PURIFICATION OF 11-BETA-HYDROXYSTEROID DEHYDROGENASE TYPE-2 FROM HUMAN PLACENTA UTILIZING A NOVEL AFFINITY LABELING TECHNIQUE

11 beta-Hydroxysteroid dehydrogenase type 2 (11 beta-HSD2) efficiently inactivates potent glucocorticoid hormones (cortisol and corticostero ne), leaving aldosterone unmetabolized. Abundant 11 beta-HSD2 activity in human placenta plays a central role in controlling fetal glucocort icoid exposure, which if excessive is harmful and may predispose to lo w birth weight and hypertension in adulthood. Similar 11 beta-HSD2 act ivity in the distal nephron protects mineralocorticoid receptors from glucocorticoids and appears to be important in normal blood pressure c ontrol. We have purified human placental 11 beta-HSD2 16000-fold, to h omogeneity, and determined over 100 residues of the internal amino aci d sequence. Purification was assisted by a novel technique allowing hi ghly specific (single spot on two-dimensional electrophoresis) photoaf finity labelling of active 11 beta-HSD2 in crude tissue extracts by it s glucocorticoid substrates. This work reveals that 11 beta-HSD2 is a member of the short-chain alcohol dehydrogenase superfamily (apparent monomer M(r) similar to 40000). It is a very basic (apparent pI = 9.1) intrinsic membrane protein, requiring as yet undefined membrane const ituents for full stability. Affinity chromatography and affinity label ling studies suggest that 11 beta-HSD2 has a compulsory ordered mechan ism, with NAD(+) binding first, followed by a conformational change al lowing glucocorticoid binding with high affinity.