Purification of 11 beta-hydroxysteroid dehydrogenase type 2 from human placenta utilizing a novel affinity labelling technique.
11 beta-hydroxysteroid dehydrogenase type 2 (11 beta-HSD2) efficiently inactivates potent glucocorticoid hormones (cortisol and corticosterone), leaving aldosterone unmetabolized. Abundant 11 beta-HSD2 activity in human placenta plays a central role in controlling fetal glucocorticoid exposure, which if excessive is harmful and may predispose to low birth weight and hypertension in adulthood. Similar 11 beta-HSD2 activity in the distal nephron protects mineralocorticoid receptors from glucocorticoids and appears to be important in normal blood pressure control. We have purified human placental 11 beta-HSD2 16000-fold, to homogeneity, and determined over 100 residues of the internal amino acid sequence. Purification was assisted by a novel technique allowing highly specific (single spot on two-dimensional electrophoresis) photoaffinity labelling of active 11 beta-HSD2 in crude tissue extracts by its glucocorticoid substrates. This work reveals that 11 beta-HSD2 is a member of the short-chain alcohol dehydrogenase superfamily (apparent monomer M(r) approximately 40,000). It is a very basic (apparent pI = 9.1) intrinsic membrane protein, requiring as yet undefined membrane constituents for full stability. Affinity chromatography and affinity labelling studies suggest that 11 beta-HSD2 has a compulsory ordered mechanism, with NAD+ binding first, followed by a conformational change allowing glucocorticoid binding with high affinity.
Brown
R W
RW
University Department of Medicine, Western General Hospital, Edinburgh, Scotland, UK.
Chapman
K E
KE
Murad
P
P
Edwards
C R
CR
Seckl
J R
JR
D042842:11-beta-Hydroxysteroid Dehydrogenases; D000345:Affinity Labels; D000595:Amino Acid Sequence; D002846:Chromatography, Affinity; D015180:Electrophoresis, Gel, Two-Dimensional; D005260:Female; D005938:Glucocorticoids / Q000378:metabolism; D006801:Humans; D006913:Hydroxysteroid Dehydrogenases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D007526:Isoelectric Point; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010920:Placenta / Q000201:enzymology*; D011247:Pregnancy; D012995:Solubility; D013347:Subcellular Fractions / Q000201:enzymology
D000345:Affinity Labels; D005938:Glucocorticoids; D006913:Hydroxysteroid Dehydrogenases; D042842:11-beta-Hydroxysteroid Dehydrogenases