RESUMO
Five stilbenoids, E-resveratrol, E-piceatannol, (+) E-(epsilon)-viniferin, (+)-ampelopsin A and vitisin C were isolated from methyl tert-butyl ether (MtBE) stem extract of Vitis vinifera (Chardonnay cv). Their purification on a preparative scale was obtained by centrifugal partition chromatography (CPC) using quaternary Arizona solvent systems composed of n-heptane/ethyl acetate/methanol/water. We tested 23 Arizona solvent systems to partition the extract and found that systems K and M (Hept/EtOAc/MeOH/water, 1:2:1:2 and 5:6:5:6, respectively; v/v) were the best to separate the stilbenes mentioned above. This support-free liquid-liquid chromatographic procedure made it possible to isolate ampelopsin A from V. vinifera for the first time. The antiamyloidogenic activity of the isolated stilbenes was evaluated versus beta-amyloid fibrils. E-resveratrol and (+)-ampelopsin A were found to be the most active compounds with 63 and 46% inhibition at 10microM, respectively. These findings suggest that E-resveratrol and (+)-ampelopsin A may function as attractive new candidates for protecting against brain cell dysfunction in vivo in AD by inhibiting the aggregation of Abeta.
Assuntos
Centrifugação/métodos , Cromatografia Líquida/métodos , Caules de Planta/química , Estilbenos/isolamento & purificação , Vitis/química , Extratos Vegetais/química , Extratos Vegetais/isolamento & purificação , Estilbenos/químicaRESUMO
Dietary polyphenols have received attention for their anti-oxidative, anti-carcinogenic and anti-neurodegenerative effects. Polyphenols bind to proteins leading to the formation of soluble or insoluble protein-polyphenol complexes which could significantly influence their biological activities. NMR and molecular modeling studies were performed to investigate the influence of the bulk, flexibility and hydrophobicity of polyphenols on the association with bradykinin, the peptide model. Our results show that the strength of the interactions could be positively correlated with polyphenol hydrophobicity and a comparison between pentagalloylglucose and vescalagin indicated that flexibility might play a positive role in the interaction with peptides and proteins.
Assuntos
Bradicinina/química , Bradicinina/metabolismo , Flavonoides/química , Flavonoides/metabolismo , Fenóis/química , Fenóis/metabolismo , Compostos de Bifenilo/química , Catecóis/química , Taninos Hidrolisáveis/química , Interações Hidrofóbicas e Hidrofílicas , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Maleabilidade , Polifenóis , Conformação Proteica , PrótonsRESUMO
Polyphenols are known for their impact on health and one of their major properties is the formation of complexes with proteins. To investigate the involvement of polyphenol-protein complexes in health, the interactions between bioactive polyphenols and neurotensin were examined by structural NMR and molecular modeling. Neurotensin is a linear bioactive tridecapeptide and polyphenols seem to affect the NT metabolism. We studied the polyphenols resveratrol and its glucoside the piceid in order to observe the possible role of glucose group and the penta-O-galloyl-D-glucopyranose (PGG). NMR data and molecular modeling showed that interaction occurred with the three polyphenols involving hydrophobic stacking and hydrogen bonds. Moreover, the peptide primary sequence plays a role in the specificity of complex formation.
Assuntos
Flavonoides/química , Neurotensina/química , Fenóis/química , Sequência de Aminoácidos , Glucosídeos/química , Taninos Hidrolisáveis/química , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Peptídeos/química , Polifenóis , Proteínas/química , Prótons , Resveratrol , Estilbenos/químicaRESUMO
Bradykinin is a bioactive hormone involved in a variety of physiological processes. In various solvents, this peptide adopts beta-turn structures. The C-terminal turn is a structural feature for the receptor affinity of agonists and antagonists while the N-terminal turn might be important for antagonistic activities. Polyphenols like dimeric proanthocyanidin B3 interact with the peptide. Thus to investigate the effects of polyphenols on bradykinin activity and structure, we studied the interaction in the structuring solvent DMSO which can be a close mimic of aqueous physiological environments like receptor-binding sites. Bradykinin alone presented a folded structure with two turns. B3 interacted with the peptide C-terminus and involved the loss of the bend structure of this region, while the N-terminus turn was maintained. Numerous studies have shown that polyphenolic molecules can act upon various biological targets, and the formation of this type of complex might be one of the possible modes of action.
Assuntos
Bradicinina/química , Flavonoides/química , Fenóis/química , Antocianinas/química , Sítios de Ligação , Dimerização , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Peptídeos/química , Polifenóis , Dobramento de Proteína , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , TemperaturaRESUMO
The solution structure of a new B-chain mutant of bovine insulin, in which the cysteines B7 and B19 are replaced by two serines, has been determined by circular dichroism, 2D-NMR and molecular modeling. This structure is compared with that of the oxidized B-chain of bovine insulin [Hawkins et al. (1995) Int. J. Peptide Protein Res.46, 424-433]. Circular dichroism spectroscopy showed in particular that a higher percentage of helical secondary structure for the B-chain mutant is estimated in trifluoroethanol solution in comparison with the oxidized B-chain. 2D-NMR experiments confirmed, among multiple conformations, that the B-chain mutant presents defined secondary structures such as a alpha-helix between residues B9 and B19, and a beta-turn between amino acids B20 and B23 in aqueous trifluoroethanol. The 3D structures, which are consistent with NMR data and were obtained using a simulated annealing protocol, showed that the tertiary structure of the B-chain mutant is better resolved and is more in agreement with the insulin crystal structure than the oxidized B-chain structure described by Hawkins et al. An explanation could be the presence of two sulfonate groups in the oxidized insulin B-chain. Either by their charges and/or their size, such chemical groups could play a destructuring effect and thus could favor peptide flexibility and conformational averaging. Thus, this study provides new insights on the folding of isolated B-chains.
Assuntos
Insulina/genética , Mutação , Estrutura Secundária de Proteína , Ácidos Sulfônicos/química , Sequência de Aminoácidos , Animais , Bovinos , Dicroísmo Circular , Insulina/síntese química , Insulina/química , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Dados de Sequência Molecular , Dobramento de Proteína , Estrutura Terciária de ProteínaRESUMO
Polyphenols from the ethyl acetate extracts of red wine were successfully fractionated using a four-step process (solvent extraction, ion-exchange column chromatography, centrifugal partition chromatography, and semipreparative HPLC), which resulted in the isolation of 22 compounds belonging to different classes of polyphenols (stilbenes, cinnamic acids, flavonoids). Five of them are red wine constituents reported for the first time. The newly isolated compounds include resveratrol dimers, dihydroflavonols, and a cinnamic derivative.
Assuntos
Flavonoides , Fenóis/isolamento & purificação , Polímeros/isolamento & purificação , Vinho/análise , Cromatografia/métodos , França , Modelos Moleculares , Conformação Molecular , Fenóis/química , Polímeros/química , Polifenóis , SolventesRESUMO
Epidemiological studies have shown that the incidence of some cardiovascular degenerative diseases appears to be lower in populations with regular but moderate drinking of red wine rich in polyphenols. One of the most important properties of polyphenols is to form complexes with proteins. The linear nonapeptide hormone bradykinin (H-Arg1-Pro2-Pro3-Gly4-Phe5-Ser6-Pro7-Phe8-Arg9-OH) is involved in a variety of physiological processes such as the cardiovascular processes. Thus, the goal of this work was to study the effects of tannins on the peptide structure by NMR investigations and molecular modeling. The results of these investigations show that in the presence of catechin, the peptide conformation is not affected and is in a random coil structure. On the contrary, the peptide structure is modified by the addition of dimeric proanthocyanidin B3 (catechin 4alpha-->8 catechin). The dimer leads to the formation of a large flexible turn between the 6-9 residues. Thus, the biological activities of bradykinin in the presence of polyphenols could be affected.
Assuntos
Bradicinina/química , Flavonoides , Modelos Moleculares , Fenóis/química , Polímeros/química , Proantocianidinas , Antocianinas/química , Bradicinina/metabolismo , Catequina/química , Simulação por Computador , Taninos Hidrolisáveis/química , Taninos Hidrolisáveis/metabolismo , Espectroscopia de Ressonância Magnética , Fenóis/metabolismo , Polímeros/metabolismo , Prolina/química , Conformação ProteicaRESUMO
Nitrogen metabolism was monitored in suspension cultured cells of Nicotiana plumbaginifolia Viv. using nuclear magnetic resonance (NMR) spectroscopy following the feeding of (15NH4)2SO4 and K15NO3. By using two-dimensional 15N-1H NMR with heteronuclear single-quantum-coherence spectroscopy and heteronuclear multiple-bond-coherence spectroscopy sequences, an enhanced resolution of the incorporation of 15N label into a range of compounds could be detected. Thus, in addition to the amino acids normally observed in one-dimensional 15N NMR (glutamine, aspartate, alanine), several other amino acids could be resolved, notably serine, glycine and proline. Furthermore, it was found that the peak normally assigned to the non-protein amino-acid gamma-aminobutyric acid in the one-dimensional 15N NMR spectrum was resolved into a several components. A peak of N-acetylated compounds was resolved, probably composed of the intermediates in arginine biosynthesis, N-acetylglutamate and N-acetylornithine and, possibly, the intermediate of putrescine degradation into gamma-aminobutyric acid, N-acetylputrescine. The occurrence of 15N-label in agmatine and the low detection of labelled putrescine indicate that crucial intermediates of the pathway from glutamate to polyamines and/or the tobacco alkaloids could be monitored. For the first time, labelling of the peptide glutathione and of the nucleotide uridine could be seen.
Assuntos
Aminoácidos/metabolismo , Nicotiana/metabolismo , Plantas Tóxicas , Sulfato de Amônio/metabolismo , Células Cultivadas , Espectroscopia de Ressonância Magnética , Nitratos/metabolismo , Isótopos de Nitrogênio , Compostos de Potássio/metabolismo , Ácido gama-Aminobutírico/metabolismoRESUMO
The metabolism of labelled pyruvate followed by 13C NMR and the measure of glutamine synthetase (GS) showed, according to previous results, a high activity of this enzyme in suspension cells of Nicotiana plumbaginifolia. This activity could derive glutamate from the alkaloid synthesizing pathways. However, a recent work showed that the rate of the GS gene transcription was inversely proportional to the Gln/Glu ratio. The measures of Gln and Glu concentrations in Nicotiana plumbaginifolia cells revealed that high GS activity correlates with the weak value of Gln/Glu ratio. Therefore, the hypothesis of GS dysfunctioning for the non-biosynthesis of alkaloids in N. plumbaginifolia suspension cells can be discarded. This conclusion is strengthened by the results obtained when using a GS inhibitor.
Assuntos
Alcaloides/biossíntese , Datura stramonium/metabolismo , Glutamato-Amônia Ligase/metabolismo , Ácido Glutâmico/biossíntese , Glutamina/biossíntese , Nicotiana/metabolismo , Proteínas de Plantas/metabolismo , Plantas Medicinais , Plantas Tóxicas , Ácido gama-Aminobutírico/biossíntese , Células Cultivadas , Datura stramonium/genética , Indução Enzimática , Inibidores Enzimáticos/farmacologia , Regulação da Expressão Gênica de Plantas , Glutamato-Amônia Ligase/antagonistas & inibidores , Glutamato-Amônia Ligase/genética , Espectroscopia de Ressonância Magnética , Metionina Sulfoximina/farmacologia , Proteínas de Plantas/antagonistas & inibidores , Proteínas de Plantas/genética , Especificidade da Espécie , Suspensões , Nicotiana/genéticaRESUMO
In an attempt to explain the relationship between conformations of peptide substrates of thermolysin in natural form and the experimental enzymatic cleavages, five peptides of various length were studied in two solvents H2O and glycerol, which may mimic the catalytic environmental conditions. As NMR failed to define sufficiently rough constraints to ensure a convergence of a refinement process for such short and flexible peptides, the conformational space was first searched using the MCMM method. The generated structures were then clustered in families using a 0.3A rmsd criterion and the derived structural characteristics were compared to the experimental NMR parameters. In a first approach, the NMR consistent conformations were compared with the structure of a thermolysin bound peptidic inhibitor ZG(P)LL to characterize the free-ligand predisposition to be cleaved. Further molecular dynamic calculations were performed at 300 K on the conformations corresponding to families in agreement with the ZG(P)LL structure in order to obtain information on their stability and on the trajectories of the torsion angles involved in the active site recognition. In conclusion, for four studied peptides, some conformations were found to be in agreement with 5 of the 8 cleavages experimentally observed.
Assuntos
Glicerol/química , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Peptídeos/química , Termolisina/química , Água/química , Cinética , Conformação Proteica , Temperatura , Fatores de TempoRESUMO
Genetic hemochromatosis (GH) is believed to be a disease restricted to those of European ancestry. In northwestern Europe, >80% of GH patients are homozygous for one mutation, the substitution of tyrosine for cysteine at position 282 (C282Y) in the unprocessed protein. In a proportion of GH patients, two mutations are present, C282Y and H63D. The clinical significance of this second mutation is such that it appears to predispose 1%-2% of compound heterozygotes to expression of the disease. The distribution of the two mutations differ, C282Y being limited to those of northwestern European ancestry and H63D being found at allele frequencies>5%, in Europe, in countries bordering the Mediterranean, in the Middle East, and in the Indian subcontinent. The C282Y mutation occurs on a haplotype that extends =6 Mb, suggesting that this mutation has arisen during the past 2,000 years. The H63D mutation is older and does not occur on such a large extended haplotype, the haplotype in this case extending =700 kb. Here we report the finding of the H63D and C282Y mutations on new haplotypes. In Sri Lanka we have found H63D on three new haplotypes and have found C282Y on one new haplotype, demonstrating that these mutations have arisen independently on this island. These results suggest that the HFE gene has been the subject of selection pressure. These selection pressures could be due to infectious diseases, environmental conditions, or other genetic disorders such as anemia.
Assuntos
Testes Genéticos , Antígenos HLA/genética , Hemocromatose/genética , Antígenos de Histocompatibilidade Classe I/genética , Proteínas de Membrana , Mutação/genética , Alelos , Substituição de Aminoácidos/genética , Sudeste Asiático/etnologia , Feminino , França , Frequência do Gene/genética , Genótipo , Haplótipos/genética , Proteína da Hemocromatose , Humanos , Masculino , Mianmar/etnologia , Linhagem , Filogenia , Polimorfismo Genético/genética , Seleção Genética , Sri LankaRESUMO
Glycerol, employed to mimic biological media with restricted water activity, has been shown to modify the activity of subtilisin BPN', an endopeptidase, towards the oxidized insulin B-chain, a well-studied substrate (FEBS Lett., 279 (1991) 123-131). Without minimizing the role of the microenvironment on the enzyme, we have studied the effect of glycerol addition on the structure of the enzyme substrate by homonuclear NMR spectroscopy and simulated annealing. Our results show that, in water, the oxidized insulin B-chain tertiary structure loses its central helix (residues B9-B19) and presents a folded structure with a flexible turn (residues B18-B24) in the beta-turn region of the insulin B-chain; whereas, in glycerol, the peptide is more rigid and is not folded. Moreover, in our experimental conditions, glycerol favors beta-strand secondary structure formation. Following these results, hypotheses about the differences observed in enzymatic activity on this substrate in glycerol have been postulated.
Assuntos
Glicerol/química , Insulina/química , Subtilisinas/química , Sequência de Aminoácidos , Espectroscopia de Ressonância Magnética , Dados de Sequência Molecular , Oxirredução , Conformação Proteica , Estrutura Secundária de Proteína , Relação Estrutura-Atividade , Água/químicaAssuntos
Bacillus/enzimologia , Glicerol/farmacologia , Conformação Proteica , Termolisina/química , Termolisina/metabolismo , Sítios de Ligação , Meios de Cultura , Modelos Moleculares , Método de Monte Carlo , Ressonância Magnética Nuclear Biomolecular , Oligopeptídeos/química , Oligopeptídeos/metabolismo , Especificidade por SubstratoRESUMO
Synthesis and use of various substrates permit an improved approach to thermolysin-peptide recognition and elucidation of several new criteria affecting enzyme specificity. Nature and position of the recognized residue, role of adjacent amino acids, lateral chain hydrophobicity, and volume and length of peptides were all considered. Hydrolysis reactions were also carried out in the presence of glycerol; the effect of microenvironment modifications was quantitative, for example in inducing variations in catalytic reaction rates, and also qualitative, such as in influencing affinity.
Assuntos
Termolisina/metabolismo , Soluções Tampão , Relação Dose-Resposta a Droga , Glicerol/farmacologia , Hidrólise , Cinética , Oligopeptídeos/síntese química , Oligopeptídeos/metabolismo , Especificidade por Substrato , Termolisina/efeitos dos fármacosRESUMO
We studied the action of urea on the spin-spin relaxation rate of 2,3-diphosphoglycerate (2,3-DPG) phosphorus atoms in normal and uremic erythrocytes. At concentrations from 10 to 60 mM, urea increased the relaxation rates of 2,3-DPG P-3 phosphorus atoms. This evidenced a stronger binding of 2,3-DPG to hemoglobin (Hb), suggesting that the deoxyform of Hb was stabilized. This hypothesis was confirmed by measurements of the association constant of oxygen to hemoglobin (K) in normal erythrocytes in presence of urea concentrations in the range of those observed in uremic patients (30 mM). Indeed, the observed decrease in K suggests that the T structure of hemoglobin is stabilized. By contrast, with higher urea concentrations (120 mM), measurements of P50 showed an increase in the hemoglobin affinity for oxygen (decrease in P50). Moreover, the relaxation rates of 2,3-DPG P-3 phosphorus atoms were not modified, which is consistent with the simultaneous increase of K. This may be attributed to the formation of carbamylated hemoglobin in presence of urea. These results suggest two opposite effects of urea on Hb-O2 affinity: the first reinforces 2,3-DPG-Hb binding and leads to a decrease in O2 affinity; the second, mediated by carbamylation of Hb, hinders the binding of 2,3-DPG and increases the O2 affinity. These findings are consistent with the fact that, despite the presence of carbamylated hemoglobin, uremic patients do not present increased Hb-O2 affinity.
Assuntos
Anemia/sangue , Hemoglobinas/metabolismo , Falência Renal Crônica/sangue , Oxigênio/metabolismo , Ureia/farmacologia , 2,3-Difosfoglicerato , Ácidos Difosfoglicéricos/metabolismo , HumanosRESUMO
Beyond their classical action on calcium channels, some calcium channel blockers also exhibit a calcium anti-ionophoretic effect. We studied this effect on respiratory control and Ca2+ fluxes in a mitochondrial model to compare calcium antagonists chosen among three clinical classes: vascular, cardiac, and mixed effects. Synthetic calcium ionophore A23187 decreases respiratory control and modifies Ca2+ fluxes. We show that calcium antagonists partially restore the parameters altered by A23187. By calculating the percentage of restoration, we found that vascular drugs exhibit a strong anti-ionophoretic effect, cardiac drugs exert no significant effect, and mixed calcium antagonists exert an intermediate effect. Thus, it appears possible to link the intensity of calcium anti-ionophoretic effect with the clinical interest of a calcium antagonist.
Assuntos
Bloqueadores dos Canais de Cálcio/farmacologia , Cálcio/metabolismo , Mitocôndrias Hepáticas/metabolismo , Animais , Calcimicina/farmacologia , Técnicas In Vitro , Iontoforese , Mitocôndrias Hepáticas/efeitos dos fármacos , Oxirredução , Consumo de Oxigênio/efeitos dos fármacos , RatosRESUMO
Using 31P-nuclear magnetic resonance (NMR) measurements of relaxation rate for 2,3 diphosphoglycerate (DPG) phosphorus atoms, we showed previously that in uraemic red blood cells the DPG-haemoglobin binding is stronger, thus stabilizing the deoxyhaemoglobin form and hence facilitating oxygen release. Here we verified if these modifications of spatial environment of DPG remain in uraemic patients treated by human recombinant erythropoietin (rHuEpo). Simultaneously we measured the intraerythrocytic ATP concentration (ATPi) and pH (pHi) of patients. Our results show a slight decrease on pHi and ATPi values during rHuEpo treatment. For the DPG relaxation rates, we observed a very weak but statistically significant increase 6 months after the beginning of treatment, but we cannot attribute a physiopathological significance to these results because of the lack of accuracy of the NMR determination of relaxation rate in red blood cells. Therefore, the DPG-haemoglobin binding is always stronger than in normal subjects.
Assuntos
Anemia/tratamento farmacológico , Ácidos Difosfoglicéricos/sangue , Eritropoetina/uso terapêutico , Hemoglobinas/metabolismo , Uremia/sangue , 2,3-Difosfoglicerato , Trifosfato de Adenosina/sangue , Adulto , Idoso , Anemia/sangue , Anemia/etiologia , Eritrócitos/metabolismo , Feminino , Humanos , Espectroscopia de Ressonância Magnética , Masculino , Pessoa de Meia-Idade , Ligação Proteica , Proteínas Recombinantes/uso terapêuticoRESUMO
The middle-molecular-weight uremic toxins which accumulate in uremic plasma seem to be associated with various uremic disorders such as uremic neuropathy and defects in the sodium pump. By a multi-step chromatographic method, two fractions of these toxins were isolated and studied because one inhibits microtubule formation in vitro (fraction 2-5), and the other impairs the sodium pump in living erythrocytes (fraction 2-3). An additional chromatographic method allows the separation of these fractions and isolation of two components: fractions 2-3-V and 2-5-III. Analyses by UV and 1H NMR spectrometry identified these compounds as two different ascorbic acid derivatives. 2-3-V is not yet totally identified and 2-5-III corresponds to ascorbic acid 2-sulfate. These two metabolites exert no toxic effects but they have the same chromatographic behavior as uremic toxins.