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Fyzikálně-chemická charakterizace peptidů a bílkovin kapilárními elektromigračními metodami
Kašička, Václav ; Koval, Dušan ; Šolínová, Veronika ; Sázelová, Petra ; Prusík, Zdeněk
High-performance capillary electromigration methods, zone electrophoresis, isotachophoresis, isoelectric focusing, affinity electrophoresis and electrokinetic chromatography, are presented as powerful tools for physicochemical characterization of peptides and proteins.
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Synthesis of unsymmetrical derivatives of azaphthalocyanines and their complex formation with pyridine
Žižkovská, Jana ; Zimčík, Petr (advisor) ; Miletín, Miroslav (referee)
1. Summary Synthesis of unsymmetrical derivatives of azaphthalocyanines and their complex formation with pyridine Mgr. Jana Žižkovská Department of Pharmaceutical Chemistry and Drug Control Faculty of Pharmacy in Hradec Králové, Charles University in Prague I prepared the mixture of six possible products (AAAA, AAAB, ABAB, AABB, ABBB, BBBB) using a statistical condensation starting from two different precursors - 5,6- bis(diethyamino)pyrazine-2,3-dicarbonitrile (A) and 5-diethylamino-6-[2-(4,4'- dimethoxytrifenymethoxy)-ethylamino]pyrazine-2,3-dicarbonitrile (B). Only compound of AAAB type was isolated by column chromatography from the mixture, purified and was characterized by NMR, IR, MS, UV-VIS spectroscopy. The precursor (B) was changed - 5-diethylamino-6-[2-(4,4'-dimethoxytrifenymethoxy)- ethylmethylamino]pyrazine-2,3-dicarbonitrile - and the statistical condensation was done the same way. Unfortunately the desired AAAB product was unstable and therefore the work was finished. In another part of this work I have studied a formation of proton-transfer complex with two molecules of pyridine. Three different AzaPc with hydroxy groups on periphery were used in this study. The influence of hydroxy groups on the rate constant was not confirmed. Then I used only one AzaPc -...
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Sperm protein profiles of different mammalian species
Pohlová, Alžběta ; Zigo, Michal ; Jonáková, Věra ; Postlerová, Pavla
Proteins are a substantial equipment of the spermatic cell; therefore, the characterization of sperm proteins is crucial for explanation of molecular mechanisms in the reproduction process. We isolated sperm proteins from different mammalian species - pig, bull, human, mouse, dog and cat. Extracted proteins were separated by SDS-electrophoresis and protein/glycoprotein profiles from epididymal or ejaculated sperm were compared. Additionally, we tested cross-reactivity of antibodies prepared to sperm boar proteins on spermatozoa of other mammalian species using immunofluorescent technique. Our future plan is to compare the protein profiles of sperm during their functional development (epididymal, ejaculated, capacitated) in various mammalian species and identify species-specific sperm proteins with zona pellucida binding activity.
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Role of Psb28 proteins in the biogenesis of the Photosystem II complex in the cyanobacterium Synechocystis sp. PCC 6803
BEČKOVÁ, Martina
The thesis focuses on the role of Psb28 proteins, namely the Psb28-1 and its homolog Psb28-2, in the biogenesis of the Photosystem II complex (PSII) in the cyanobacterium Synechocystis PCC 6803. The aims of this work were to localize the proteins within the cells, and to determine their function. A fraction of both Psb28 proteins was identified in the monomeric PSII core complexes but most proteins were found in the unassembled protein fraction associated with thylakoid membranes. Psb28-1 was mostly detected as a dimer while Psb28-2 as a monomer. Psb28-1 also differed from Psb28-2 by its higher affinity to the PSII core complex lacking CP43 antenna. Characterization of Psb28-less mutants suggested regulatory function of the proteins in PSII biogenesis in connection with chlorophyll biosynthetic pathway. Analysis of preparations isolated using FLAG-tagged versions of Psb28 proteins showed their association with Photosystem II - Photosystem I supercomplexes, especially under increased irradiance, and supported a role of Photosystem I in the PSII biogenesis.
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Biochemical methods as tool for study of reproductive proteins
Postlerová, Pavla ; Zigo, Michal ; Pohlová, Alžběta ; Jonáková, Věra
Study of molecular mechanisms in reproduction is essential for the understanding of this outstanding process. Our lab studies proteins secreted by reproductive organs and sperm using various biochemical methods for a long time. We have expertise in protein extraction from spermatic cells using different approaches, and by kits for proteins from the sperm surface and distinct subcellular compartments. The proteins of reproductive organ fluids are separated by chromatographic methods, such as size exclusion chromatography, high-performance liquid chromatography with reverse phase (RP-HPLC) and affinity chromatography on matrices with various ligands. Proteins are subjected to SDS- or 2D-electrophoresis for their characterization and comparison of various extraction methods, different mammalian species, and sperm in different functional development. Electrophoretically separated proteins may be transferred onto nitrocellulose membrane (Western blot) for antibody detection or binding studies with lectin-labelled ligands (lectins, polysaccharides, zona pellucida glycoproteins). We use immunoprecipitation method with specific antibody for protein determination followed by the MALDI identification. Proteins are localized by immunofluorescent techniques on/in spermatic cells and tissue sections of reproductive organs. Isolation of proteins from reproductive tissues and fluids, and the antibody detection is crucial for the studying of reproductive protein origin.
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Possible role of spermatogenic protein glyceraldehyde-3-phosphate dehydrogenase (GAPDHS) in mammalian sperm
Margaryan, Hasmik ; Dorosh, Andriy ; Čapková, Jana ; Postlerová, Pavla ; Philimonenko, Anatoly ; Hozák, Pavel ; Pěknicová, Jana
Sperm proteins are important for the structure and function of these specific, highly differentiated cells. Certain of these proteins play a role in sperm-egg recognition during primary or secondary binding at zona pellucida glycoprotein matrix. The aim of this study was to characterize the acrosomal sperm protein recognized by a monoclonal antibody (MoAb) Hs-8, prepared in our laboratory by immunization of BALB/c mice with human ejaculated sperm, and to test the possible role of this protein in gamete interaction. MoAb Hs-8 specifically labelled a 45 kDa protein from the sperm extract in the immunoblotting test. Sequence analysis identified this Hs-8 protein as GAPDHS. In order to perform a control tests, a commercial mouse anti-GAPDHS MoAb was applied. Both antibodies showed similar staining patterns using immunofluorescence labelling, transmission electron microscopy and immunoblot analysis. Moreover, both Hs-8 and commercial anti-GAPDHS antibodies blocked the secondary sperm-zona pellucida binding. Generally, GAPDHS was considered mainly as sperm-specific glycolytic enzyme involved in energy production during spermatogenesis and sperm motility and its role in the sperm head was unknown. In this study, we confirmed the potential additional role of GAPDHS as a binding protein that is involved in the sperm-zona pellucida interaction.
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