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Abundance of NADP-dependent enzymes in plants
Kovaľová, Terézia ; Hýsková, Veronika (advisor) ; Ryšlavá, Helena (referee)
NADPH is an important molecule in plant metabolism. It is an indispensable substrate for many processes; participates in redox balance of the cell and it is a part of antioxidant defense system in plants. In this work, activities of selected NADP-dependent enzymes producing NADPH, NADP-malic enzyme (oxalacetate decarboxylating) (NADP-ME, EC 1.1.1.40), NADP-isocitrate dehydrogenase (decarboxylating) (NADP-ICDH, EC 1.1.1.42), glukosa-6-phosphate dehydrogenase (G6PDH, EC 1.1.1.49) and shikimate dehydrogenase (SDH, EC 1.1.1.25) in various plants and their parts (leaves, fruits, seeds) were established and compared. The aim of this work was to select suitable model plants for testing the effect of abiotic stress on NADP-dependent enzymes and also to select the best source for purification of these enzymes. Cucumber (Cucumis sativa L.), pea (Pisum sativum L.), or water-lily (Nymphaea) were chosen as suitable water-receptive model plants and Capsella bursa-pastoris as xerophilic plant from 19 analyzed plants. From group of 39 plants the most suitable for purification of NADP-ME were roots of Chinese radish (Raphanus sativus var. Major), which were simultaneously a rich source of other analyzed NADP-enzymes. The best source of NADP-ICDH was poppy (Papaver somniferum), or quinoa Chenopodium quinoa. For...
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HPLC determination of gallic acid as a possible product of enzymatic reaction of shikimic acid, NADP+ and SDH.
Smolejová, Jana ; Červený, Václav (advisor) ; Nesměrák, Karel (referee)
This diploma thesis deals with the development of an HPLC method for the determination of selected compounds participating in enzymatic reaction leading to the formation of gallic acid. The analysed reaction mixture contains the following reagents: shikimic acid, NADP+ and shikimatedehydrogenase (SDH) extracted from parsley; the presumed product of the reaction is gallic acid. Two chromatographic methods for the determination of the above mentioned compounds were developed using C18 HPLC column and porous graphitic carbon Hypercarb column. Molecular absorption spectrometric detection in the UV range was used in all measurements. Separation on the C18 column was found particularly suitable for analysing the composition of the end products of the reaction. Because of the NADP+ and shikimic acid peak overlap it is necessary to observe absorbance at 212 and 260 nm. Shikimic acid and NADP+ can be quantified due to the fact that shikimic acid does not absorb at 260 nm while NADP+ absorb radiation at both wavelengths. Separation via Hypercarb column was found particularly suitable for analysing the process of the reaction; additional products or intermediates can be seen in chromatograms compared to the C18 method. Determination with Hypercarb column is characterized by higher sensitivity and lower limit...
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Abundance of NADP-dependent enzymes in plants
Kovaľová, Terézia ; Hýsková, Veronika (advisor) ; Ryšlavá, Helena (referee)
NADPH is an important molecule in plant metabolism. It is an indispensable substrate for many processes; participates in redox balance of the cell and it is a part of antioxidant defense system in plants. In this work, activities of selected NADP-dependent enzymes producing NADPH, NADP-malic enzyme (oxalacetate decarboxylating) (NADP-ME, EC 1.1.1.40), NADP-isocitrate dehydrogenase (decarboxylating) (NADP-ICDH, EC 1.1.1.42), glukosa-6-phosphate dehydrogenase (G6PDH, EC 1.1.1.49) and shikimate dehydrogenase (SDH, EC 1.1.1.25) in various plants and their parts (leaves, fruits, seeds) were established and compared. The aim of this work was to select suitable model plants for testing the effect of abiotic stress on NADP-dependent enzymes and also to select the best source for purification of these enzymes. Cucumber (Cucumis sativa L.), pea (Pisum sativum L.), or water-lily (Nymphaea) were chosen as suitable water-receptive model plants and Capsella bursa-pastoris as xerophilic plant from 19 analyzed plants. From group of 39 plants the most suitable for purification of NADP-ME were roots of Chinese radish (Raphanus sativus var. Major), which were simultaneously a rich source of other analyzed NADP-enzymes. The best source of NADP-ICDH was poppy (Papaver somniferum), or quinoa Chenopodium quinoa. For...
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