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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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Preparation and preliminary characterization of the eukaryotic initiation factor 2α and its heme regulated kinase
Ovad, Tomáš ; Martínková, Markéta (advisor) ; Stráňava, Martin (referee)
Heme sensor proteins perform a variety of important functions in both prokaryotic and eukaryotic organisms. Heme-regulated inhibitor (HRI) is an example of a eukaryotic heme-sensor protein, which catalyzes the phosphorylation of the α subunit of the eukaryotic initiation factor 2 (eIF2α). In this bachelor thesis, the pET-21c(+)/eIF2α plasmid was amplified and its authenticity for the eIF2α expression was verified with the use of two independent methods. Next, HRI and eIF2α were produced using the recombinant expression in E. coli BL-21(DE3) cells transformed with the pET- 21c(+)/eIF2α and pET-21c(+)/HRI plasmid, respectively. Both proteins were then isolated from the cells and purified with the use of affinity chromatography and gel permeation chromatography. eIF2α was obtained in sufficient yield (560 μg out of 1 l of TB medium) and purity (90%). A lower yield (25 μg out of 1 l of TB medium) and purity (20%) was reached in the case of HRI. On the other hand, the authenticity of the HRI product was confirmed using spectrophotometric characterization and its enzyme activity was verified as well. Pilot experiments showed that GTP may replace ATP in the process of eIF2α phosphorylation, while UTP and CTP may not.
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