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Effect of cytochrome b5 on enzyme kinetics of Sudan I hydroxylation catalyzed by human cytochrome P450 1A1
Netolický, Jakub ; Martínek, Václav (advisor) ; Černá, Věra (referee)
Cytochromes P450 are the major xenobiotics converting enzymes. They are classified as mixed function monooxygenases (MFO). Isoform 1A1 is a extrahepatic form found mainly in the lung and other tissues. It is strongly induced by polycyclic aromatic hydrocarbons and their derivatives via the Ah receptor. As a marker reaction for this enzyme can be used hydroxylation of Sudan I, which has previously been widely used as a azo dye in industry, but since 1980s it is banned for coloring food and cosmetics for its negative influence on the organism. NADPH:cytochrome P450 reductase is the major electron donor for cytochrome P450 catalyzed monooxygenation reactions. Another electron carrier for cytochrome P450 catalyzed reactions is cytochrome b5. It was shown that cytochrome b5 can stimulate, inhibit or have no effect on P450 catalyzed reactions. This thesis aims to evaluate the influence of the ration between NADPH:cytochrome P450 reductase and cytochrome b5 on cytochrome P450 1A1 catalyzed Sudan I hydroxylation. The main goal is to characterize the influence of electron donor and electron transfer ratios on hydroxylation of Sudan I, and to determine the kinetic parameters KM and VMAX for selected protein ratios. Partial aims of the thesis were to characterize the recombinant proteins used in this study...
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Molar ratio between cytochrome b5 and its reductases affects activity of cytochrome P450 1A1 towards Sudan I
Netolický, Jakub ; Martínek, Václav (advisor) ; Dračínská, Helena (referee)
Cytochromes P450 are an evolutionary very old group of enzymes. It spread into many isoforms that can be found in animals, plants, fungi, bacteria, and some viruses. They play a major role in the first phase of the biotransformation of drugs, environmental pollutants and other xenobiotics. Also for this reason, they belong among the most researched enzymes. Cytochrome P450 for its function requires an electron donor, such as NADPH:cytochrome P450 oxidoreductase and cytochrome b5. The alternative reductase involved in this process is NADH:cytochrome b5 oxidoreductase, which is able to reduce cytochrome b5. In a eukaryotic cell, all these membrane proteins are found in the endoplasmic reticulum membrane, where they can naturally interact. This work evaluates the activity of human recombinant cytochrome P450 1A1 against the carcinogenic azo dye Sudan I, specifically it focuses on mapping the formation of major metabolites in relation to the ratio of cytochrome b5 to NADPH:cytochrome P450 oxidoreductase as well as to NADH:cytochrome b5 oxidoreductase. Keywords: cytochrome P450 1A1, NADPH:cytochrome P450 oxidoreductase, cytochrome b5, NADH:cytochrome b5 oxidoreductase, Sudan I, HPLC [In Czech]
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Co-operativity of cytochrome P450 system and its impact on drug and carcinogen metabolism
Holý, Petr ; Hodek, Petr (advisor) ; Chmelík, Josef (referee)
The system of mixed-function oxidases (MFO system) has a significant role in metabolism of many endogenous compounds, as well as xenobiotics (for ex. karcinogens, drugs). Membrane-bound haemoproteins called cytochromes P450 are a vital part of that system. Reactions catalyzed by cytochromes P450 are influenced by another protein of the MFO system, cytochrome b5. The mechanism of this cyt b5 agency has not yet been fully described. One of methods used for study of this protein-protein interaction is covalent cross- linking. By replacing one of three methionines in the cyt b5 structure by a photo-reactive analogue (photo-methionine), an analogue of cyt b5 (photo-cyt b5) can be obtained. When activated by UV radiation, the protein covalently bonds cytochrome P450 in a membrane environment. This paper focuses on expression and isolation of a recombinant cyt b5 analogue with only one methionine position (96) in the protein structure and substitution by photo-methionine. Protein was purified in a yiedl of 6 mg from 1 liter of baterial suspension. Analysis by mass spectrometry (MALDI-TOF/TOF) showed methionine to have been substituted by the photo-reactive analogue in approx. 30 %. Photo-cyt b5 was used to fixate transient protein-protein interactions with cytochrome P450 2B4 (CYP2B4). Photo-cyt b5 was...
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