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The isolation of carbonyl reducing enzymes by a new affinity adsorbent

Autoři: Wsól Vladimír | Andrýs Rudolf | Škarydová Lucie | Holubová Lucie | Bílková Zuzana

Rok: 2012

Druh publikace: ostatní - přednáška nebo poster

Strana od-do: nestránkováno

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Jazyk	Název	Abstrakt	Klíčová slova
eng	The isolation of carbonyl reducing enzymes by a new affinity adsorbent	Carbonyl reducing enzymes play an important role in metabolic pathways of various eobiotic substrates as steroids or prostaglandins. They also contribute to development of some diseases like hormone-dependent cancers, metabolic syndrome or Cushing’s syndrome. Many of these enzymes have been only poorly characterized or totally uncharacterized yet. Carbonyl reducing enzymes are usually in quite low concentrations in tissues so their isolation with classical techniques is complicated. It is difficult to obtain fraction with sufficient purity and amount of desired enzyme. The aim of the study was the preparation of an affinity adsorbent for selective isolation of carbonyl reducing enzymes from natural samples. Besides of eobiotics many xenobiotic substances e.g. doxorubicin, daunorubicin or haloperidol are good substrates for carbonyl reducing enzymes too. So the idea of this project was utilization of suitable xenobiotic substrate bearing a carbonyl moiety as a ligand for the affinity adsorbent. Oracin is a potential anticancer drug that has been shown as a substrate for many well-known carbonyl reducing enzymes so it seems to be universal ligand for development of the affinity adsorbent. The molecule of oracin have been modified and built on various carries in different ways.. Four types of magnetic microparticles, SiMAG-NH2, SiMAG-COOH, SiMAG-PGL and magnetic macroporous bead cellulose have been used as carriers. After series of tests, optimal binding method of oracin on suitable carrier was chosen for further studies. The selectivity of prepared affinity adsorbent to carbonyl reducing enzymes was tested on model enzymes AKR1C3 and CBR1. These enzymes were selectively captured from prepared samples on the affinity adsorbent and then eluted. It is clear that the new affinity adsorbent is well working with reductases AKR1C3 and CBR1 and it is ready for use in isolation process of different carbonyl reducing enzymes from natural samples.	affinity chromatoghraphy, carbonyl reducing enzymes, oracin, magnetic particles

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