Carbonyl-reducing enzymes are important in both metabolism of endogenous substances and biotransformation of xenobiotics. Because sufficient amounts of native enzymes must be obtained to study their roles in metabolism, an efficient purification strategy is very important. Oracin (6-[2-(2-hydroxyethyl)aminoethyl]-5,11-dioxo-5,6-dihydro-11H-indeno[1,2-c] isoquinoline) is a prospective anticancer drug and one of the xenobiotic substrates for carbonyl-reducing enzymes. A new purification strategy based on molecular recognition of carbonyl-reducing enzymes with oracin as a ligand is reported here. The type of covalent bond, ligand molecules orientation, and their distance from the backbone of the solid matrix for good stearic accessibility were taken into account during the designing of the carrier. The carriers based on magnetically active microparticles were tested by recombinant enzymes AKR1C3 and CBR1. The SiMAG-COOH magnetic microparticles with N-alkylated oracin and BAPA as spacer arm provide required parameters: proper selectivity and specificity enabling to isolate the target enzyme in sufficient quantity, purity, and activity.
- MeSH
- alkoholoxidoreduktasy izolace a purifikace MeSH
- biotest MeSH
- chromatografie afinitní MeSH
- elektroforéza v polyakrylamidovém gelu MeSH
- enzymatické testy metody MeSH
- enzymy izolace a purifikace MeSH
- ethanolaminy chemie MeSH
- isochinoliny chemie MeSH
- ligandy MeSH
- magnetismus * MeSH
- mikrosféry MeSH
- molekulární struktura MeSH
- protinádorové látky chemie MeSH
- Schiffovy báze chemie MeSH
- vysokoúčinná kapalinová chromatografie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Carbonyl reducing enzymes play important roles in the biotransformation and detoxification of endo- and xenobiotics. They are grouped into two protein superfamilies, the short-chain dehydrogenases (SDR) and aldo-keto reductases (AKR), and usually are present in the cytoplasm of a cell. So far, only one membraneous carbonyl reductase has been described, 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1), which is located in the endoplasmic reticulum and which significantly contributes to the metabolism of a variety of carbonyl containing drugs and toxicants. Oracin is a new and prospective anticancer drug bearing a prochiral carbonyl moiety. The main metabolic pathway of oracin is carbonyl reduction to 11-dihydrooracin (DHO) which, however, eliminates the therapeutic potential of the drug, because the two DHO enantiomers formed have significantly less anti-tumor activities. Therefore, the oracin inactivating enzymes should urgently be identified to search for specific inhibitors and to enhance the chemotherapeutic efficacy. Interestingly, the calculation of enzyme specific activities and stereospecificities of (+)-DHO and (-)-DHO formation strongly suggested the existence of a second, hitherto unknown microsomal oracin carbonyl reductase in human liver. Therefore, the aim of the present study was to provide proof for the existence of this new enzyme and to develop a purification method for further characterization. First, we succeeded in establishing a gentle solubilization technique which provided a favourable detergent surrounding during the further purification procedure by stabilizing the native form of this fragile protein. Second, we could partially purify this new microsomal carbonyl reductase by a two step separation on Q-sepharose followed by Phenyl-sepharose. The enzyme turned out to be NADPH specific, displaying kinetic values for oracin carbonyl reduction of K(m)=42 microM and V(max)=813 nmol/(30 min x mg protein). Compared to the microsomal fraction, the enzyme specific activity towards oracin could be enhanced 73-fold, while the stereospecificity of (+)-DHO formation shifted from 40% to 86%. Considering these data for 11beta-HSD1, as described in previous reports, it is clear that the microsomal carbonyl reductase investigated in the present study is new and has a great potential to significantly impair the chemotherapy with the new anticancer drug oracin. 2009 Elsevier Ireland Ltd
- MeSH
- alkoholoxidoreduktasy chemie izolace a purifikace MeSH
- buněčná membrána enzymologie účinky léků MeSH
- chemorezistence MeSH
- chromatografie agarózová MeSH
- elektroforéza v polyakrylamidovém gelu MeSH
- ethanolaminy metabolismus MeSH
- financování organizované MeSH
- isochinoliny metabolismus MeSH
- jaterní mikrozomy enzymologie účinky léků MeSH
- kinetika MeSH
- lidé MeSH
- protinádorové látky metabolismus MeSH
- rozpustnost MeSH
- stereoizomerie MeSH
- substrátová specifita MeSH
- vysokoúčinná kapalinová chromatografie MeSH
- Check Tag
- lidé MeSH
