Inhibitors of the shikimate pathway are widely used as herbicides, antibiotics, and anti-infectious drugs. However, the regulation of the shikimic pathway is complex, and little is known about the feedback regulation of the shikimate dehydrogenase (SDH, EC 1.1.1.25) in plants. Thus, the aim of this study was to elucidate the kinetic mechanism of SDH purified from the root of Petroselinum crispum (parsley), to determine all possible reaction products and to identify phenylpropanoid compounds that affect its activity. Our results showed that the bisubstrate reaction catalyzed by P. crispum SDH follows a sequential ordered mechanism, except for three dead-end complexes. The main and lateral reactions of SDH were monitored by mass spectrometry, thereby detecting protocatechuic acid as a byproduct. Gallic acid was formed non-enzymatically, whereas quinate was not detected. Several polyphenolic compounds inhibited SDH activity, especially tannic, caffeic and chlorogenic acids, with IC50 0.014 mM, 0.15 mM, and 0.19 mM, respectively. The number of hydroxyl groups influenced their inhibition effect on SDH, and p-coumaric, t-ferulic, sinapic, syringic and salicylic acids were less effective SDH inhibitors. Nevertheless, one branch of the phenylpropanoid pathway may affect SDH activity through feedback regulation.
- MeSH
- alkoholoxidoreduktasy * MeSH
- katalýza MeSH
- kyselina shikimová MeSH
- petržel (rod) * MeSH
- Publikační typ
- časopisecké články MeSH
The anticonvulsant drug carbamazepine is considered as an indicator of sewage water pollution: however, its uptake by plants and effect on metabolism have not been sufficiently documented, let alone its metabolite (10,11-epoxycarbamazepine). In a model system of sterile, hydroponically cultivated Zea mays (as C4 plant) and Helianthus annuus (as C3 plant), the uptake and effect of carbamazepine and 10,11-epoxycarbamazepine were studied in comparison with those of acetaminophen and ibuprofen. Ibuprofen and acetaminophen were effectively extracted from drug-supplemented media by both plants, while the uptake of more hydrophobic carbamazepine was much lower. On the other hand, the carbamazepine metabolite, 10,11-epoxycarbamazepine, was, unlike sunflower, willingly taken up by maize plants (after 96 h 88 % of the initial concentration) and effectively stored in maize tissues. In addition, the effect of the studied pharmaceuticals on the plant metabolism (enzymes of Hatch-Slack cycle, peroxidases) was followed. The activity of bound peroxidases, which could cause xylem vessel lignification and reduction of xenobiotic uptake, was at the level of control plants in maize leaves contrary to sunflower. Therefore, our results indicate that maize has the potential to remove 10,11-epoxycarbamazepine from contaminated soils.
- MeSH
- antikonvulziva analýza metabolismus MeSH
- biodegradace MeSH
- Helianthus účinky léků růst a vývoj metabolismus MeSH
- hydroponie MeSH
- karbamazepin analogy a deriváty analýza metabolismus MeSH
- kukuřice setá účinky léků růst a vývoj metabolismus MeSH
- látky znečišťující půdu analýza metabolismus MeSH
- listy rostlin účinky léků růst a vývoj metabolismus MeSH
- vysokoúčinná kapalinová chromatografie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Geografické názvy
- Česká republika MeSH
Drought stress is one of the most frequent forms of abiotic stresses, which occurs under condition of limited water availability. In this work, the possible participation of phosphoenolpyruvate carboxylase (EC 4.1.1.31; PEPC), NADP-malic enzyme (EC 1.1.1.40; NADP-ME), and pyruvate, phosphate dikinase (EC 2.7.9.1; PPDK) in response to drought of tobacco plants (Nicotiana tabacum L., cv. W38) was investigated. Enzyme specific activities in tobacco leaves of drought stressed plants were significantly increased after 11 days of stress, PEPC 2.3-fold, NADP-ME 3.9-fold, and PPDK 2.7-fold compared to control plants. The regulation of PEPC and NADP-ME activities were studied on transcriptional level by the quantitative RT PCR and on translational level - immunochemically. The amount of NADP-ME protein and transcription of mRNA for chloroplastic NADP-ME isoform were increased indicating their enhanced synthesis de novo. On the other hand, mRNA for cytosolic isoform of NADP-ME was decreased. The changes in PEPC protein and PEPC mRNA were not substantial. Therefore regulation of PEPC activity by phosphorylation was evaluated and found to be involved in the stress response. During recovery, activities of the tested enzymes returned close to their basal levels.
- MeSH
- aklimatizace MeSH
- chloroplasty metabolismus MeSH
- fosfoenolpyruvátkarboxylasa genetika metabolismus MeSH
- fyziologický stres fyziologie MeSH
- kvantitativní polymerázová řetězová reakce MeSH
- listy rostlin enzymologie MeSH
- malátdehydrogenasa genetika metabolismus MeSH
- období sucha * MeSH
- pyruvátfosfátdikinasa genetika metabolismus MeSH
- regulace genové exprese u rostlin MeSH
- rostlinné proteiny genetika metabolismus MeSH
- tabák enzymologie genetika MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The kinetic properties of β-N-acetylhexosaminidase purified from tobacco (Nicotiana tabacum L.) leaves have been investigated. In addition to chromogenic pNP derivates, N,N'-diacetylchitobiose and N,N',N″-triacetylchitotriose were also used as substrates of β-N-acetylhexosaminidase. The highest reaction rate and the affinity for the substrate were observed for pNP-GlcNAc; however, an excess of this substrate inhibits the reaction. The reaction rate with pNP-GalNAc as the substrate was found to be about 85% of that obtained with pNP-GlcNAc. The hydrolysis of acetylated chitooligomers by β-N-acetylhexosaminidase followed by separation and quantification using capillary electrophoresis was slower compared to pNP-GlcNAc. The pH optimum of β-N-acetylhexosaminidase for individual substrates was found at 4.3-5.0 and the temperature optimum was 50-55 °C. Gel permeation chromatography and red native electrophoresis determined the relative molecular weight as 280 000 and the isoelectric point as 5.3. The inhibition of β-N-acetylhexosaminidase by monosaccharides GlcN, GalN, GlcNAc, GalNAc in combination with substrates pNP-GlcNAc and pNP-GalNAc was studied and the type of inhibition and the inhibition constants were determined.
- MeSH
- acetylgalaktosamin analogy a deriváty metabolismus MeSH
- acetylglukosamin analogy a deriváty metabolismus farmakologie MeSH
- beta-N-acetylhexosaminidasy antagonisté a inhibitory chemie izolace a purifikace metabolismus MeSH
- disacharidy metabolismus MeSH
- hydrolýza MeSH
- inhibitory enzymů farmakologie MeSH
- kinetika MeSH
- koncentrace vodíkových iontů MeSH
- listy rostlin enzymologie MeSH
- molekulová hmotnost MeSH
- substrátová specifita MeSH
- tabák enzymologie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31) from mature maize seeds (Zea mays L.) was purified to homogeneity and a final specific activity of 13.3 μmol min⁻¹ mg⁻¹. Purified PEPC was treated with phosphatase from bovine intestinal mucosa or protein kinase A to study its apparent phosphorylation level. Kinetic parameters of the enzyme reaction catalyzed by phosphorylated and dephosphorylated forms under different conditions were compared, as well as an effect of modulators. The enzyme dephosphorylation resulted in the change of hyperbolic kinetics to the sigmoidal one (with respect to PEP), following with the decrease of maximal reaction rate and the increase of sensitivity to L-malate inhibition. The hyperbolic kinetics of native PEPC present in dry maize seeds was not changed after the protein kinase A treatment, while it was converted to the sigmoidal one after dephosphorylation. Level of PEPC phosphorylation was not affected during seed imbibition.
- MeSH
- fosfoenolpyruvátkarboxylasa antagonisté a inhibitory izolace a purifikace metabolismus MeSH
- fosforylace MeSH
- kinetika MeSH
- kukuřice setá enzymologie MeSH
- maláty farmakologie MeSH
- proteinkinasy závislé na cyklickém AMP farmakologie MeSH
- semena rostlinná enzymologie MeSH
- skot MeSH
- zvířata MeSH
- Check Tag
- skot MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
