Добірка наукової літератури з теми "Complexes de type galactose oxydase"

The thermodynamics of the binding of derivatives of galactose and lactose to a 14 kDa beta-galactoside-binding lectin (L-14) from sheep spleen has been studied in 10 nM phosphate/150 mM NaCl/10 mM beta-mercaptoethanol buffer, pH 7.4, and in the temperature range 285-300 K using titration calorimetry. The single-site binding constants of various sugars for the lectin were in the following order: N-acetyl-lactosamine thiodigalactoside > 4-methylumbelliferyl lactoside > lactose > 4-methylumbelliferyl alpha-D-galactoside > methyl-alpha-galactose > methyl-beta-galactose. Reactions were essentially enthalpically driven with the binding enthalpies ranging from -53.8 kJ/mol for thiodigalactoside at 301 K to -2.2 kJ/mol for galactose at 300 K, indicating that hydrogen-bonding and van der Waals interactions provide the major stabilization for these reactions. However, the binding of 4-methylumbelliferyl-alpha-D-galactose displays relatively favourable entropic contributions, indicating the existence of a non-polar site adjacent to the galactose-binding subsite. From the increments in the enthalpies for the binding of lactose, N-acetyl-lactosamine and thiodigalactoside relative to methyl-beta-galactose, the contribution of glucose binding in the subsite adjacent to that for galactose shows that glucose makes a major contribution to the stability of L-14 disaccharide complexes. Observation of enthalpy-entropy compensation for the recognition of saccharides such as lactose by L-14 and the absence of it for monosaccharides such as galactose, together with the lack of appreciable changes in the heat capacity (delta Cp), indicate that reorganization of water plays an important role in these reactions.

CEL-IV is a C-type lectin isolated from a sea cucumber, Cucumaria echinata. This lectin is composed of four identical C-type carbohydrate-recognition domains (CRDs). X-ray crystallographic analysis of CEL-IV revealed that its tetrameric structure was stabilized by multiple interchain disulfide bonds among the subunits. Although CEL-IV has the EPN motif in its carbohydrate-binding sites, which is known to be characteristic of mannose binding C-type CRDs, it showed preferential binding of galactose and N-acetylgalactosamine. Structural analyses of CEL-IV-melibiose and CEL-IV-raffinose complexes revealed that their galactose residues were recognized in an inverted orientation compared with mannose binding C-type CRDs containing the EPN motif, by the aid of a stacking interaction with the side chain of Trp-79. Changes in the environment of Trp-79 induced by binding to galactose were detected by changes in the intrinsic fluorescence and UV absorption spectra of WT CEL-IV and its site-directed mutants. The binding specificity of CEL-IV toward complex oligosaccharides was analyzed by frontal affinity chromatography using various pyridylamino sugars, and the results indicate preferential binding to oligosaccharides containing Galβ1–3/4(Fucα1–3/4)GlcNAc structures. These findings suggest that the specificity for oligosaccharides may be largely affected by interactions with amino acid residues in the binding site other than those determining the monosaccharide specificity.

Eight novel carbohydrate-tethered trithiolato dinuclear ruthenium(II)-arene complexes were synthesized using CuAAC ‘click’ (Cu(I)-catalyzed azide-alkyne cycloaddition) reactions, and there in vitro activity against transgenic T. gondii tachyzoites constitutively expressing β-galactosidase (T. gondii β-gal) and in non-infected human foreskin fibroblasts, HFF, was determined at 0.1 and 1 µM. When evaluated at 1 µM, seven diruthenium-carbohydrate conjugates strongly impaired parasite proliferation by >90%, while HFF viability was retained at 50% or more, and they were further subjected to the half-maximal inhibitory concentration (IC50) measurement on T. gondii β-gal. Results revealed that the biological activity of the hybrids was influenced both by the nature of the carbohydrate (glucose vs. galactose) appended on ruthenium complex and the type/length of the linker between the two units. 23 and 26, two galactose-based diruthenium conjugates, exhibited low IC50 values and reduced effect on HFF viability when applied at 2.5 µM (23: IC50 = 0.032 µM/HFF viability 92% and 26: IC50 = 0.153 µM/HFF viability 97%). Remarkably, compounds 23 and 26 performed significantly better than the corresponding carbohydrate non-modified diruthenium complexes, showing that this type of conjugates are a promising approach for obtaining new antiparasitic compounds with reduced toxicity.

Abstract The Cu(II)-phenoxyl radical formed during the catalytic cycle of galactose oxidase (GO) attracted much attention, and the structures and properties of a number of metal-phenoxyl radical complexes have been studied. Some of the functional model systems of GO reported previously have shown that the Cu complexes oxidize primary alcohols to aldehydes and that the Cu(II)-phenoxyl radical species is formed in the catalytic cycle. Many Cu(II)-phenoxyl radical species have been produced by one-electron oxidation of the Cu(II)-phenolate complexes. On the other hand, one-electron oxidation of a Cu(II)-phenolate complex has the possibility to give different electronic structures, one of which is the Cu(III)-phenolate. From these points of view, this micro review is focused on the one-electron oxidized square-planar Cu(II) complexes of the salen-type ligands. Introduction of substituents into the phenolate moieties and conversion from a 5- to a 6-membered chelate backbone alter the electronic structure of the one-electron oxidized Cu(II) complexes and give rise to a different reactivity of benzyl alcohol oxidation. The relationship between the electronic structure and the reactivity is herein discussed.

The GalNAc/Gal-specific lectin from the sea mussel Crenomytilus grayanus (CGL) with anticancer activity represents а novel lectin family with β-trefoil fold. Earlier, the crystal structures of CGL complexes with globotriose, galactose and galactosamine, and mutagenesis studies have revealed that the lectin contained three carbohydrate-binding sites. The ability of CGL to recognize globotriose (Gb3) on the surface of breast cancer cells and bind mucin-type glycoproteins, which are often associated with oncogenic transformation, makes this compound to be perspective as a biosensor for cancer diagnostics. In this study, we describe results on in silico analysis of binding mechanisms of CGL to ligands (galactose, globotriose and mucin) and evaluate the individual contribution of the amino acid residues from carbohydrate-binding sites to CGL activity by site-directed mutagenesis. The alanine substitutions of His37, His129, Glu75, Asp127, His85, Asn27 and Asn119 affect the CGL mucin-binding activity, indicating their importance in the manifestation of lectin activity. It has been found that CGL affinity to ligands depends on their structure, which is determined by the number of hydrogen bonds in the CGL-ligand complexes. The obtained results should be helpful for understanding molecular machinery of CGL functioning and designing a synthetic analog of CGL with enhanced carbohydrate-binding properties.

Antibodies of the IgM type present in rabbit anti-epiglycanin antiserum were purified by (NH4)2SO4 precipitation and by ion-exchange, affinity and gel-filtration chromatography. After papain treatment of this fraction, followed by gel filtration, the fraction with highest apparent Mr was incubated with epiglycanin, and the antigen-antibody complexes separated by gel filtration. These were examined by electron microscopy, using rotational shadow casting, and the photographs of the complexes were mapped for the locations of the antibody molecules on the extended epiglycanin molecules. Distribution of the frequency of attachment of immunoglobulin showed a strong tendency toward binding at one end of epiglycanin, suggesting the probable presence of only one epitope, probably a glycopeptide structure containing a beta-D-galactopyranosyl-(1→3)-2-acetamido-2-deoxy-D-galactose chain.

Abstract The extracellular matrix (ECM) of cultured bovine aortic endothelial cells (BAEs) was analyzed by immunoblotting and reverse fibrin autography and shown to contain type 1 plasminogen activator inhibitor (PAI-1). Most PAI-1 in the ECM formed complexes with exogenously added tissue-type plasminogen activator (tPA), demonstrating that this PAI-1 was functionally active. The resulting tPA/PAI-1 complexes were recovered in the reaction solution, indicating that the PAI-1 in such complexes no longer bound to ECM. The PAI-1 could not be removed by incubating ECM in high salt (2 mol/L NaCl), sugars (1 mol/L galactose, 1 mol/L mannose), glycosaminoglycans (10 mmol/L heparin, 10 mmol/L dermatan sulfate), or epsilon-aminocaproic acid (0.1 mol/L). However, PAI-1 could be extracted from ECM by treatment with either arginine (0.5 mol/L) or potassium thiocyanate (2 mol/L), or by incubation under acidic conditions (pH 2.5). ECM depleted of PAI-1 by acid extraction was able to bind both the active and latent forms of PAI-1. In this instance, most of the bound PAI-1 did not form complexes with tPA, indicating that the latent form was not activated as a consequence of binding to ECM. Although the PAI-1 activity in conditioned medium decayed with a half-life (t 1/2) of less than 3 hours, the t 1/2 of ECM- associated PAI-1 was greater than 24 hours. These data suggest that PAI- 1 is produced by cultured BAEs in an active form and is then either released into the medium where it is rapidly inactivated or into the subendothelium where it binds to ECM. The specific binding of PAI-1 to ECM protects it from this inactivation.

The extracellular matrix (ECM) of cultured bovine aortic endothelial cells (BAEs) was analyzed by immunoblotting and reverse fibrin autography and shown to contain type 1 plasminogen activator inhibitor (PAI-1). Most PAI-1 in the ECM formed complexes with exogenously added tissue-type plasminogen activator (tPA), demonstrating that this PAI-1 was functionally active. The resulting tPA/PAI-1 complexes were recovered in the reaction solution, indicating that the PAI-1 in such complexes no longer bound to ECM. The PAI-1 could not be removed by incubating ECM in high salt (2 mol/L NaCl), sugars (1 mol/L galactose, 1 mol/L mannose), glycosaminoglycans (10 mmol/L heparin, 10 mmol/L dermatan sulfate), or epsilon-aminocaproic acid (0.1 mol/L). However, PAI-1 could be extracted from ECM by treatment with either arginine (0.5 mol/L) or potassium thiocyanate (2 mol/L), or by incubation under acidic conditions (pH 2.5). ECM depleted of PAI-1 by acid extraction was able to bind both the active and latent forms of PAI-1. In this instance, most of the bound PAI-1 did not form complexes with tPA, indicating that the latent form was not activated as a consequence of binding to ECM. Although the PAI-1 activity in conditioned medium decayed with a half-life (t 1/2) of less than 3 hours, the t 1/2 of ECM- associated PAI-1 was greater than 24 hours. These data suggest that PAI- 1 is produced by cultured BAEs in an active form and is then either released into the medium where it is rapidly inactivated or into the subendothelium where it binds to ECM. The specific binding of PAI-1 to ECM protects it from this inactivation.

An antibody highly specific for heat-shock protein (hsp)26, the unique small hsp of yeast, and mutants carrying a deletion of the HSP26 gene were used to examine the physical properties of the protein and to determine its intracellular distribution. The protein was found in complexes with a molecular mass of greater than 500 kD. Thus, it has all of the characteristics, including sequence homology and induction patterns, of small hsps from other organisms. When log-phase cells growing in glucose were heat shocked, hsp26 concentrated in nuclei and continued to concentrate in nuclei when these cells were returned to normal temperatures for recovery. However, hsp26 did not concentrate in nuclei under a variety of other conditions. For example, in early stationary-phase cells hsp26 is induced at normal growth temperatures. This protein was generally distributed throughout the cells, even after heat shock. Similarly, in cells genetically engineered to synthesize hsp26 in the presence of galactose, hsp26 did not concentrate in nuclei, with or without a heat shock. To determine if the failure of hsp26 to concentrate in the nucleus of these cells was due to the fact that the protein had been produced at 25 degrees C or to a difference in the physiological state of the cell, we investigated the distribution of the heat-induced protein in cells grown under several different conditions. In wild-type cells grown in galactose or acetate and in mitochondrial mutants grown in glucose or galactose, hsp26 also failed to concentrate in nuclei with a heat shock. We conclude that the intracellular location of hsp26 in yeast depends upon the physiological state of the cell and not simply upon the presence or absence of heat stress. Our findings may explain why previous investigations of the intracellular localization of small hsps in a variety of organisms have yielded seemingly contradictory results.

La galactose oxydase (GOase), une métallo-enzyme contenant du cuivre, est l'un des biocatalyseurs les plus étudiés pour l'oxydation enzymatique des glucides. Le mécanisme couramment accepté implique la forme oxydée clé (GOaseox), dans laquelle une unité glucidique (galactose) se lie au site équatorial (libre) et subit une déprotonation, suivie d'un arrachage d'atome d'hydrogène par un radical et d'un transfert d'électron supplémentaire, conduisant ainsi à la formation du produit final, l'aldéhyde, et à la réduction de la GOase. En raison de leur potentiel pour des oxydations catalytiques hautement sélectives, des modèles moléculaires du site actif de la GOase dérivés de bases de Schiff encombrées ont été développés. Cette approche biomimétique passe par la synthèse de complexes Cu(II)-phénol servant de pré-catalyseurs, qui subissent ensuite une oxydation à un électron pour devenir la forme "active" du catalyseur. Une question cruciale demeure : quels sont les facteurs qui déterminent si l’oxydation est centrée sur le ligand, produisant des radicaux Cu(II)-phénoxyle, ou vers le métal, formant des espèces Cu(III)-phénolate ? Malgré des efforts importants, une réponse définitive à cette question reste elusive.L'objectif de cette thèse est de développer des ligands redox-actifs afin de mieux comprendre les facteurs qui influencent le site d’oxydation du complexe de cuivre correspondant. La stratégie consiste à incorporer des fonctions chimiques stabilisant l'un ou l'autre tautomère de valence (Cu(II)-radical phénoxyle et Cu(III)-phénolate) et à étudier leur impact. À cet effet, plusieurs complexes ont été synthétisés et caractérisés par spectroscopie et électrochimie. Les activités catalytiques ont également été évaluées sur divers substrats contenant des groupes hydroxyle. Enfin, des calculs de chimie quantique (DFT) ont été réalisés pour aider à élucider les mécanismes catalytiques et à mieux comprendre les caractéristiques des différents complexes
Galactose Oxidase (GOase), a copper-containing metallo-enzyme, is one of the most studied biocatalysts for the enzymatic oxidation of carbohydrates. The consensus mechanism involves the key oxidized form (GOaseox), in which an the carbohydrate substrate (galactose unit) binds to the equatorial (free) site and is subsequently deprotonated. It undergoes hydrogen atom abstraction by the radical and further electron transfer to give the final product aldehyde and the reduced form of the GOase. Due to the potential for highly selective catalytic oxidations, the development of small-molecular models of the GOase active site has been carried out. Notably, sterically hindered schiff bases, which stand as one of the most representative mimics, have garnered significant attention. This biomimetic approach has extended to encompass other strategies. Within this framework, a range of Cu(II)-phenol complexes, serving as pre-catalysts, have been synthesized, subsequently undergoing one-electron oxidation to yield the "active" catalyst form. A central question then arises: What factors determine whether the oxidation pathway proceeds toward the ligand, resulting in the formation of Cu(II)-phenoxyl radicals, or toward the metal, giving rise to the Cu(III)-phenolate species? Despite substantial efforts, a definitive answer to this question has yet to be obtained.The aim of this thesis is to develop redox-active ligands aimed at understanding the factors affecting the oxidation state of copper, able to catalyze the oxidation of an alcohol into an aldehyde. The strategy is to include chemical functions that can stabilize either valence tautomer (Cu(II)-phenoxyl radical and Cu(III)-phenolate) and study their effect. For that purpose, several complexes were synthesized and characterized by different ways to understand their properties. The catalytic activities were also tested against different families of substrates comprising hydroxyl functions. Finally, quantum chemistry (DFT) calculations have been carried to help understand the characteristics of different complexes and elucidate of the catalytic mechanisms at work

Dans cette thèse, le processus d'assemblage ainsi que la maturation du cytochrome c oxydase de type cbb3 (cbb3-Cox) ont été étudiés dans la proteobactérie phototrophique pourpe non soufrée Rhodobacter capsulatus. R. capsulatus contient une chaîne de transfert d'électrons très ramifiée et represente un modèle d’organisme très utilisé dans l'étude des processus respiratoires et photosynthétiques.Les cbb3-Coxs spécifiques des bactéries représentent la deuxième catégorie la plus abondante des cytochromes c oxydases après le type Cox-aa3, mais n'ont jusqu'à présent pas été étudiées en détail. Récemment, la première structure cristalline cbb3-Cox de P. stutzeri a été obtenue, fournissant ainsi une avancée majeure invitant à des etudes plus détaillées sur le mécanisme catalytique et le processus d'assemblage. Les études sur les procédés d'assemblage et de maturation sont d'une très grande importance en raison du fait que de nombreux agents pathogènes humains tels que Helicobacter pylori, Neisseria meningitidis ou Campylobacter jejuni utilisent ce type de Cox, ce qui par conséquent pourrait amener a développer une interessante cible thérapeutique.Cbb3-Cox dans R. capsulatus est encodé par le gène opéron ccoNOQP codant quatre protéines membranaires constitutives de cbb3-Cox. CcoP et CcoO sont des cytochromes de type c, contenant des motifs périplasmiques fixés à l’hème. CcoN est la sous-unité centrale catalytique contenant deux molécules d’hèmes de type b et un ion cuivre. L’étude de la distribution de l’ion Cu à la sous-unité CcoN et l'assemblage des quatre sous-unités dans le complexe actif cbb3-Cox complexe sont les thèmes principaux de ce travail.Ici, le rôle du facteur d'assemblage putatif CcoH, sa structure et son interaction avec cbb3-Cox ont été étudiés en détail. CcoH est une petite protéine membranaire codé dans le groupe de gènes ccoGHIS situé à proximité des gènes codant cbb3-Cox. L'analyse in vivo de la formation de cbb3-Cox dans une souche ne contenant pas le facteur CcoH a montré une absence totale de cbb3-Cox. De même, la stabilité du facteur CcoH a été considérablement altérée dans une souche avec deletion du gene ccoNOQP. La dépendance mutuelle des deux protéines suggère leur interaction directe, ce qui a été confirmé par la photoréticulation directe de CcoH à la sous-unité CcoN, l’immunodétection de CcoH dans les cbb3-Cox complexes sur gels Blue Native, la co-purification par marquage CcoH-cbb3-Cox et le marquage radioactive in vitro des complexes cbb3-Cox avec CcoH.[...]
In this thesis, the assembly and maturation process of the cbb3-type cytochrome c oxidase (cbb3-Cox) was studied in the purple-non-sulphur phototrophic α-proteobacterium Rhodobacter capsulatus. R. capsulatus contains a highly branched electron-transfer chain and is a well studied model organism for investigating respiratory and photosynthetic processes.The bacteria-specific cbb3-Coxs represent the second most abundant class of cytochrome c oxidases after the aa3-type Cox, but have so far not been investigated in much detail. Recently, the first crystal structure of cbb3-Cox from P. stutzeri was obtained, providing a major breakthrough and inviting detailed studies on the catalytic mechanism and the assembly process. Studies on the assembly and maturation processes are of wide significance due to the fact that many human pathogens like Helicobacter pylori, Neisseria meningitides or Campylobacter jejuni use this type of Cox and it therefore might develop into an attractive drug-target. cbb3-Cox in R. capsulatus is encoded by the ccoNOQP gene operon which codes for four membrane proteins constituting cbb3-Cox. CcoP and CcoO are c-type cytochromes, containing periplasmic heme-binding motifs. CcoN is the central catalytic subunit which contains two b-type hemes and a copper ion. Investigating the delivery of Cu to the CcoN subunit and the assembly of all four subunits into the active cbb3-Cox complex were the main topics of this work. Here the role of the putative assembly factor CcoH, its structure and interaction with cbb3-Cox was investigated in detail. CcoH is a small membrane protein encoded in the ccoGHIS gene cluster located adjacent to the genes coding for cbb3-Cox. In vivo analysis of cbb3-Cox formation in a strain lacking ccoH showed the total absence of cbb3-Cox. Likewise, the stability of CcoH was drastically impaired in a ccoNOQP deletion strain. The mutual dependency of both proteins suggested their direct interaction, which was confirmed by site-directed photocrosslinking of CcoH to the CcoN subunit, by immunodetection of CcoH in cbb3-Cox complexes on Blue Native gels, by CcoH-cbb3-Cox co-purification and by in vitro labelling of cbb3-Cox complexes with radioactively labelled CcoH.[...]

The ECM of cultured BAEs was analyzed by immunoblotting and reverse fibrin autography and shown to contain PAI-1. PAI-1 containing ECM was incubated with tissue plasminogen activator (tPA) to determine whether the bound PAI-1 was functionally active or latent. The majority of the detectable PAI-1 in the ECM formed complexes with tPA indicating that it was active. The resulting tPA/PAI-1 complexes were released from ECM and recovered in the reaction solution indicating that the PAI-1 in such complexes had lost its ECM binding site. The PAI-1 could not be removed from ECM by incubating it in high salt (2M NaCl), sugar (galactose, mannose, 1 M), heparin (10 mg/ml) or lysine (1 M). However, PAI-1 could be extracted from ECM by treatment with arginine (0.5 M), potassium thiocyanate (2 M), or by incubation at acid pH (2.5). These treatments did not remove appreciable amounts of fibronectin or von willebrand's factor from ECM. ECM depleted of PAI-1 by acid extraction was able to bind all forms of exogenously added PAI-1 including both the active and the latent molecules. The majority of the bound PAI-1 could not form complexes with exogenously added tPA suggesting that the latent form was not activated as a consequence of this interaction. Although the PAI-1 activity in conditioned medium was unstable and decayed with a half life of less than 3 hrs, the half-life of ECM associated PAI-1 was greater than 24 h. These data suggest that PAI-1 is produced by cultured BAEs in an active form, and either released into the medium where it is rapidly inactivated, or bound to ECM. The specific binding of PAI-1 to ECM protects it from this inactivation.