|
|
Optimization of flavin monooxygenase activity assay
Hovorková, Adéla ; Dračínská, Helena (advisor) ; Heidingsfeld, Olga (referee)
Flavin monooxygenases, biotransformation enzymes catalyzing the oxidation of broad spectrum of xenobiotics, have long been overlooked compared to cytochromes P450, a larger group of biotransformation enzymes. Liver microsomes containing both flavin monooxygenases and cytochrome P450 are often used to study metabolism of xenobiotics. The catalytic aktivity of both enzymes may overlap due to the need of identical cofactors and it is therefore necessary to differentiate them appropriately. This bachelor thesis deals with the optimization of the method for the determination of enzyme activity of flavin monooxygenases in rat liver microsomes. Model reaction for optimization of the method was the oxidation of methyl p-tolyl sulfide to methyl p-tolyl sulfoxide. To determine the activity, the most suitable conditions were set: sample buffer with pH 9.5, 2 mM methyl p-tolyl sulfide and a 10 minute incubation time. It has been found that in the oxidation of methyl p-tolyl sulfide cytochromes P450 are also involved, mostly isoform 1A1. Various inhibitors of both of the above mentioned biotransformation enzymes (lipoic acid, methimazole, Brij 35 and Triton X-100) have also been tested. Brij 35 was selected as a suitable inhibitor of the catalytic action of cytochromes P450, because it had no effect on the rate...
|
|
|
The effect of tyrosinkinase inhibitors vandetanib and lenvatinib and cytotoxic alkaloid ellipticine on biotransformation enzymes
Baráčková, Petra ; Dračínská, Helena (advisor) ; Václavíková, Radka (referee)
In recent years, tyrosine kinase inhibitors have been widely used for the treatment of certain tumors as so-called targeted therapy. Many studies are concerned with their metabolism and the role of enzymes in the biotransformation process, but very little is known about the impact of tyrosine kinase inhibitors on the expression and activity of biotransformation enzymes. Nevertheless modification of the expression and activity of enzymes may cause adverse interactions of co-administered drugs and their negative impact on the human body. This diploma thesis studies the effect of tyrosine kinase inhibitors vandetanib and lenvatinib and cytotoxic alkaloid ellipticine on biotransformation enzymes in a rat model organism in vivo. The aim was to characterize the effect of the investigated compounds on gene expression, protein expression and activity of cytochromes P450 (CYP) 1A1, 1A2 and 1B1 and flavin-containing monooxygenases FMO1 and FMO3 in renal and hepatic microsomes. Microsomes and RNA were isolated from kidneys of control rats and the pretreated rats. Western blot and immunodetection was used to compare the protein expression levels of studied enzymes in kidney and liver. By reverse transcription, cDNA was prepared from isolated RNA and used as a template for quantitative PCR to compare the...
|
|
|
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 ::
