016466:CHO Cells; D003001:Cloning, Molecular; D003106:Colon / Q000737:chemistry / Q000201:enzymology*; D006224:Cricetinae; D004247:DNA / Q000032:analysis / Q000737:chemistry / Q000235:genetics; D017931:DNA Primers / Q000032:analysis / Q000737:chemistry / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic; D015723:Gene Library; D006913:Hydroxysteroid Dehydrogenases / Q000032:analysis / Q000737:chemistry / Q000235:genetics* / Q000502:physiology; D007668:Kidney / Q000737:chemistry / Q000201:enzymology*; D008168:Lung / Q000737:chemistry / Q000201:enzymology; D008969:Molecular Sequence Data; D009243:NAD / Q000502:physiology*; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000032:analysis / Q000737:chemistry / Q000235:genetics; D051381:Rats; D016415:Sequence Alignment; D014018:Tissue Distribution
D017931:DNA Primers; D012333:RNA, Messenger; D009243:NAD; D004247:DNA; D006913:Hydroxysteroid Dehydrogenases; D042842:11-beta-Hydroxysteroid Dehydrogenases