1,5-diamino-2-pentyne (DAPY) was found to be a weak substrate of grass pea (Lathyrus sativus, GPAO) and sainfoin (Onobrychis viciifolia, OVAO) amine oxidases. Prolonged incubations, however, resulted in irreversible inhibition of both enzymes. For GPAO and OVAO, rates of inactivation of 0.1-0.3 min(-1) were determined, the apparent KI values (half-maximal inactivation) were of the order of 10(-5) m. DAPY was found to be a mechanism-based inhibitor of the enzymes because the substrate cadaverine significantly prevented irreversible inhibition. The N1-methyl and N5-methyl analogs of DAPY were tested with GPAO and were weaker inactivators (especially the N5-methyl) than DAPY. Prolonged incubations of GPAO or OVAO with DAPY resulted in the appearance of a yellow-brown chromophore (lambda(max) = 310-325 nm depending on the working buffer). Excitation at 310 nm was associated with emitted fluorescence with a maximum at 445 nm, suggestive of extended conjugation. After dialysis, the color intensity was substantially decreased, indicating the formation of a low molecular mass secondary product of turnover. The compound provided positive reactions with ninhydrin, 2-aminobenzaldehyde and Kovacs' reagents, suggesting the presence of an amino group and a nitrogen-containing heterocyclic structure. The secondary product was separated chromatographically and was found not to irreversibly inhibit GPAO. MS indicated an exact molecular mass (177.14 Da) and molecular formula (C10H15N3). Electrospray ionization- and MALDI-MS/MS analyses yielded fragment mass patterns consistent with the structure of a dihydropyridine derivative of DAPY. Finally, N-(2,3-dihydropyridinyl)-1,5-diamino-2-pentyne was identified by means of 1H- and 13C-NMR experiments. This structure suggests a lysine modification chemistry that could be responsible for the observed inactivation.

• MeSH
• alkyny metabolismus   farmakologie   MeSH
• diaminy metabolismus   farmakologie   MeSH
• histaminasa antagonisté a inhibitory   metabolismus   MeSH
• inhibitory enzymů metabolismus   farmakologie   MeSH
• kinetika MeSH
• kolorimetrie MeSH
• Lathyrus enzymologie   MeSH
• oxidace-redukce MeSH
• spektrální analýza metody   MeSH
• substrátová specifita MeSH
• vysokoúčinná kapalinová chromatografie MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• 1,5-diamino-2-pentyne MeSH Prohlížeč
• alkyny MeSH
• diaminy MeSH
• histaminasa MeSH
• inhibitory enzymů MeSH

The crystal structure of the complex between human immunodeficiency virus type 1 (HIV-1) protease and a peptidomimetic inhibitor of ethyleneamine type has been refined to R factor of 0.178 with diffraction limit 2.5 A. The peptidomimetic inhibitor Boc-Phe-Psi[CH2CH2NH]-Phe-Glu-Phe-NH2 (denoted here as OE) contains the ethyleneamine replacement of the scissile peptide bond. The inhibitor lacks the hydroxyl group which is believed to mimic tetrahedral transition state of proteolytic reaction and thus is suspected to be necessary for good properties of peptidomimetic HIV-1 protease inhibitors. Despite the missing hydroxyl group the inhibition constant of OE is 1.53 nm and it remains in the nanomolar range also towards several available mutants of HIV-1 protease. The inhibitor was found in the active site of protease in an extended conformation with a unique hydrogen bond pattern different from hydroxyethylene and hydroxyethylamine inhibitors. The isostere nitrogen forms a hydrogen bond to one catalytic aspartate only. The other aspartate forms two weak hydrogen bridges to the ethylene group of the isostere. A comparison with other inhibitors of this series containing isostere hydroxyl group in R or S configuration shows different ways of accommodation of inhibitor in the active site. Special attention is devoted to intermolecular contacts between neighbouring dimers responsible for mutual protein adhesion and for a special conformation of Met46 and Phe53 side chains not expected for free protein in water solution.

• MeSH
• aminokyseliny chemie   metabolismus   MeSH
• difrakce rentgenového záření MeSH
• HIV-1 účinky léků   enzymologie   MeSH
• HIV-proteasa chemie   metabolismus   MeSH
• hydrofobní a hydrofilní interakce MeSH
• inhibitory proteas chemie   metabolismus   farmakologie   MeSH
• katalytická doména MeSH
• konformace proteinů MeSH
• lidé MeSH
• molekulární modely MeSH
• molekulární sekvence - údaje MeSH
• oligopeptidy chemie   metabolismus   farmakologie   MeSH
• sekvence aminokyselin MeSH
• statická elektřina MeSH
• vazba proteinů MeSH
• vazebná místa MeSH
• vodíková vazba MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• srovnávací studie MeSH
• Názvy látek
• aminokyseliny MeSH
• Boc-Phe-psi(CH2CH2NH)-Phe-Glu-Phe-NH2 MeSH Prohlížeč
• HIV-proteasa MeSH
• inhibitory proteas MeSH
• oligopeptidy MeSH

The tumor suppressor protein, p53, selectively binds to supercoiled (sc) DNA lacking the specific p53 consensus binding sequence (p53CON). Using p53 deletion mutants, we have previously shown that the p53 C-terminal DNA-binding site (CTDBS) is critical for this binding. Here we studied supercoil-selective binding of bacterially expressed full-length p53 using modulation of activity of the p53 DNA-binding domains by oxidation of cysteine residues (to preclude binding within the p53 core domain) and/or by antibodies mapping to epitopes at the protein C-terminus (to block binding within the CTDBS). In the absence of antibody, reduced p53 preferentially bound scDNA lacking p53CON in the presence of 3 kb linear plasmid DNAs or 20 mer oligonucleotides, both containing and lacking the p53CON. Blocking the CTDBS with antibody caused reduced p53 to bind equally to sc and linear or relaxed circular DNA lacking p53CON, but with a high preference for the p53CON. The same immune complex of oxidized p53 failed to bind DNA, while oxidized p53 in the absence of antibody restored selective scDNA binding. Antibodies mapping outside the CTDBS did not prevent p53 supercoil-selective (SCS) binding. These data indicate that the CTDBS is primarily responsible for p53 SCS binding. In the absence of the SCS binding, p53 binds sc or linear (relaxed) DNA via the p53 core domain and exhibits strong sequence-specific binding. Our results support a hypothesis that alterations to DNA topology may be a component of the complex cellular regulatory mechanisms that control the switch between latent and active p53 following cellular stress.

• MeSH
• lidé MeSH
• monoklonální protilátky metabolismus   MeSH
• mutageneze cílená MeSH
• nádorový supresorový protein p53 metabolismus   ultrastruktura   MeSH
• oxidace-redukce MeSH
• sekvenční delece MeSH
• superhelikální DNA metabolismus   ultrastruktura   MeSH
• terciární struktura proteinů MeSH
• vazba proteinů MeSH
• vazebná místa protilátek MeSH
• vazebná místa MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• srovnávací studie MeSH
• Názvy látek
• monoklonální protilátky MeSH
• nádorový supresorový protein p53 MeSH
• superhelikální DNA MeSH

The cloning of two novel genes that encode cytokinin oxidase/dehydrogenase (CKX) in barley is described in this work. Transformation of both genes into Arabidopsis and tobacco showed that at least one of the genes codes for a functional enzyme, as its expression caused a cytokinin-deficient phenotype in the heterologous host plants. Additional cloning of two gene fragments, and an in silico search in the public expressed sequence tag clone databases, revealed the presence of at least 13 more members of the CKX gene family in barley and wheat. The expression of three selected barley genes was analyzed by RT-PCR and found to be organ-specific with peak expression in mature kernels. One barley CKX (HvCKX2) was characterized in detail after heterologous expression in tobacco. Interestingly, this enzyme shows a pH optimum at 4.5 and a preference for cytokinin ribosides as substrates, which may indicate its vacuolar targeting. Different substrate specificities, and the pH profiles of cytokinin-degrading enzymes extracted from different barley tissues, are also presented.

• MeSH
• 2,6-dichlorindofenol chemie   metabolismus   MeSH
• Arabidopsis enzymologie   genetika   růst a vývoj   MeSH
• benzochinony chemie   metabolismus   MeSH
• cytokininy chemie   metabolismus   MeSH
• exprese genu MeSH
• geneticky modifikované rostliny MeSH
• ječmen (rod) enzymologie   genetika   MeSH
• klonování DNA MeSH
• koncentrace vodíkových iontů MeSH
• molekulární sekvence - údaje MeSH
• oxidoreduktasy biosyntéza   genetika   metabolismus   MeSH
• pšenice enzymologie   genetika   MeSH
• rekombinantní proteiny genetika   metabolismus   MeSH
• rostlinné geny genetika   MeSH
• sekvence aminokyselin MeSH
• sekvenční homologie aminokyselin MeSH
• sekvenční seřazení MeSH
• substrátová specifita MeSH
• tabák enzymologie   genetika   růst a vývoj   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• 2,6-dichlorindofenol MeSH
• benzochinony MeSH
• cytokinin oxidase MeSH Prohlížeč
• cytokininy MeSH
• oxidoreduktasy MeSH
• rekombinantní proteiny MeSH
• ubiquinone-O MeSH Prohlížeč

The LmbB1 protein, participating in the biosynthesis of lincomycin, was heterologously expressed in Escherichia coli, purified in its active form, and characterized as a dimer of identical subunits. Methods for purification and analysis of the LmbB1 reaction product were developed. Molecular mass and fragmentation pattern of the product revealed by capillary electrophoresis-mass spectrometry were in agreement with its proposed structure, 4-(3-carboxy-3-oxo-propenyl)-2,3-dihydro-1H-pyrrole-2-carboxylic acid. The LmbB1 is therefore a dioxygenase catalysing the 2,3-extradiol cleavage of the l-3,4-dihydroxyphenyl alanine aromatic ring. The final LmbB1 reaction product, a unique compound found in biosynthesis of lincomycin and expected in anthramycins, arises through subsequent cyclization of the primary cleavage product, 2,3-secodopa. A possible role of LmbB1 in 2,3-secodopa cyclization and alternative ways of the cyclization in the formation of biosynthetically related compounds, muscaflavin and stizolobinic acid, are discussed.

• MeSH
• chromatografie iontoměničová MeSH
• cyklizace MeSH
• dihydroxyfenylalanin metabolismus   MeSH
• elektroforéza kapilární MeSH
• Escherichia coli genetika   metabolismus   MeSH
• estradiol metabolismus   MeSH
• hmotnostní spektrometrie MeSH
• linkomycin biosyntéza   chemie   izolace a purifikace   MeSH
• molekulární struktura MeSH
• Streptomyces metabolismus   MeSH
• techniky dvojhybridového systému MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• dihydroxyfenylalanin MeSH
• estradiol MeSH
• linkomycin MeSH

The structure of photosystem II (PSII) complex isolated from thylakoid membranes of the red alga Porphyridium cruentum was investigated using electron microscopy followed by single particle image analysis. The dimeric complexes observed contain all major PSII subunits (CP47, CP43, D1 and D2 proteins) as well as the extrinsic proteins (33 kDa, 12 kDa and the cytochrome c(550)) of the oxygen-evolving complex (OEC) of PSII, encoded by the psbO, psbU and psbV genes, respectively. The single particle analysis of the top-view projections revealed the PSII complex to have maximal dimensions of 22 x 15 nm. The analysis of the side-view projections shows a maximal thickness of the PSII complex of about 9 nm including the densities on the lumenal surface that has been attributed to the proteins of the OEC complex. These results clearly demonstrate that the red algal PSII complex is structurally very similar to that of cyanobacteria and to the PSII core complex of higher plants. In addition, the arrangement of the OEC proteins on the lumenal surface of the PSII complex is consistent to that obtained by X-ray crystallography of cyanobacterial PSII.

• MeSH
• bakteriální proteiny chemie   genetika   metabolismus   MeSH
• bílkoviny řas chemie   genetika   metabolismus   ultrastruktura   MeSH
• fotosystém II - proteinový komplex chemie   genetika   metabolismus   ultrastruktura   MeSH
• kvarterní struktura proteinů * MeSH
• kyslík metabolismus   MeSH
• makromolekulární látky MeSH
• molekulární modely MeSH
• podjednotky proteinů chemie   genetika   metabolismus   MeSH
• Porphyridium chemie   cytologie   metabolismus   MeSH
• tylakoidy chemie   ultrastruktura   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• bakteriální proteiny MeSH
• bílkoviny řas MeSH
• fotosystém II - proteinový komplex MeSH
• kyslík MeSH
• makromolekulární látky MeSH
• podjednotky proteinů MeSH

Human glutamate carboxypeptidase II (GCPII) is a co-catalytic metallopeptidase and its putative catalytic domain is homologous to the aminopeptidases from Vibrio proteolyticus and Streptomyces griseus. In humans, the enzyme is expressed predominantly in the nervous system and the prostate. The prostate form, termed prostate-specific membrane antigen, is overexpressed in prostate cancer and is used as a diagnostic marker of the disease. Inhibition of the form of GCPII expressed in the central nervous system has been shown to protect against ischemic injury in experimental animal models. Human GCPII consists of 750 amino acids, and six individual domains were predicted to constitute the protein structure. Here, we report the analysis of the contribution of these putative domains to the structure/function of recombinant human GCPII. We cloned 13 mutants of human GCPII that are truncated or extended at one or both the N- and C-termini of the GCPII sequence. The clones were used to generate stably transfected Drosophila Schneider's cells, and the expression and carboxypeptidase activities of the individual protein products were determined. The extreme C-terminal region of human GCPII was found to be critical for the hydrolytic activity of the enzyme. The deletion of as few as 15 amino acids from the C-terminus was shown to completely abolish the enzymatic activity of GCPII. Furthermore, the GCPII carboxypeptidase activity was abrogated upon removal of more than 60 amino acid residues from the N-terminus of the protein. Overall, these results clearly show that amino acid segments at the N- and C-termini of the ectodomain of GCPII are essential for its carboxypeptidase activity and/or proper folding.

• MeSH
• aminokyseliny chemie   metabolismus   MeSH
• antigeny povrchové chemie   metabolismus   MeSH
• DNA primery MeSH
• elektroforéza v polyakrylamidovém gelu MeSH
• glutamátkarboxypeptidasa II chemie   metabolismus   MeSH
• katalýza MeSH
• lidé MeSH
• sbalování proteinů * MeSH
• sekvence nukleotidů MeSH
• western blotting MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• aminokyseliny MeSH
• antigeny povrchové MeSH
• DNA primery MeSH
• FOLH1 protein, human MeSH Prohlížeč
• glutamátkarboxypeptidasa II MeSH

Two soluble enzymes (FerA and FerB) catalyzing the reduction of a number of iron(III) complexes by NADH, were purified to near homogeneity from the aerobically grown iron-limited culture of Paracoccus denitrificans using a combination of anion-exchange chromatography (Sepharose Q), chromatofocusing (Mono P), and gel permeation chromatography (Superose 12). FerA is a monomer with a molecular mass of 19 kDa, whereas FerB exhibited a molecular mass of about 55 kDa and consists of probably two identical subunits. FerA can be classified as an NADH:flavin oxidoreductase with a sequential reaction mechanism. It requires the addition of FMN or riboflavin for activity on Fe(III) substrates. In these reactions, the apparent substrate specificity of FerA seems to stem exclusively from different chemical reactivities of Fe(III) compounds with the free reduced flavin produced by the enzyme. Observations on reducibility of Fe(III) chelated by vicinal dihydroxy ligands support the view that FerA takes part in releasing iron from the catechol type siderophores synthesized by P. denitrificans. Contrary to FerA, the purified FerB contains a noncovalently bound redox-active FAD coenzyme, can utilize NADPH in place of NADH, does not reduce free FMN at an appreciable rate, and gives a ping-pong type kinetic pattern with NADH and Fe(III)-nitrilotriacetate as substrates. FerB is able to reduce chromate, in agreement with the fact that its N-terminus bears a homology to the previously described chromate reductase from Pseudomonas putida. Besides this, it also readily reduces quinones like ubiquinone-0 (Q0) or unsubstituted p-benzoquinone.

• MeSH
• aniontoměniče MeSH
• chromatografie iontoměničová MeSH
• elektroforéza v polyakrylamidovém gelu MeSH
• FMN-reduktasa chemie   izolace a purifikace   metabolismus   MeSH
• hmotnostní spektrometrie MeSH
• izoenzymy chemie   izolace a purifikace   metabolismus   MeSH
• katalýza MeSH
• kinetika MeSH
• molekulární sekvence - údaje MeSH
• molekulová hmotnost MeSH
• Paracoccus denitrificans enzymologie   MeSH
• rozpustnost MeSH
• sekvence aminokyselin MeSH
• sekvenční homologie aminokyselin MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• aniontoměniče MeSH
• ferric citrate iron reductase MeSH Prohlížeč
• FMN-reduktasa MeSH
• izoenzymy MeSH

Lectin from females of the important sand fly vector, Phlebotomus duboscqi (Diptera: Psychodidae), was isolated by immunoaffinity chromatography using a minicolumn with immobilized anti-lectin immunoglobulins. Carbohydrate-binding specificity of active fractions corresponded to that of midgut and salivary gland lysates. Haemagglutination was inhibited by d-glucosamine, d-galactosamine and d-mannosamine. The homogeneity and molecular mass of the purified lectin was examined by SDS/PAGE in both reducing and nonreducing conditions. The active fractions showed one band strongly stained by Coomassie blue or silver nitrate; the molecular mass of the lectin was 42 kDa under nonreducing and 44 kDa under reducing conditions. SDS/PAGE of active fractions from the gel filtration revealed four to six protein bands, but the 42/44-kDa protein present in all active fractions was the only component reacting with specific antibodies in Western blots. Localization of the lectin in the gut of females was studied using indirect immunofluorescence on sections. The positive reaction of specific antibodies was localized in the lumen and along the microvillar surfaces of epithelial cells. The lectin was partially sequenced and characterized by MS. Peptide maps were obtained by MALDI-TOF MS, and several sequence tags were identified from tandem mass spectra on an ion trap. These sequences displayed high similarity to salivary protein precursors previously identified in a cDNA library of the sand flies Phlebotomus papatasi and Lutzomyia longipalpis. Two main hypotheses on the role of female lectin in Leishmania development are discussed.

• MeSH
• chromatografie afinitní MeSH
• elektroforéza v polyakrylamidovém gelu MeSH
• erytrocyty metabolismus   MeSH
• fluorescenční mikroskopie MeSH
• gelová chromatografie MeSH
• genová knihovna MeSH
• hemaglutininy metabolismus   MeSH
• imunoglobuliny metabolismus   MeSH
• komplementární DNA metabolismus   MeSH
• králíci MeSH
• lektiny chemie   metabolismus   MeSH
• Phlebotomus metabolismus   MeSH
• spektrometrie hmotnostní - ionizace laserem za účasti matrice MeSH
• terciární struktura proteinů MeSH
• trávicí systém metabolismus   MeSH
• vysokoúčinná kapalinová chromatografie MeSH
• western blotting MeSH
• zvířata MeSH
• Check Tag
• králíci MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• hemaglutininy MeSH
• imunoglobuliny MeSH
• komplementární DNA MeSH
• lektiny MeSH

The PsbH protein, a small subunit of the photosystem II complex (PSII), was identified as a 6-kDa protein band in the PSII core and subcore (CP47-D1-D2-cyt b-559) from the wild-type strain of the cyanobacterium Synechocystis PCC 6803. The protein was missing in the D1-D2-cytochrome b-559 complex and also in all PSII complexes isolated from IC7, a mutant lacking the psbH gene. The following properties of PSII in the mutant contrasted with those in wild-type: (a) CP47 was released during nondenaturing electrophoresis of the PSII core isolated from IC7; (b) depletion of CO2 resulted in a reversible decrease of the QA- reoxidation rate in the IC7 cells; (c) light-induced decrease in PSII activity, measured as 2,5-dimethyl-benzoquinone-supported Hill reaction, was strongly dependent on the HCO3- concentration in the IC7 cells; and (d) illumination of the IC7 cells lead to an extensive oxidation, fragmentation and cross-linking of the D1 protein. We did not find any evidence for phosphorylation of the PsbH protein in the wild-type strain. The results showed that in the PSII complex of Synechocystis attachment of CP47 to the D1-D2 heterodimer appears weakened and binding of bicarbonate on the PSII acceptor side is destabilized in the absence of the PsbH protein.

• MeSH
• fosfoproteiny genetika   MeSH
• fosforylace MeSH
• fotosyntetické reakční centrum - proteinové komplexy genetika   metabolismus   MeSH
• fotosystém II - proteinový komplex * MeSH
• hydrogenuhličitany metabolismus   MeSH
• oxidační stres MeSH
• sinice genetika   metabolismus   MeSH
• trypsin metabolismus   MeSH
• vazba proteinů MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• fosfoproteiny MeSH
• fotosyntetické reakční centrum - proteinové komplexy MeSH
• fotosystém II - proteinový komplex * MeSH
• hydrogenuhličitany MeSH
• photosystem II, psbH subunit MeSH Prohlížeč
• trypsin MeSH