|
|
Cytochrome P-450: Study of structure and interactions using chemical modification, photo-initiated cross-linking and mass spectrometry
Ječmen, Tomáš ; Šulc, Miroslav (advisor) ; Petrák, Jiří (referee) ; Šebela, Marek (referee)
ABSTRACT Mixed function oxygenase system participates in biosynthesis of endogenous and metabolism of exogenous substances (e.g. drugs or chemical procarcinogens) in an organism. Substrates are biotransformed by terminal oxygenases - cytochromes P450 (P450). Catalytic properties of certain P450s (e.g. studied isoform 2B4) are altered in the presence of a redox partner - cytochrome b5 (cyb5). Both cytochromes are anchored by hydrophobic domains in a lipid membrane of endoplasmic reticulum whereas their catalytic domains are exposed to cytosol. Two zero-length cross-linking approaches were employed to extend present knowledge of P450 2B4 and cyb5 protein structure and protein-protein interactions: (1) interlinking of carboxylate and primary amine groups of amino acids by water soluble 1- ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), and (2) photo-initiated cross-linking by photo-labile methionine analog (pMet), which links to any amino acid after activation by UV-irradiation, either in hydrophilic or hydrophobic environment. pMet was incorporated to methionine site(s) of cyb5 during recombinant expression in E. coli, which was carried out in limit medium supplemented with amino acid analog. Optimization of experimental conditions led to ~20-30% substitution of the natural amino acid. Covalent...
|
|
|
Possible role of flavine monooxygenases in xenobiotic metabolism
Cikánová, Magdaléna ; Hudeček, Jiří (advisor) ; Hodek, Petr (referee)
Presented bachelor thesis is an overview of the most important facts about flavin-containing monooxygenases (FMOs) found in literary sources. FMOs are enzymes, which participate on oxygenation of many foreign compounds which are soft nucleophils, thus compounds containing mostly nitrogen in their molecules as well as sulfur and sometimes also phosphorus and selenium. FMOs use molecular oxygen in their catalytic cycle, which is similar to catalytic cycle of cytochome P- 450: one molecule of oxygen is activated by its reduction in presence of NADPH, one atom of oxygen is incorporated to substrate molecule, meanwhile the second one is reduced to water. In these days FMOs in mammals, bacteria, plants and yeast were described. Up to date five human genes FMOs (FMO1-FMO5) were detected. By three of those (FMO1-FMO3) genetic polymorphism was proofed.
|
|
| |
|
| |
|
| |
|
| |
guest ::
