RESUMO
Purpose: This work investigates the effect of resveratrol and melatonin on structural and functional changes of two enzymes, lactate dehydrogenase (LDH) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), exposed to radiation-induced reactive oxygen species.Materials and methods: Solutions of dehydrogenases with or without antioxidants (resveratrol or melatonin) were irradiated with X-rays under the atmosphere of air and at room temperature (21 ± 2 °C). In order to determine the protective effect of melatonin and resveratrol in radiation-induced damage to GAPDH and LDH spectroscopy and HPLC methods were used. Furthermore, plausible binding sites of melatonin or resveratrol to the GAPDH or LDH molecule were analysed.Results and conclusions: Resveratrol shows better protective properties in the inactivation of GAPDH when compared to melatonin. LDH does not contain âSH groups in its active site, and is not inactivated by water radiolysis products other than hydroxyl radicals or the secondary radicals of the studied low-molecular-weight compounds. Resveratrol and melatonin protected the structure of LDH to a greater extent than GAPDH. This difference can be attributed to the fact that LDH potentially binds more resveratrol or melatonin molecules (27 binding sites for resveratrol and 40 for melatonin) than GAPDH (10 binding sites for resveratrol and 18 for melatonin).
Assuntos
Gliceraldeído-3-Fosfato Desidrogenases/metabolismo , L-Lactato Desidrogenase/metabolismo , Melatonina/administração & dosagem , Proteção Radiológica/métodos , Resveratrol/administração & dosagem , Ar , Animais , Antioxidantes/química , Domínio Catalítico , Cisteína/química , Relação Dose-Resposta à Radiação , Concentração de Íons de Hidrogênio , Cinética , Modelos Moleculares , Coelhos , Espécies Reativas de Oxigênio , Temperatura , Raios XAssuntos
Gliceraldeído-3-Fosfato Desidrogenases/metabolismo , L-Lactato Desidrogenase/metabolismo , Melatonina/farmacologia , Óxido Nítrico/farmacologia , Estilbenos/farmacologia , Animais , Antioxidantes/farmacologia , Ditiotreitol/farmacologia , Ativação Enzimática/efeitos dos fármacos , Glutationa/farmacologia , Gliceraldeído-3-Fosfato Desidrogenases/química , Glicólise , Cinética , L-Lactato Desidrogenase/química , Óxido Nítrico/metabolismo , Oxirredução , Coelhos , ResveratrolRESUMO
The presented study analyzed potential binding sites of 3,5,4'-trihydroxystilbene (resveratrol, RSV) and its derivative, trans-3,3',5,5'-tetrahydroxy-4'-methoxystilbene (THMS) to glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The effects of stilbene analogs on the structure of GAPDH were determined by fluorescence spectroscopy and ζ potential measurements. To what extent the studied compounds affect the activity of the enzyme was also assessed. A computational ligand-docking study showed that there are 11 potential binding sites of RSV and 8 such sites of THMS in the GAPDH molecule. While resveratrol does not significantly affect the activity of the dehydrogenase upon binding to it, THMS leads to approximately 10% inactivation of this enzyme. THMS has no effect on GAPDH inactivation induced by the superoxide anion radical, in contrast to resveratrol, which increases dehydrogenase inactivation.
Assuntos
Gliceraldeído-3-Fosfato Desidrogenases/química , Gliceraldeído-3-Fosfato Desidrogenases/metabolismo , Simulação de Acoplamento Molecular , Estilbenos/metabolismo , Estilbenos/farmacologia , Antioxidantes/metabolismo , Antioxidantes/farmacologia , Domínio Catalítico/efeitos dos fármacos , Ligantes , Polifenóis/metabolismo , Polifenóis/farmacologia , Resveratrol , Superóxidos/farmacologia , TriptofanoRESUMO
Cellulose is a linear extracellular polysaccharide. It is synthesized by membrane-embedded glycosyltransferases that processively polymerize UDP-activated glucose. Polymer synthesis is coupled to membrane translocation through a channel formed by the cellulose synthase. Although eukaryotic cellulose synthases function in macromolecular complexes containing several different enzyme isoforms, prokaryotic synthases associate with additional subunits to bridge the periplasm and the outer membrane. In bacteria, cellulose synthesis and translocation is catalyzed by the inner membrane-associated bacterial cellulose synthase (Bcs)A and BcsB subunits. Similar to alginate and poly-ß-1,6 N-acetylglucosamine, bacterial cellulose is implicated in the formation of sessile bacterial communities, termed biofilms, and its synthesis is likewise stimulated by cyclic-di-GMP. Biochemical studies of exopolysaccharide synthesis are hampered by difficulties in purifying and reconstituting functional enzymes. We demonstrate robust in vitro cellulose synthesis reconstituted from purified BcsA and BcsB proteins from Rhodobacter sphaeroides. Although BcsA is the catalytically active subunit, the membrane-anchored BcsB subunit is essential for catalysis. The purified BcsA-B complex produces cellulose chains of a degree of polymerization in the range 200-300. Catalytic activity critically depends on the presence of the allosteric activator cyclic-di-GMP, but is independent of lipid-linked reactants. Our data reveal feedback inhibition of cellulose synthase by UDP but not by the accumulating cellulose polymer and highlight the strict substrate specificity of cellulose synthase for UDP-glucose. A truncation analysis of BcsB localizes the region required for activity of BcsA within its C-terminal membrane-associated domain. The reconstituted reaction provides a foundation for the synthesis of biofilm exopolysaccharides, as well as its activation by cyclic-di-GMP.
Assuntos
Biofilmes , Celulose/biossíntese , Glucosiltransferases/metabolismo , Rhodobacter sphaeroides/enzimologia , Transporte Biológico/fisiologia , Western Blotting , Eletroforese em Gel de Poliacrilamida , Escherichia coli , Cromatografia Gasosa-Espectrometria de Massas , Subunidades Proteicas/metabolismoRESUMO
Cellulose, the most abundant biological macromolecule, is an extracellular, linear polymer of glucose molecules. It represents an essential component of plant cell walls but is also found in algae and bacteria. In bacteria, cellulose production frequently correlates with the formation of biofilms, a sessile, multicellular growth form. Cellulose synthesis and transport across the inner bacterial membrane is mediated by a complex of the membrane-integrated catalytic BcsA subunit and the membrane-anchored, periplasmic BcsB protein. Here we present the crystal structure of a complex of BcsA and BcsB from Rhodobacter sphaeroides containing a translocating polysaccharide. The structure of the BcsA-BcsB translocation intermediate reveals the architecture of the cellulose synthase, demonstrates how BcsA forms a cellulose-conducting channel, and suggests a model for the coupling of cellulose synthesis and translocation in which the nascent polysaccharide is extended by one glucose molecule at a time.