Introduction of an afinity separation to purification protocol for human membrane carbonyl-reducing enzymes

Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Radka Štýbnarová Supervisor: RNDr. Lucie Zemanová, Ph.D. Title of diploma thesis: Implementation of affinity separation step into the purification protocol of human membrane-bound carbonyl-reducing enzymes Carbonyl reducing enzymes play important role in the metabolism of various eobiotic (e.g. steroids, prostaglandins) and xenobiotic (e.g. doxorubicin, daunorubicin, oracin, NNK, haloperidol) compounds. Due to their substrate specificity, they also play a role in development of some diseases like hormone-dependent cancers and metabolic syndrome. While cytosolic carbonyl reducing enzymes are well characterised the knowledge about membrane-bound types is quite poor because their study is demanding. Actually, until today there are only three described microsomal carbonyl reducing enzymes participating in the metabolism of xenobiotic compounds (11β-HSD1, DHRS7 and DHRS3) of which only the 11β-HSD1 is well characterized. However, based on the research of anticancer drug oracin reduction stereospecificity, there were predicted other microsomal carbonyl reducing enzymes involved in its metabolism and inactivation. In order to isolate these "unknown" enzymes the in-house developed affinity..