A novel high molecular weight glutenin subunit encoded by the Glu-1B locus was identified in the French genotype Bagou, which we named 1B × 6.5. This subunit differed in SDS-PAGE from well-known 1B × 6 and 1B × 7 subunits, which are also encoded at this locus. Subunit 1B × 6.5 has a theoretical molecular weight of 88,322.83 Da, which is more mobile than 1B × 6 subunit, and isoelectric point (pI) of about 8.7, which is lower than that for 1B × 6 subunit. The specific primers were designed to amplify and sequence 2476 bp of the Glu-1B locus from genotype Bagou. A high level of similarity was found between the sequence encoding 1B × 6.5 and other x-type encoding alleles of this locus.

• Klíčová slova
• 1-D electrophoresis, 2-D electrophoresis, HMW glutenin, mass spectrometry, wheat,
• Publikační typ
• časopisecké články MeSH

Proteins play a major role in the three-dimensional organization of nuclear genome and its function. While histones arrange DNA into a nucleosome fiber, other proteins contribute to higher-order chromatin structures in interphase nuclei, and mitotic/meiotic chromosomes. Despite the key role of proteins in maintaining genome integrity and transferring hereditary information to daughter cells and progenies, the knowledge about their function remains fragmentary. This is particularly true for the proteins of condensed chromosomes and, in particular, chromosomes of plants. Here, we purified barley mitotic metaphase chromosomes by a flow cytometric sorting and characterized their proteins. Peptides from tryptic protein digests were fractionated either on a cation exchanger or reversed-phase microgradient system before liquid chromatography coupled to tandem mass spectrometry. Chromosomal proteins comprising almost 900 identifications were classified based on a combination of software prediction, available database localization information, sequence homology, and domain representation. A biological context evaluation indicated the presence of several groups of abundant proteins including histones, topoisomerase 2, POLYMERASE 2, condensin subunits, and many proteins with chromatin-related functions. Proteins involved in processes related to DNA replication, transcription, and repair as well as nucleolar proteins were found. We have experimentally validated the presence of FIBRILLARIN 1, one of the nucleolar proteins, on metaphase chromosomes, suggesting that plant chromosomes are coated with proteins during mitosis, similar to those of human and animals. These results improve significantly the knowledge of plant chromosomal proteins and provide a basis for their functional characterization and comparative phylogenetic analyses.

• Klíčová slova
• FIBRILLARIN 1, barley, chromatin, flow cytometric sorting, mass spectrometry, mitotic chromosome, perichromosomal layer, protein prediction,
• Publikační typ
• časopisecké články MeSH

Carnivorous plants within the order Caryophyllales use jasmonates, a class of phytohormone, in the regulation of digestive enzyme activities. We used the carnivorous butterwort Pinguicula × Tina from the order Lamiales to investigate whether jasmonate signaling is a universal and ubiquitous signaling pathway that exists outside the order Caryophyllales. We measured the electrical signals, enzyme activities, and phytohormone tissue levels in response to prey capture. Mass spectrometry was used to identify proteins in the digestive secretion. We identified eight enzymes in the digestive secretion, many of which were previously found in other genera of carnivorous plants. Among them, alpha-amylase is unique in carnivorous plants. Enzymatic activities increased in response to prey capture; however, the tissue content of jasmonic acid and its isoleucine conjugate remained rather low in contrast to the jasmonate response to wounding. Enzyme activities did not increase in response to the exogenous application of jasmonic acid or coronatine. Whereas similar digestive enzymes were co-opted from plant defense mechanisms among carnivorous plants, the mode of their regulation differs. The butterwort has not co-opted jasmonate signaling for the induction of enzyme activities in response to prey capture. Moreover, the presence of alpha-amylase in digestive fluid of P. × Tina, which has not been found in other genera of carnivorous plants, might indicate that non-defense-related genes have also been co-opted for carnivory.

• Klíčová slova
• Pinguicula, Butterwort, carnivorous plant, digestive enzymes, electrical signals, jasmonic acid, protease, variation potential,
• MeSH
• cyklopentany MeSH
• hluchavkotvaré * MeSH
• masožravci * MeSH
• oxylipiny MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• cyklopentany MeSH
• jasmonic acid MeSH Prohlížeč
• oxylipiny MeSH

Cyanobacteria represent a bacterial phyllum characteristic by the ability to photosynthesize. They are potentially applicable for the production of useful compounds but may also cause poisoning or at least health problems as they can produce cyanotoxins. The introduction of a fast methodology is important not only for fundamental taxonomic purposes, but also for reliable identifications in biological studies. In this work, we have used matrix-assisted laser desorption/ionization time-of-flight mass spectrometry of intact cells to study Chroococcidiopsis strains. A library of the obtained reference mass spectra containing characteristic peptide/protein profiles was examined by software tools to characterize similarities and differences applicable for diagnostics and taxonomy. Both a similarity tree and heat map constructed from the mass spectrometric data proved consistent with 16S rRNA sequencing results. We show as novelty that a binary matrix combining ferulic and sinapinic acids performs well in acquiring reproducible mass spectra of cyanobacteria. Using the matrix solvent, a protein extraction from cells was done. After polyacrylamide gel electrophoresis, the separated protein fractions were in-gel digested and the resulting peptides analyzed by liquid chromatography coupled with tandem mass spectrometry. For the first time, photosystem protein components, phycobilisome proteins, electron transport proteins, nitrogen-metabolism and nucleic acids binding-proteins, cytochromes plus other enzymes and various uncharacterized proteins could be assigned to characteristic peaks in the mass spectrometric profiles and some of them suggested as markers in addition to 30S and 50S ribosomal proteins known from previous studies employing intact cell mass spectrometry of microorganisms.

• MeSH
• bakteriální proteiny analýza   izolace a purifikace   MeSH
• elektroforéza v polyakrylamidovém gelu MeSH
• fylogeneze MeSH
• peptidy analýza   izolace a purifikace   MeSH
• RNA ribozomální 16S genetika   MeSH
• sinice chemie   klasifikace   genetika   MeSH
• spektrometrie hmotnostní - ionizace laserem za účasti matrice metody   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• bakteriální proteiny MeSH
• peptidy MeSH
• RNA ribozomální 16S MeSH

Almost 25 years ago, an enzyme named zeatin cis-trans isomerase from common bean has been described by Bassil et al. (1993). The partially purified enzyme required an external addition of FAD and dithiothreitol for the conversion of cis-zeatin to its trans- isomer that occurred only under light. Although an existence of this important enzyme involved in the metabolism of plant hormones cytokinins was generally accepted by plant biologists, the corresponding protein and encoding gene have not been identified to date. Based on the original paper, we purified and identified an enzyme from maize, which shows the described zeatin cis-trans isomerase activity. The enzyme belongs to nucleotide pyrophosphatase/phosphodiesterase family, which is well characterized in mammals, but less known in plants. Further experiments with the recombinant maize enzyme obtained from yeast expression system showed that rather than the catalytic activity of the enzyme itself, a non-enzymatic flavin induced photoisomerization is responsible for the observed zeatin cis-trans interconversion in vitro. An overexpression of the maize nucleotide pyrophosphatase/phosphodiesterase gene led to decreased FAD and increased FMN and riboflavin contents in transgenic Arabidopsis plants. However, neither contents nor the ratio of zeatin isomers was altered suggesting that the enzyme is unlikely to catalyze the interconversion of zeatin isomers in vivo. Using enhanced expression of a homologous gene, functional nucleotide pyrophosphatase/phosphodiesterase was also identified in rice.

• Klíčová slova
• flavins, isomerization, maize, nucleotide pyrophosphatase/phosphodiesterase, zeatin,
• Publikační typ
• časopisecké články MeSH

Plant ALDH10 family members are aminoaldehyde dehydrogenases (AMADHs), which oxidize ω-aminoaldehydes to the corresponding acids. They have been linked to polyamine catabolism, osmoprotection, secondary metabolism (fragrance), and carnitine biosynthesis. Plants commonly contain two AMADH isoenzymes. We previously studied the substrate specificity of two AMADH isoforms from peas (PsAMADHs). Here, two isoenzymes from tomato (Solanum lycopersicum), SlAMADHs, and three AMADHs from maize (Zea mays), ZmAMADHs, were kinetically investigated to obtain further clues to the catalytic mechanism and the substrate specificity. We also solved the high resolution crystal structures of SlAMADH1 and ZmAMADH1a because these enzymes stand out from the others regarding their activity. From the structural and kinetic analysis, we can state that five residues at positions 163, 288, 289, 444, and 454 (PsAMADHs numbering) can, directly or not, significantly modulate AMADH substrate specificity. In the SlAMADH1 structure, a PEG aldehyde derived from the precipitant forms a thiohemiacetal intermediate, never observed so far. Its absence in the SlAMADH1-E260A structure suggests that Glu-260 can activate the catalytic cysteine as a nucleophile. We show that the five AMADHs studied here are capable of oxidizing 3-dimethylsulfoniopropionaldehyde to the cryo- and osmoprotectant 3-dimethylsulfoniopropionate. For the first time, we also show that 3-acetamidopropionaldehyde, the third aminoaldehyde besides 3-aminopropionaldehyde and 4-aminobutyraldehyde, is generally oxidized by AMADHs, meaning that these enzymes are unique in metabolizing and detoxifying aldehyde products of polyamine degradation to nontoxic amino acids. Finally, gene expression profiles in maize indicate that AMADHs might be important for controlling ω-aminoaldehyde levels during early stages of the seed development.

• MeSH
• aldehydoxidoreduktasy chemie   genetika   metabolismus   MeSH
• aldehydy chemie   MeSH
• chemické modely MeSH
• fylogeneze MeSH
• fyziologie rostlin MeSH
• kinetika MeSH
• krystalografie rentgenová metody   MeSH
• kukuřice setá enzymologie   MeSH
• mutageneze cílená MeSH
• NAD chemie   MeSH
• polyethylenglykoly chemie   MeSH
• regulace genové exprese enzymů * MeSH
• regulace genové exprese u rostlin * MeSH
• rostliny enzymologie   MeSH
• semena rostlinná metabolismus   MeSH
• Solanum lycopersicum enzymologie   MeSH
• substrátová specifita MeSH
• vazba proteinů MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• aldehydoxidoreduktasy MeSH
• aldehydy MeSH
• NAD MeSH
• polyethylenglykoly MeSH

The aim of this work was to prepare recombinant amine oxidase from Aspergillus niger after overexpressing in yeast. The yeast expression vector pDR197 that includes a constitutive PMA1 promoter was used for the expression in Saccharomyces cerevisiae. Recombinant amine oxidase was extracted from the growth medium of the yeast, purified to homogeneity and identified by activity assay and MALDI-TOF peptide mass fingerprinting. Similarity search in the newly published A. niger genome identified six genes coding for copper amine oxidase, two of them corresponding to the previously described enzymes AO-I a methylamine oxidase and three other genes coding for FAD amine oxidases. Thus, A. niger possesses an enormous metabolic gear to grow on amine compounds and thus support its saprophytic lifestyle.

• MeSH
• aminy metabolismus   MeSH
• Aspergillus niger enzymologie   genetika   MeSH
• chromatografie DEAE-celulózová MeSH
• fungální proteiny chemie   genetika   metabolismus   MeSH
• histaminasa chemie   genetika   metabolismus   MeSH
• hmotnostní spektrometrie MeSH
• kinetika MeSH
• klonování DNA MeSH
• molekulární sekvence - údaje MeSH
• rekombinantní proteiny chemie   genetika   metabolismus   MeSH
• Saccharomyces cerevisiae genetika   metabolismus   MeSH
• sekvence aminokyselin MeSH
• sekvenční seřazení MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• aminy MeSH
• fungální proteiny MeSH
• hexylamine MeSH Prohlížeč
• histaminasa MeSH
• rekombinantní proteiny MeSH

Aminoaldehydes are products of polyamine degradation and are known to be reactive metabolites that are toxic to living cells at high concentrations. These compounds are catabolized by aminoaldehyde dehydrogenases, which are enzymes that contain a nicotinamide adenine dinucleotide coenzyme. Aminoaldehyde dehydrogenase from Pisum sativum was overexpressed in Escherichia coli, purified and crystallized using the hanging-drop method. A complete data set was collected to 2.8 A resolution at 100 K. Crystals belong to the monoclinic space group P2(1), with unit-cell parameters a = 86.4, b = 216.6, c = 205.4 A, beta = 98.1 degrees. Molecular replacement was performed and led to the identification of six dimers per asymmetric unit.

• MeSH
• aldehyddehydrogenasa chemie   izolace a purifikace   metabolismus   MeSH
• DNA primery MeSH
• elektroforéza v polyakrylamidovém gelu MeSH
• hrách setý enzymologie   MeSH
• klonování DNA MeSH
• konformace proteinů MeSH
• krystalizace MeSH
• krystalografie rentgenová MeSH
• rekombinantní proteiny chemie   izolace a purifikace   metabolismus   MeSH
• sekvence nukleotidů MeSH
• spektrometrie hmotnostní - ionizace laserem za účasti matrice MeSH
• western blotting MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• aldehyddehydrogenasa MeSH
• DNA primery MeSH
• rekombinantní proteiny MeSH