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Effect of cytochrome b5 on activity of cytochromes P450
Ličko, Vojtech ; Indra, Radek (advisor) ; Feglarová, Tereza (referee)
ABSTRACT Cytochrome b5 (CYB5) is heme protein capable of reduction of cytochromes P450 (CYP) or some other enzymes. However, his regulative capability was also observed by his apo form, i.e. in absence of heme prosthetic group in the active center. CYB5 can accept electron from cytochrome b5 reductase (CYB5R) or from cytochrome P450 reductase (CYPOR). CYPOR by itself is reduced by NADPH and is also able to forward electron to CYP independently of CYB5. CYB5R on the other hand is reduced by NADH. Efficiency of CYB5 to accept and forward an electron was studied in vitro with five different substrates - testosterone, Sudan I, aristolochic acid I (AAI), ellipticine and vandetanib. These substrates were chosen considering their characteristic reactions, which are catalyzed by their respective isoforms of CYP. The experiments with these substrates were carried out in the medium with recombinant CYPs prepared in insect cells or E. coli or in the medium with hepatic microsomes isolated from different organisms. Rats, from which the majority of these microsomes was isolated, were premedicated by different CYP inducers. The experiments were carried out in medium with NADH or NADPH in order to assess the capability of CYB5 to reduce CYP independently of CYPOR. The capability of CYB5 and CYB5R to act as a...
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The influence of cytochrome b5 on naphthalene oxidation catalyzed by human cytochrome P450 1A2
Stránský, Jaroslav ; Martínek, Václav (advisor) ; Moserová, Michaela (referee)
Cytochromes P450 (CYP) form a crucial group of oxidoreductase-class enzymes participating in the biotransformation of numerous endo- and exogenous compounds (xenobiotics). A negative aspect to the activity of this enzyme, however, can be the bioactivation, a process during which an originally only slightly toxic compound is transformed into a substance of much higher toxicity. Cytochrome P450 has therefore long been standing at the forefront of biochemical and medicinal interest. For the proper function of cytochrome P450 a supply of electrons into a catalytic cycle is necessary. This is usually taken care of by NADPH:cytochrome P450 oxidoreductase. Cytochrome b5, an enzyme present in the membrane of the endoplasmic reticulum of eukaryotic cells along with NADPH:cytochrome P450 oxidoreductase and cytochrome P450, however, can also act as an alternative electron donor. The participation of cytochrome b5 in the catalytic cycle of cytochrome P450 have a potential to modulate this cycle. Over the course of evolution, many isoforms of cytochrome P450 have developed, each possessing different substrate specifity, catalytic properties, distributions within various tissues etc. This bachelor thesis is focused on the isoform 1A2 (CYP1A2) which represents nearly 10 % of all human hepatic cytochrome P450s....
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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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Preparation of cytochrome b5 mutants containing photoreactive amino acids, and their crosslinking with the interaction partners
Landl, David ; Ječmen, Tomáš (advisor) ; Kukačka, Zdeněk (referee)
Cytochrome b5 is an electron transport protein of a clinically prominent mixed-function oxygenase (MFO) system. This system participates in the first stage of xenobiotic biotransformation. The hydrofility of xenobiotics is increased by introduction of an oxygen atom into their structure. The MFO system also activates or deactivates certain drugs and carcinogens. Cytochrome b5 affects reactions catalyzed by the terminal oxygenases of the system - cytochromes P450. Electrons are donated to cytochrome b5 by redox partners NADH:cytochrome b5 reductase and NADPH:cytochrome P450 reductase. The aim of this work was to demonstrate capability of photo-crosslinking approach to fixate transient interactions within MFO system. Covalent complexes obtained by this technique could be further analyzed by mass spectrometry, which can provide structural information about the binding sites of the proteins. We prepared a mutant cytochrome b5 comprising photo-reactive methionine analogue in the position 41 of the sequence. We expressed the protein employing E. coli B834 (DE3) auxotrophic strain in 300 ml of the limit medium supplemented with L-2- amino-5,5-azi-hexanoic acid (photo-methionine). The rate of the unnatural amino acid incorporation was determined by mass spectrometry and it was about 40 % after 16 hours of...
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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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(Construction of deletion mutants of human cytochrome b5 using gene synthesis)
Kotlánová, Iveta ; Martínek, Václav (advisor) ; Moserová, Michaela (referee)
Cytochrome b5 is a small amphipathic protein. The human form is anchored to the outer membrane of the endoplasmic reticulum and mitochondria, a free form is located in red blood cells. It consists of two domains: a large hydrophilic domain binds heme, a small hydrophobic domain anchors cytochrome b5 to the microsomal membrane. Both domains are connected by linker chain of about 15 amino acids, which gives a flexibility to the protein. Its length plays an important role in transferring electrons to cytochrome P450. If the linker domain is too short, cytochrome b5 is not able to tranfer electrons to cytochrome P450 and not participates in the reactions of MFO system. Other functions are preserved. The aim of this study was to design and build 4 deletion mutants of cytochrome b5 using gene synthesis. The linker domain contains long and short deletions, which are expected to have distortion interaction with cytochrome P450. Part of this thesis was the expression of heterologous proteins by cells of Escherichia coli strain XL10-Gold and DH5α. As expression vectors for the transformation were used plasmids pET- 30a(+) and pET-22b. DNA from cells was isolated and the accuracy of the genetic code was verified using the sequencing. Keywords: cytochrome b5, heterologous expression, gene synthesis (In Czech)
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The comparison of properties of cell lines resistant to ellipticine, doxorubicin, and cisplatin
Černá, Tereza ; Poljaková, Jitka (advisor) ; Eckschlager, Tomáš (referee)
7 Abstract Neuroblastoma is the most common extracranial solid tumor of childhood. Despite advances in cancer diagnosis and therapy, the treatment of some forms of neuroblastoma is still complicated. One of the major complications of the chemotherapy is a developed drug resistance. This master thesis deals with the effect of cytostatics on protein and gene expression of selected proteins, which may contribute to chemoresistance of the human neuroblastoma cell line UKF-NB-4. The sensitive line UKF-NB-4 and the resistant line UKF-NB-4CDDP , UKF-NB-4DOXO and UKF-NB-4ELLI were exposed to cisplatin, doxorubicin, ellipticine for 24, 48 and 72 hours. The Western blot analysis showed that cytostatic agents cisplatin, doxorubicin or ellipticine added to the sensitive neuroblastoma cell line UKF-NB-4 in amounts which are added to resistant neuroblastoma cell lines in order to maintain resistance induced expression of p53 and reduced expression of retinoblastoma protein pRb after 72 hours of cultivation. Differences in the expression of RAS protein, cytochrome P450 1A1, 3A4 and cytochrome b5 has not been shown. Changes in the expression of the studied proteins in resistant lines UKF-NB-4CDDP , UKF-NB-4DOXO and UKF-NB-4ELLI cultured with and without cytostatic agents were not detected by the Western blot analysis....
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The mechanism of action of anticancer drug ellipticin in target tissues of its effect
Mrízová, Iveta
Ellipticine is an alkaloid isolated from Apocynaceae plants exhibiting significant antitumor and anti-HIV activities. Cytochromes P450 (CYP) and peroxidases are the enzymes participating in metabolism of ellipticine. This process provides activation and detoxication metabolites of ellipticine. The CYP enzymes, which participate in oxidation of ellipticine in different tissues (liver, lung and kidney) of rat, a model organism simulating the fate of ellipticine in humans have already been identified. In this work, the effects of ellipticine on contents and catalytic activities of CYPs and other components of the mixed-function oxidase (MFO) system in this animal system were studied. For detection of contents of CYPs and other components of the MFO system, spectroscopic and electrochemical methods were used. To determine catalytic activities of CYPs and NADPH:cytochrome P450 reductase, reactions with specific substrates of these enzymes were utilized. The results found in this study demonstrate that expression and catalytic activity of CYP1A is induced by ellipticine in all of the tested organs (liver, kidney and lung) of rats treated with the drug. Moreover in liver, the cytochrome b5 expression is also induced. In addition, in this organ, expression and catalytic activity of CYP3A was increased by...
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Study of effect of carcinogenic benzo[a]pyrene on cytochromes P450 and cytochrome b5 expression in organs of the rat
Blecha, Tomáš ; Moserová, Michaela (advisor) ; Dračínská, Helena (referee)
Polycyclic aromatic hydrocarbons are a large group of organic compounds which consist solely of carbon and hydrogen atoms, two or more fused aromatic rings. They represent group of persistent organic pollutants (POPs) present in all components and fields of the environment. Benzo[a]pyrene is characteristic compound of polycyclic aromatic hydrocarbons formed by five fused aromatic rings. Benzo[a]pyrene is procarcinogen with genotoxic effects, which is metabolically activated by variety of enzyme systems such as cytochrome P450 and epoxide hydrolase to reactive metabolite of BaP-7,8-diol-9,10-epoxide (BPDE) and 9-hydroxy-4,5-epoxy-BaP. These reactive metabolites can form covalent DNA adducts. In the present work, we studied the influence of carcinogenic benzo[a]pyrene on the expression of cytochrome P450 (CYP) 1A1, 1A2 and cytochrome b5 in livers, kidneys and lungs of laboratory rats. Total RNA was isolated and afterwards converted into cDNA with the participation of random primers. Using polymerase chain reaction in real time (RT-PCR) the relatively gene expression of CYP1A1, CYP1A2 and cytochrome b5 was quantified in the organs of rats treated with BaP and control (untreated) animals to a reference gene (β-actin). It was found that benzo[a]pyrene significantly increases expression of CYP1A1 and...
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Mechanism of enzymatic activation of carcinogens and drugs by the system of cytochrome P450
Indra, Radek ; Stiborová, Marie (advisor) ; Souček, Pavel (referee) ; Koblihová, Jitka (referee)
13 Abstract An environmental pollutant and a human carcinogen benzo[a]pyrene (BaP) is after its activation with cytochrome P450 (CYP) able to covalently bind to DNA. In the thesis, one of the target was to investigate an influence of individual components of mixed function monooxygenase (MFO) system on metabolism of benzo[a]pyrene and generation of adducts of activated BaP with DNA. The study was particularly focused to increase our knowledge on the effect of cyt b5 on metabolism of BaP by cytochrome P450 1A1 (CYP1A1) and its potential to serve as a donor of electrons during the reaction cycle of this cytochrome P450. The effect of cyt b5 on generation of BaP metabolites and adducts of BaP with DNA was investigated. In addition the effect of two different expression systems for cytochrome P450 1A1 (prokaryotic and eukaryotic) was also studied. The influence of cyt b5 on oxidation another xenobiotic compound, a plant alkaloid ellipticine that exhibit antitumor activities, was also investigated. Its pharmacological efficiency, as well as side effects depends on its metabolic activation by cytochrome P450. CYP3A4 is very important for ellipticine activation and therefore this enzyme was used in our experiments. Furthermore, a suitability of rat as a model organism mimicking the metabolic fate of BaP...
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