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Phosphorylation of eukaryotic initiation factor 2α by alternative phosphate sources catalyzed by heme-regulated kinase: kinetic analysis
Ovad, Tomáš ; Martínková, Markéta (advisor) ; Heidingsfeld, Olga (referee)
master thesis Ing. Bc. Tomáš Ovad Phosphorylation of eukaryotic initiation factor 2α by alternative phosphate sour- ces catalyzed by heme-regulated kinase: kinetic analysis This master thesis focuses on the phosphorylation of the eukaryotic initiation factor 2α (eIF2α) by the heme-regulated inhibitor (HRI), a key reaction in the regulation of eukary- otic protein synthesis. Kinetic parameters of this reaction in the presence of ATP as the phosphate donor were determined previously. However, there are no reports on the potential of alternative NTPs (GTP, UTP, CTP) to serve as the sources of phosphate for this reaction. In this thesis, the wild-type HRI enzyme was produced by heterologous expression in E. coli and its kinase activity was assayed in the presence of ATP, GTP, UTP, and CTP. Kinetic parameters and heme half-maximal inhibitory concentrations for the HRI kinase reaction with the use of each NTP as a phosphate donor were determined. To validate these kinetic experiments, contaminations by ATP in the solutions of GTP, UTP, and CTP were excluded with the aid of ion-pair reversed-phase high-performance liquid chromatography. It has been shown that ATP is the most efficient phosphate donor for the HRI kinase reaction, although the remaining NTPs (GTP, UTP, CTP) may be utilized as the sources...
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Study of retention and separation of peptides in HPLC
Boudová, Hana ; Kalíková, Květa (advisor) ; Kubíčková, Anna (referee)
This diploma thesis deals with the study of retention and separation of structurally different peptides and digest products of cytochrome c using reverse phase and mixed modes of liquid chromatography. As the model peptides, five unmodified dipeptides, nine dipeptides containing a protective benzoyl group on the N-terminal amino acid and eight therapeutic peptides containing five to ten amino acids were selected. The chromatographic behaviour of the model peptides was tested on three different columns: XBridge C18, XSelect CSH C18 and Atlantis Premier BEH C18 AX under different conditions. The influence of the pH of the aqueous part of the mobile phase, i.e. 2,1; 3,0; 4,7 and 6,8, and the amount of acetonitrile in the mobile phase on retention and separation of model peptides was tested. The effect of the concentration of ammonium acetate buffer, pH = 6,8 in the mobile phase on the retention and peak symmetry in the concentration range of 10-50 mmol L-1 was assessed. The chromatographic conditions for the baseline separation of 20 different model peptides were optimized. The conditions were as follows: XSelect CSH C18 column, mobile phase composed of acetonitrile/10mM ammonium acetate, pH = 6,8 (v/v) under gradient elution. The suitable chromatographic conditions for the analysis and identification...
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Cytochrome c and its role in apoptosis
Rajsiglová, Lenka ; Kalous, Martin (advisor) ; Švadlenka, Jan (referee)
Cell energetic metabolism and cell survival are strictly controlled by pathways in which cytochrome molecules play a central role, in particular cytochrome c. It is localized in the mitochondrial intermembrane space with other molecules cooperating in keeping energetic metabolism. Permeabilization of outer mitochondrial membrane by proteins from Bcl-2 family or changes in Ca2+ levels causes cytochrome c release into cytosol. In cytosol cytochrome c interacts with other pro-apoptotic proteins (Apaf-1, procaspase-9) cooperating to form apoptosome and phosphatidylserine. As a result of these interactions, the cell is going to apoptosis. This bachelor thesis summarizes the current state of knowledge of these processes. In the first part it focuses on the biosynthesis of cytochrome c, further on the mechanisms of its releasing from mitochondria and its interactions with other proteins within apoptosis including options of regulation of these processes.
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Cytochrome c and its role in apoptosis
Rajsiglová, Lenka ; Kalous, Martin (advisor) ; Švadlenka, Jan (referee)
Cell energetic metabolism and cell survival are strictly controlled by pathways in which cytochrome molecules play a central role, in particular cytochrome c. It is localized in the mitochondrial intermembrane space with other molecules cooperating in keeping energetic metabolism. Permeabilization of outer mitochondrial membrane by proteins from Bcl-2 family or changes in Ca2+ levels causes cytochrome c release into cytosol. In cytosol cytochrome c interacts with other pro-apoptotic proteins (Apaf-1, procaspase-9) cooperating to form apoptosome and phosphatidylserine. As a result of these interactions, the cell is going to apoptosis. This bachelor thesis summarizes the current state of knowledge of these processes. In the first part it focuses on the biosynthesis of cytochrome c, further on the mechanisms of its releasing from mitochondria and its interactions with other proteins within apoptosis including options of regulation of these processes.
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