The purification of 11 beta-hydroxysteroid dehydrogenase from mouse liver microsomes.
11 beta-Hydroxysteroid dehydrogenase (11 beta-HSD) is a microsomal enzyme complex responsible for the interconversion of active 11-hydroxy glucocorticoids to inactive 11-oxo metabolites. It has long been controversially discussed whether 11-dehydrogenation and 11-oxoreduction are catalysed by a single bidirectional enzyme or if the 11 beta-HSD system comprises 2 kinetically distinct microsomal enzyme activities, 11-dehydrogenase and 11-oxoreductase. However, 11-oxoreduction of homogeneously purified 11 beta-HSD could not be demonstrated under in vitro conditions until today. We have purified 11 beta-HSD from mouse liver microsomes to homogeneity by a purification method which affords a gentle membrane protein solubilization as well as providing a favourable detergent surrounding during the various chromatographic steps. Following 11-dehydrogenation of corticosterone and 11-oxoreduction of dehydrocorticosterone simultaneously throughout the entire purification procedure we could demonstrate that 11 beta-HSD retains both oxidative and reductive activities in almost the same ratio, which is also true for the homogeneously purified enzyme. Deducing from the coincidentally increasing specific activities of 11-dehydrogenation and 11-oxoreduction the conclusion can be drawn that both activities reside within the same protein. Furthermore, in addition to NADP(H) also NAD(H) can serve as cosubstrate, which is mainly true for the oxidative direction. In conclusion, our results provide evidence that the oxidative and reductive behaviour of 11 beta-HSD can be explained by the concept of a unique, reversible oxidoreductase thus disproving the two enzyme theory.
Maser
E
E
Department of Pharmacology and Toxicology, School of Medicine, Philipps-University Marburg, Germany.
Bannenberg
G
G
D042842:11-beta-Hydroxysteroid Dehydrogenases; D000818:Animals; D002241:Carbohydrates / Q000032:analysis; D002845:Chromatography; D003345:Corticosterone / Q000378:metabolism; D003902:Detergents; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D006854:Hydrocortisone / Q000378:metabolism; D006913:Hydroxysteroid Dehydrogenases / Q000302:isolation & purification* / Q000378:metabolism; D007700:Kinetics; D051379:Mice; D008862:Microsomes, Liver / Q000201:enzymology*; D009243:NAD / Q000378:metabolism; D009249:NADP / Q000378:metabolism; D010084:Oxidation-Reduction; D013194:Staining and Labeling; D013379:Substrate Specificity
D002241:Carbohydrates; D003902:Detergents; D009243:NAD; D009249:NADP; D006913:Hydroxysteroid Dehydrogenases; D042842:11-beta-Hydroxysteroid Dehydrogenases; D003345:Corticosterone; D006854:Hydrocortisone