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Title: Purification and cDNA cloning of human placental 11β-hydroxysteroid dehydrogenase
Author: Brown, Roger W.
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 1996
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The work in this thesis began by partially purifying the 11B-HSD activity from human placenta and comparing it to that from rat liver. The activities differed substantially in all parameters measured and did not merely reflect species differences as rat placental 11B-HSD was similar to the human placental isoform. This clearly showed, for the first time, that human tissues contained an 11B-HSD isoform (which we designated 11B-HSD2) distinct from that encoded by the previously known 'liver-type' isoform (11B-HSD1). Moreover this 11B-HSD2 isoform was an exclusive dehydrogenase, and kinetic measurements indicated it had over one hundred fold higher affinity for glucocorticoids and thus was much better suited to the tissue-specific eradication of glucocorticoids attributed to the crucial 'protective' 11B-HSD activities in placenta and kidney. Purification of human placental 11B-HSD2 16000-fold, to homogeneity, was achieved by subcellular fractionation, detergent solubilisation, AMP-affinity chromatography and 2-D electrophoresis. Placental 11B-HSD2 is ≈40 kDa protein, with a basic pl and is N-terminally blocked. Over 100 residues of internal amino acid sequence were obtained by digestion of the homogenous protein and sequencing of several of the resulting tryptic peptides. Purification was assisted by a novel technique allowing highly specific (single spot on 2-dimensional electrophoresis) photoaffinity labelling of active 11B-HSD2 in crude tissue extracts by its glucocorticoid substrates. This work reveals 11B-HSD2 is a novel member of the short chain alcohol dehydrogenase superfamily (apparent monomer Mr 40000). It is a very basic (apparent pl = 9.1) intrinsic membrane protein requiring, as yet undefined, membrane constituents for full stability. Affinity chromatography and affinity labelling studies suggest 11B-HSD2 has a compulsory ordered mechanism, with NAD binding first, followed by conformational change allowing glucocorticoid binding with high affinity. Amino acid sequence from homogenous human placental 11B-HSD2 was used to isolate an 1897bp cDNA encoding this enzyme (predicted Mr 44126, predicted pl 9.9).
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available