title: Characterisation of an associate 17-beta-hydroxysteroid dehydrogenase activity and affinity labelling of the 3-alpha-hydroxysteroid dehydrogenase of Pseudomonas testosteroni.
abstract: The 3-alpha-hydroxysteroid dehydrogenase and the 3-beta-hydroxysteroid dehydrogenase of Pseudomonas testosteroni were purified to homogeneity by polyaerylamide gel electrophoresis using the following stages: DEAE cellulose chromatography, affinity chromatography on oestrone-aminocaproate sepharose and Sephadex gel filtration. The pure 3-alpha-hydroxysteroid dehydrogenase was completely devoid of 3-beta-hydroxysteroid dehydrogenase activity but could oxidize estradiol 17-beta at an appreciable rate. This activity accounts for about 40 per cent of the total 17-beta-estradiol dehydrogenase of the crude bacterial extract. Affinity labelling of pure 3-alpha-hydroxysteroid dehydrogenase was carried out using 5-beta-pregnane 3,20-dione-12-alpha-iodoacetate and 5-alpha-androstane 3-one-17-beta-bromoacetate. With both reagents, inactivation was obtained only in the presence of coenzyme, the substrate protected against inactivation and the enzyme was fully inhibited with covalent binding of 1 mole of reagent per mole of subunit suggesting an active site directed inhibition. Histidine and methionine were identified as the labelled aminoacid residues.
mesh_terms: D015067:17-Hydroxysteroid Dehydrogenases / Q000378:metabolism*; D015096:3-Hydroxysteroid Dehydrogenases / Q000302:isolation & purification / Q000378:metabolism*; D000345:Affinity Labels; D002846:Chromatography, Affinity; D007700:Kinetics; D011549:Pseudomonas / Q000201:enzymology*; D013379:Substrate Specificity
chemical_list: D000345:Affinity Labels; D015067:17-Hydroxysteroid Dehydrogenases; D015096:3-Hydroxysteroid Dehydrogenases