Purification of 11 beta-hydroxysteroid dehydrogenase type 2 from human placenta utilizing a novel affinity labelling technique

R W Brown, K E Chapman, P Murad, C R W Edwards, J R Seckl

Research output: Contribution to journalArticlepeer-review

Abstract

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) similar to 40000). 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.

Original languageEnglish
Pages (from-to)997-1005
Number of pages9
JournalBiochemical Journal
Volume313
Publication statusPublished - 1 Feb 1996

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