RESUMO
The oxidative phase of the pentose phosphate pathway (PPP) involving the enzymes glucose-6-phosphate dehydrogenase (G6PDH), 6-phosphogluconolactonase (6PGL), and 6-phosphogluconate dehydrogenase (6PGDH), is critical to NADPH generation within cells, with these enzymes catalyzing the conversion of glucose-6-phosphate (G6P) into ribulose-5-phosphate (Ribu5-P). We have previously studied peroxyl radical (ROOâ¢) mediated oxidative inactivation of E. coli G6PDH, 6PGL, and 6PGDH. However, these data were obtained from experiments where each enzyme was independently exposed to ROOâ¢, a condition not reflecting biological reality. In this work we investigated how NADPH production is modulated when these enzymes are jointly exposed to ROOâ¢. Enzyme mixtures (1:1:1 ratio) were exposed to ROO⢠produced from thermolysis of 100 mM 2,2'-azobis(2-methylpropionamidine) dihydrochloride (AAPH). NADPH was quantified at 340 nm, and protein oxidation analyzed by liquid chromatography with mass spectrometric detection (LC-MS). The data obtained were rationalized using a mathematical model. The mixture of non-oxidized enzymes, G6P and NADP+ generated â¼175 µM NADPH. Computational simulations showed a constant decrease of G6P associated with NADPH formation, consistent with experimental data. When the enzyme mixture was exposed to AAPH (3 h, 37 °C), lower levels of NADPH were detected (â¼100 µM) which also fitted with computational simulations. LC-MS analyses indicated modifications at Tyr, Trp, and Met residues but at lower concentrations than detected for the isolated enzymes. Quantification of NADPH generation showed that the pathway activity was not altered during the initial stages of the oxidations, consistent with a buffering role of G6PDH towards inactivation of the oxidative phase of the pathway.
Assuntos
Escherichia coli , Glucosefosfato Desidrogenase , NADP , Oxirredução , Via de Pentose Fosfato , Fosfogluconato Desidrogenase , Glucosefosfato Desidrogenase/metabolismo , Fosfogluconato Desidrogenase/metabolismo , NADP/metabolismo , Escherichia coli/metabolismo , Escherichia coli/genética , Ribulosefosfatos/metabolismo , Glucose-6-Fosfato/metabolismo , Peróxidos/metabolismo , Hidrolases de Éster CarboxílicoRESUMO
Activation of phosphoenolpyruvate carboxylase (PEPC) enzymes by glucose 6-phosphate (G6P) and other phospho-sugars is of major physiological relevance. Previous kinetic, site-directed mutagenesis and crystallographic results are consistent with allosteric activation, but the existence of a G6P-allosteric site was questioned and competitive activation-in which G6P would bind to the active site eliciting the same positive homotropic effect as the substrate phosphoenolpyruvate (PEP)-was proposed. Here, we report the crystal structure of the PEPC-C4 isozyme from Zea mays with G6P well bound into the previously proposed allosteric site, unambiguously confirming its existence. To test its functionality, Asp239-which participates in a web of interactions of the protein with G6P-was changed to alanine. The D239A variant was not activated by G6P but, on the contrary, inhibited. Inhibition was also observed in the wild-type enzyme at concentrations of G6P higher than those producing activation, and probably arises from G6P binding to the active site in competition with PEP. The lower activity and cooperativity for the substrate PEP, lower activation by glycine and diminished response to malate of the D239A variant suggest that the heterotropic allosteric activation effects of free-PEP are also abolished in this variant. Together, our findings are consistent with both the existence of the G6P-allosteric site and its essentiality for the activation of PEPC enzymes by phosphorylated compounds. Furthermore, our findings suggest a central role of the G6P-allosteric site in the overall kinetics of these enzymes even in the absence of G6P or other phospho-sugars, because of its involvement in activation by free-PEP.
Assuntos
Glucose-6-Fosfato/química , Fosfoenolpiruvato Carboxilase/química , Fosfoenolpiruvato/química , Proteínas de Plantas/química , Zea mays/enzimologia , Regulação Alostérica , Domínio Catalítico , Glucose-6-Fosfato/metabolismo , Cinética , Fosfoenolpiruvato/metabolismo , Fosfoenolpiruvato Carboxilase/genética , Fosfoenolpiruvato Carboxilase/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Zea mays/genéticaRESUMO
Diabetic retinopathy is thought to be trigger by glucose- induced oxidative stress which leads to an increase of the mitochondrial permeability through opening the permeability transition pore (MTP). In several cell types, hexokinases interact with the mitochondria regulating MTP opening, avoiding cytochrome c release. We studied HK I mitochondrial proportion in control and streptozotocin-induced diabetic rat retinas. In the normal retina, 50% of HK I was linked to mitochondria, proportion that did not change up to 60 days of diabetes. Mitochondria from normal and diabetic rat retinas showed a limited swelling, and similar cytochrome c levels. G-6-P and glycogen content increased 3-6-fold in diabetic rat retinas, while lactate content did not vary. Results suggest that mitochondrial bound HK produce G-6-P and drove it to glycogen synthesis, controlling ROS production and lactate toxicity.
Assuntos
Diabetes Mellitus/induzido quimicamente , Retinopatia Diabética/metabolismo , Hexoquinase/metabolismo , Retina/metabolismo , Animais , Citocromos c/metabolismo , Diabetes Mellitus/metabolismo , Modelos Animais de Doenças , Feminino , Glucose-6-Fosfato/metabolismo , Mitocôndrias/metabolismo , Ratos , EstreptozocinaRESUMO
In general, eukaryotic glucose-6-phosphate dehydrogenases (G6PDHs) are structurally stabilized by NADP+. Here we show by spectrofluorometric analysis, thermal and urea denaturation, and trypsin proteolysis, that a different mechanism stabilizes the enzyme from Pseudomonas aeruginosa (PaG6PDH) (EC 1.1.1.363). The spectrofluorometric analysis of the emission of 8-anilino-1-naphthalenesulfonic acid (ANS) indicates that this stabilization is the result of a structural change in the enzyme caused by G6P. The similarity between the Kd values determined for the PaG6PDH-G6P complex (78.0⯱â¯7.9⯵M) and the K0.5 values determined for G6P (57.9⯱â¯2.5 and 104.5⯱â¯9.3⯵M in the NADP+- and NAD+-dependent reactions, respectively) suggests that the structural changes are the result of G6P binding to the active site of PaG6PDH. Modeling of PaG6PDH indicated the residues that potentially bind the ligand. These results and a phylogenetic analysis of the amino acid sequences of forty-four G6PDHs, suggest that the stabilization observed for PaG6PDH could be a characteristic that distinguishes this and other G6PDHs that use NAD+ and NADP+ from those that use NADP+ only or preferentially, such as those found in eukaryotes. This characteristic could be related to the metabolic roles these enzymes play in the organisms to which they belong.
Assuntos
Glucosefosfato Desidrogenase/metabolismo , Pseudomonas aeruginosa/enzimologia , Sequência de Aminoácidos , Naftalenossulfonato de Anilina/química , Sítios de Ligação , Domínio Catalítico , Glucose-6-Fosfato/química , Glucose-6-Fosfato/metabolismo , Glucosefosfato Desidrogenase/classificação , Glucosefosfato Desidrogenase/genética , Cinética , Simulação de Dinâmica Molecular , NAD/metabolismo , NADP/química , NADP/metabolismo , Filogenia , Ligação Proteica , Desnaturação Proteica , Estabilidade Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificaçãoRESUMO
BACKGROUND: Efficient xylose fermentation still demands knowledge regarding xylose catabolism. In this study, metabolic flux analysis (MFA) and metabolomics were used to improve our understanding of xylose metabolism. Thus, a stoichiometric model was constructed to simulate the intracellular carbon flux and used to validate the metabolome data collected within xylose catabolic pathways of non-Saccharomyces xylose utilizing yeasts. RESULTS: A metabolic flux model was constructed using xylose fermentation data from yeasts Scheffersomyces stipitis, Spathaspora arborariae, and Spathaspora passalidarum. In total, 39 intracellular metabolic reactions rates were utilized validating the measurements of 11 intracellular metabolites, acquired by mass spectrometry. Among them, 80% of total metabolites were confirmed with a correlation above 90% when compared to the stoichiometric model. Among the intracellular metabolites, fructose-6-phosphate, glucose-6-phosphate, ribulose-5-phosphate, and malate are validated in the three studied yeasts. However, the metabolites phosphoenolpyruvate and pyruvate could not be confirmed in any yeast. Finally, the three yeasts had the metabolic fluxes from xylose to ethanol compared. Xylose catabolism occurs at twice-higher flux rates in S. stipitis than S. passalidarum and S. arborariae. Besides, S. passalidarum present 1.5 times high flux rate in the xylose reductase reaction NADH-dependent than other two yeasts. CONCLUSIONS: This study demonstrated a novel strategy for metabolome data validation and brought insights about naturally xylose-fermenting yeasts. S. stipitis and S. passalidarum showed respectively three and twice higher flux rates of XR with NADH cofactor, reducing the xylitol production when compared to S. arborariae. Besides then, the higher flux rates directed to pentose phosphate pathway (PPP) and glycolysis pathways resulted in better ethanol production in S. stipitis and S. passalidarum when compared to S. arborariae.
Assuntos
Fermentação , Análise do Fluxo Metabólico/métodos , Metaboloma , Metabolômica/métodos , Saccharomycetales/metabolismo , Frutosefosfatos/metabolismo , Glucose-6-Fosfato/metabolismo , Glicólise , Malatos/metabolismo , Espectrometria de Massas/métodos , Modelos Biológicos , Via de Pentose Fosfato , Ribulosefosfatos/metabolismo , Saccharomycetales/classificação , Leveduras/classificação , Leveduras/metabolismoRESUMO
Glucose-6-phosphate dehydrogenase (G6PDH) is the key enzyme supplying reducing power (NADPH) to the cells, by oxidation of glucose-6-phosphate (G6P), and in the process providing a precursor of ribose-5-phosphate. G6PDH is also a virulence factor of pathogenic trypanosomatid parasites. To uncover the biochemical and structural features that distinguish TcG6PDH from its human homolog, we have solved and analyzed the crystal structures of the G6PDH from Trypanosoma cruzi (TcG6PDH), alone and in complex with G6P. TcG6PDH crystallized as a tetramer and enzymatic assays further indicated that the tetramer is the active form in the parasite, in contrast to human G6PDH, which displays higher activity as a dimer. This quaternary structure was shown to be particularly stable. The molecular reasons behind this disparity were unveiled by structural analyses: a TcG6PDH-specific residue, R323, is located at the dimer-dimer interface, critically contributing with two salt bridges per subunit that are absent in the human enzyme. This explains why TcG6PDH dimerization impaired enzyme activity. The parasite protein is also distinct in displaying a 37-amino-acid extension at the N-terminus, which comprises the non-conserved C8 and C34 involved in the covalent linkage of two neighboring protomers. In addition, a cysteine triad (C53, C94 and C135) specific of Kinetoplastid G6PDHs proved critical for stabilization of TcG6PDH active site. Based on the structural and biochemical data, we posit that the N-terminal region and the catalytic site are highly dynamic. The unique structural features of TcG6PDH pave the way toward the design of efficacious and highly specific anti-trypanosomal drugs.
Assuntos
Glucose-6-Fosfato/metabolismo , Glucosefosfato Desidrogenase/metabolismo , Proteínas de Protozoários/metabolismo , Trypanosoma cruzi/metabolismo , Doença de Chagas/parasitologia , Cristalografia por Raios X , Glucosefosfato Desidrogenase/química , Humanos , Modelos Moleculares , Conformação Proteica , Multimerização Proteica , Proteínas de Protozoários/química , Trypanosoma cruzi/químicaRESUMO
Optimization of cellulose enzymatic hydrolysis is crucial for cost-effective bioethanol production from lignocellulosic biomass. Enzyme immobilization in solid support allows enzyme recycling for reuse, lowering hydrolysis costs. Graphene is a nanomaterial isolated in 2004, which possesses exceptional properties for biomolecule immobilization. This study evaluates the potential for ß-glucosidase recycling by immobilization on graphene nanosheets. Data reported here demonstrated that graphene-immobilized ß-glucosidase can be recycled for at least eight cycles. Immobilization did not change the optimal temperature of catalysis and improved enzymatic stability upon storage. The role of glucose-6-phosphate on immobilized enzyme was also investigated, demonstrating that glucose-6-phosphate acts as a mixed-type activator and improves storage stability of immobilized enzyme. Complete cellulose hydrolysis using graphene-immobilized ß-glucosidase in the presence of glucose-6-phosphate resulted in greatly improved hydrolysis rates, demonstrating the potential of this strategy for biomass hydrolysis.
Assuntos
Bacillus/enzimologia , Celulose/metabolismo , Enzimas Imobilizadas/metabolismo , Glucose-6-Fosfato/metabolismo , Grafite/química , Nanoestruturas/química , beta-Glucosidase/metabolismo , Bacillus/química , Bacillus/metabolismo , Estabilidade Enzimática , Enzimas Imobilizadas/química , Hidrólise , beta-Glucosidase/químicaRESUMO
The enzyme glucose-6-phosphate dehydrogenase from Trypanosoma cruzi (TcG6PDH) catalyses the first step of the pentose phosphate pathway (PPP) and is considered a promising target for the discovery of a new drug against Chagas diseases. In the present work, we describe the crystal structure of TcG6PDH obtained in a ternary complex with the substrate ß-d-glucose-6-phosphate (G6P) and the reduced 'catalytic' cofactor NADPH, which reveals the molecular basis of substrate and cofactor recognition. A comparison with the homologous human protein sheds light on differences in the cofactor-binding site that might be explored towards the design of new NADP(+) competitive inhibitors targeting the parasite enzyme.
Assuntos
Doença de Chagas/tratamento farmacológico , Coenzimas/química , Glucosefosfato Desidrogenase/química , Conformação Proteica , Trypanosoma cruzi/enzimologia , Sequência de Aminoácidos/genética , Animais , Sítios de Ligação/efeitos dos fármacos , Doença de Chagas/genética , Coenzimas/metabolismo , Cristalografia por Raios X , Inibidores Enzimáticos/farmacologia , Glucose-6-Fosfato/metabolismo , Glucosefosfato Desidrogenase/genética , Glucosefosfato Desidrogenase/metabolismo , Humanos , NADP/metabolismo , Via de Pentose Fosfato/genética , Especificidade por Substrato , Trypanosoma cruzi/patogenicidadeRESUMO
BACKGROUND: Mycobacterium tuberculosis is a pathogenic prokaryote adapted to survive in hostile environments. In this organism and other Gram-positive actinobacteria, the metabolic pathways of glycogen and trehalose are interconnected. RESULTS: In this work we show the production, purification and characterization of recombinant enzymes involved in the partitioning of glucose-1-phosphate between glycogen and trehalose in M. tuberculosis H37Rv, namely: ADP-glucose pyrophosphorylase, glycogen synthase, UDP-glucose pyrophosphorylase and trehalose-6-phosphate synthase. The substrate specificity, kinetic parameters and allosteric regulation of each enzyme were determined. ADP-glucose pyrophosphorylase was highly specific for ADP-glucose while trehalose-6-phosphate synthase used not only ADP-glucose but also UDP-glucose, albeit to a lesser extent. ADP-glucose pyrophosphorylase was allosterically activated primarily by phosphoenolpyruvate and glucose-6-phosphate, while the activity of trehalose-6-phosphate synthase was increased up to 2-fold by fructose-6-phosphate. None of the other two enzymes tested exhibited allosteric regulation. CONCLUSIONS: Results give information about how the glucose-1-phosphate/ADP-glucose node is controlled after kinetic and regulatory properties of key enzymes for mycobacteria metabolism. GENERAL SIGNIFICANCE: This work increases our understanding of oligo and polysaccharides metabolism in M. tuberculosis and reinforces the importance of the interconnection between glycogen and trehalose biosynthesis in this human pathogen.
Assuntos
Glucofosfatos/metabolismo , Glicogênio/biossíntese , Redes e Vias Metabólicas , Mycobacterium tuberculosis/metabolismo , Trealose/biossíntese , Regulação Alostérica , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Glucose-1-Fosfato Adenililtransferase/genética , Glucose-1-Fosfato Adenililtransferase/metabolismo , Glucose-6-Fosfato/metabolismo , Glucosiltransferases/genética , Glucosiltransferases/metabolismo , Glicogênio Sintase/genética , Glicogênio Sintase/metabolismo , Cinética , Modelos Biológicos , Mycobacterium tuberculosis/enzimologia , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , UTP-Glucose-1-Fosfato Uridililtransferase/genética , UTP-Glucose-1-Fosfato Uridililtransferase/metabolismoRESUMO
PURPOSE: The present study analyzes the effect of the replacement of dietary casein by soy protein on the mechanisms underlying dyslipidemia, liver steatosis and altered glucose and lipid metabolism in the skeletal muscle which developed in rats fed long-term a sucrose-rich diet (SRD). METHODS: Wistar rats were fed a SRD for 4 months. From months 4 to 8, half the animals continued with the SRD, and the other half were fed a SRD in which the source of protein casein was replaced by soy. The control group received a diet with cornstarch as source of carbohydrate. RESULTS: Compared to SRD-fed animals, the rats fed soy showed: A--in the liver: reduction of triglyceride and cholesterol storage and decreased steatosis; normalization of mature forms of the protein mass levels of SREBP-1 and the activities of lipogenic enzymes, while the protein mass level of PPAR-α and fatty acid oxidase activity increased. B-in the gastrocnemius muscle: normalization of the enhanced lipid storage and the altered glucose oxidation, improving glucose phosphorylation; decreasing protein mass level of nPKCθ in the membrane fraction; reversion of the impaired insulin-stimulated glucose transporter Glut-4, and glucose-6-phosphate and glycogen concentrations. Besides, dyslipidemia and glucose homeostasis returned to control values. CONCLUSIONS: This study provides new information concerning some key mechanisms related to the effect of dietary soy on hepatic lipid metabolism and insulin action in the skeletal muscle in the presence of pre-existing dyslipidemia and insulin resistance induced by a SRD.
Assuntos
Sacarose Alimentar/efeitos adversos , Dislipidemias/dietoterapia , Resistência à Insulina , Fígado/metabolismo , Músculo Esquelético/metabolismo , Proteínas de Soja/administração & dosagem , Animais , Glicemia/metabolismo , Colesterol/sangue , Sacarose Alimentar/administração & dosagem , Ácidos Graxos não Esterificados/sangue , Fígado Gorduroso/dietoterapia , Transportador de Glucose Tipo 4/metabolismo , Glucose-6-Fosfato/metabolismo , Glicogênio/metabolismo , Insulina/sangue , Metabolismo dos Lipídeos , Masculino , PPAR alfa/metabolismo , Ratos , Ratos Wistar , Proteína de Ligação a Elemento Regulador de Esterol 1/metabolismo , Triglicerídeos/sangue , Aumento de PesoRESUMO
Wolbachia pipientis, a maternally transmitted bacterium that colonizes arthropods, may affect the general aspects of insect physiology, particularly reproduction. Wolbachia is a natural endosymbiont of Aedes fluviatilis, whose effects in embryogenesis and reproduction have not been addressed so far. In this context, we investigated the correlation between glucose metabolism and morphological alterations during A. fluviatilis embryo development in Wolbachia-positive (W+) and Wolbachia-negative (W-) mosquito strains. While both strains do not display significant morphological and larval hatching differences, larger differences were observed in hexokinase activity and glycogen contents during early and mid-stages of embryogenesis, respectively. To investigate if glycogen would be required for parasite-host interaction, we reduced Glycogen Synthase Kinase-3 (GSK-3) levels in adult females and their eggs by RNAi. GSK-3 knock-down leads to embryonic lethality, lower levels of glycogen and total protein and Wolbachia reduction. Therefore, our results suggest that the relationship between A. fluviatilis and Wolbachia may be modulated by glycogen metabolism.
Assuntos
Aedes/embriologia , Aedes/microbiologia , Glicogênio/metabolismo , Interações Hospedeiro-Parasita/fisiologia , Simbiose/fisiologia , Wolbachia/fisiologia , Aedes/metabolismo , Animais , Embrião não Mamífero/microbiologia , Desenvolvimento Embrionário/fisiologia , Feminino , Glucose-6-Fosfato/metabolismo , Quinase 3 da Glicogênio Sintase/genética , Masculino , Filogenia , Wolbachia/metabolismoRESUMO
In Saccharomyces cerevisiae addition of glucose inhibits oxygen consumption, i.e. S. cerevisiae is Crabtree-positive. During active glycolysis hexoses-phosphate accumulate, and probably interact with mitochondria. In an effort to understand the mechanism underlying the Crabtree effect, the effect of two glycolysis-derived hexoses-phosphate was tested on the S. cerevisiae mitochondrial unspecific channel (ScMUC). Glucose-6-phosphate (G6P) promoted partial opening of ScMUC, which led to proton leakage and uncoupling which in turn resulted in, accelerated oxygen consumption. In contrast, fructose-1,6-bisphosphate (F1,6BP) closed ScMUC and thus inhibited the rate of oxygen consumption. When added together, F1,6BP reverted the mild G6P-induced effects. F1,6BP is proposed to be an important modulator of ScMUC, whose closure contributes to the "Crabtree effect".
Assuntos
Frutosedifosfatos/metabolismo , Glucose/metabolismo , Consumo de Oxigênio , Canais de Potássio/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Glucose-6-Fosfato/metabolismo , Glicólise , Ativação do Canal Iônico , Potencial da Membrana Mitocondrial , Dilatação MitocondrialRESUMO
The role of tertiary conformational changes associated to ligand binding was explored using the allosteric enzyme glucosamine-6-phosphate (GlcN6P) deaminase from Escherichia coli (EcGNPDA) as an experimental model. This is an enzyme of amino sugar catabolism that deaminates GlcN6P, giving fructose 6-phosphate and ammonia, and is allosterically activated by N-acetylglucosamine 6-phosphate (GlcNAc6P). We resorted to the nanoencapsulation of this enzyme in wet silica sol-gels for studying the role of intrasubunit local mobility in its allosteric activation under the suppression of quaternary transition. The gel-trapped enzyme lost its characteristic homotropic cooperativity while keeping its catalytic properties and the allosteric activation by GlcNAc6P. The nanoencapsulation keeps the enzyme in the T quaternary conformation, making possible the study of its allosteric activation under a condition that is not possible to attain in a soluble phase. The involved local transition was slowed down by nanoencapsulation, thus easing the fluorometric analysis of its relaxation kinetics, which revealed an induced-fit mechanism. The absence of cooperativity produced allosterically activated transitory states displaying velocity against substrate concentration curves with apparent negative cooperativity, due to the simultaneous presence of subunits with different substrate affinities. Reaction kinetics experiments performed at different tertiary conformational relaxation times also reveal the sequential nature of the allosteric activation. We assumed as a minimal model the existence of two tertiary states, t and r, of low and high affinity, respectively, for the substrate and the activator. By fitting the velocity-substrate curves as a linear combination of two hyperbolic functions with Kt and Kr as KM values, we obtained comparable values to those reported for the quaternary conformers in solution fitted to MWC model. These results are discussed in the background of the known crystallographic structures of T and R EcGNPDA conformers. These results are consistent with the postulates of the Tertiary Two-States (TTS) model.
Assuntos
Aldose-Cetose Isomerases/química , Proteínas de Escherichia coli/química , Conformação Proteica , Estrutura Terciária de Proteína , Aldose-Cetose Isomerases/genética , Aldose-Cetose Isomerases/metabolismo , Algoritmos , Regulação Alostérica , Sítio Alostérico , Sítios de Ligação , Biocatálise , Dicroísmo Circular , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Frutosefosfatos/metabolismo , Géis/química , Glucosamina/análogos & derivados , Glucosamina/metabolismo , Glucose-6-Fosfato/análogos & derivados , Glucose-6-Fosfato/metabolismo , Cinética , Modelos Químicos , Modelos Moleculares , Mutagênese Sítio-Dirigida , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Dióxido de Silício/química , Espectrometria de FluorescênciaRESUMO
This work reports the effect of dietary Salba (chia) seed rich in n-3 α-linolenic acid on the morphological and metabolic aspects involved in adipose tissue dysfunction and the mechanisms underlying the impaired glucose and lipid metabolism in the skeletal muscle of rats fed a sucrose-rich diet (SRD). Rats were fed a SRD for 3 months. Thereafter, half the rats continued with SRD while in the other half, corn oil (CO) was replaced by chia seed for 3 months (SRD+chia). In control group, corn starch replaced sucrose. The replacement of CO by chia seed in the SRD reduced adipocyte hypertrophy, cell volume and size distribution, improved lipogenic enzyme activities, lipolysis and the anti-lipolytic action of insulin. In the skeletal muscle lipid storage, glucose phosphorylation and oxidation were normalized. Chia seed reversed the impaired insulin stimulated glycogen synthase activity, glycogen, glucose-6-phosphate and GLUT-4 protein levels as well as insulin resistance and dyslipidemia.
Assuntos
Tecido Adiposo/efeitos dos fármacos , Suplementos Nutricionais , Dislipidemias/dietoterapia , Músculo Esquelético/efeitos dos fármacos , Salvia/química , Sementes/química , Ácido alfa-Linolênico/administração & dosagem , Adipócitos/efeitos dos fármacos , Adipócitos/metabolismo , Adipócitos/patologia , Tecido Adiposo/metabolismo , Tecido Adiposo/patologia , Animais , Tamanho Celular , Óleo de Milho/administração & dosagem , Dislipidemias/induzido quimicamente , Dislipidemias/metabolismo , Dislipidemias/patologia , Glucose/metabolismo , Transportador de Glucose Tipo 4/metabolismo , Glucose-6-Fosfato/metabolismo , Glicogênio Sintase/metabolismo , Insulina/farmacologia , Resistência à Insulina , Metabolismo dos Lipídeos/efeitos dos fármacos , Masculino , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Fosforilação Oxidativa/efeitos dos fármacos , Ratos , Ratos Wistar , Sementes/metabolismo , Sacarose/administração & dosagem , Sacarose/efeitos adversosRESUMO
Here we set out to evaluate the role of hexokinase and glycogen synthase in the control of glycogen synthesis in vivo. We used metabolic control analysis (MCA) to determine the flux control coefficient for each of the enzymes involved in the pathway. Acute microinjection experiments in frog oocytes were specifically designed to change the endogenous activities of the enzymes, either by directly injecting increasing amounts of a given enzyme (HK, PGM and UGPase) or by microinjection of a positive allosteric effector (glc-6P for GS). Values of 0.61 ± 0.07, 0.19 ± 0.03, 0.13 ± 0.03, and -0.06 ± 0.08 were obtained for the flux control coefficients of hexokinase EC 2.7.1.1 (HK), phosphoglucomutase EC 5.4.2.1 (PGM), UDPglucose pyrophosphorylase EC 2.7.7.9 (UGPase) and glycogen synthase EC 2.4.1.11 (GS), respectively. These values satisfy the summation theorem since the sum of the control coefficients for all the enzymes of the pathway is 0.87. The results show that, in frog oocytes, glycogen synthesis through the direct pathway is under the control of hexokinase. Phosphoglucomutase and UDPG-pyrophosphorylase have a modest influence, while the control exerted by glycogen synthase is null.
Assuntos
Glicogênio Sintase/fisiologia , Glicogênio/biossíntese , Hexoquinase/fisiologia , Oócitos/enzimologia , Animais , Anuros , Vias Biossintéticas , Células Cultivadas , Feminino , Glucose-6-Fosfato/metabolismo , Microinjeções , Oócitos/metabolismo , Fosfoglucomutase/fisiologiaRESUMO
In Escherichia coli, the pentose phosphate pathway is one of the main sources of NADPH. The first enzyme of the pathway, glucose-6-phosphate dehydrogenase (G6PDH), is generally considered an exclusive NADPH producer, but a rigorous assessment of cofactor preference has yet to be reported. In this work, the specificity constants for NADP and NAD for G6PDH were determined using a pure enzyme preparation. Absence of the phosphate group on the cofactor leads to a 410-fold reduction in the performance of the enzyme. Furthermore, the contribution of the phosphate group to binding of the transition state to the active site was calculated to be 3.6 kcal·mol(-1). In order to estimate the main kinetic parameters for NAD(P) and NAD(P)H, we used the classical initial-rates approach, together with an analysis of reaction time courses. To achieve this, we developed a new analytical solution to the integrated Michaelis-Menten equation by including the effect of competitive product inhibition using the ω-function. With reference to relevant kinetic parameters and intracellular metabolite concentrations reported by others, we modeled the sensitivity of reduced cofactor production by G6PDH as a function of the redox ratios of NAD/NADH (rR(NAD)) and NADP/NADPH (rR(NADP)). Our analysis shows that NADPH production sharply increases within the range of thermodynamically feasible values of rR(NADP), but NADH production remains low within the range feasible for rR(NAD). Nevertheless, we show that certain combinations of rR(NADP) and rR(NAD) sustain greater levels of NADH production over NADPH.
Assuntos
Escherichia coli/enzimologia , Glucose-6-Fosfato/química , Glucosefosfato Desidrogenase/metabolismo , Modelos Biológicos , NADP/metabolismo , NAD/metabolismo , Ligação Competitiva , Glucose-6-Fosfato/metabolismo , Cinética , OxirreduçãoRESUMO
Retinal tissue is exceptional because it shows a high level of energy metabolism. Glycogen content represents the only energy reserve in retina, but its levels are limited. Therefore, elucidation of the mechanisms controlling glycogen content in retina will allow us to understand retina response under local energy demands that can occur under normal and pathological conditions. Thus, we studied retina glycogen levels under different experimental conditions and correlated them with glucose-6-phosphate (G-6-P) content and glycogen synthase (GS) activity. Glycogen and G-6-P content were studied in ex vivo retinas from normal, fasted, streptozotocin-treated, and insulin-induced hypoglycemic rats. Expression levels of GS and its phosphorylated form were also analyzed. Ex vivo retina from normal rats showed low G-6-P (14±2 pmol/mg protein) and glycogen levels (43±3 nmol glycosyl residues/mg protein), which were increased 6 and 3 times, respectively, in streptozotocin diabetic rats. While no changes in phosphorylated GS levels were observed in any condition tested, a positive correlation was found between G-6-P levels with GS activity and glycogen content. The results indicated that in vivo, retina glycogen may act as an immediately accessible energy reserve and that its content was controlled primarily by G-6-P allosteric activation of GS. Therefore, under hypoglycemic situations retina energy supply is strongly compromised and could lead to the alterations observed in type 1 diabetes.
Assuntos
Glicogênio Sintase/metabolismo , Glicogênio/metabolismo , Retina/enzimologia , Regulação Alostérica/efeitos dos fármacos , Animais , Glicemia/efeitos dos fármacos , Glicemia/metabolismo , Glucose-6-Fosfato/metabolismo , Glicogênio Fosforilase/metabolismo , Humanos , Insulina/administração & dosagem , Insulina/farmacologia , Fosforilação/efeitos dos fármacos , Ratos , Ratos Long-Evans , Retina/efeitos dos fármacosRESUMO
The tellurium oxyanion tellurite induces oxidative stress in most microorganisms. In Escherichia coli, tellurite exposure results in high levels of oxidized proteins and membrane lipid peroxides, inactivation of oxidation-sensitive enzymes and reduced glutathione content. In this work, we show that tellurite-exposed E. coli exhibits transcriptional activation of the zwf gene, encoding glucose 6-phosphate dehydrogenase (G6PDH), which in turn results in augmented synthesis of reduced nicotinamide adenine dinucleotide phosphate (NADPH). Increased zwf transcription under tellurite stress results mainly from reactive oxygen species (ROS) generation and not from a depletion of cellular glutathione. In addition, the observed increase of G6PDH activity was paralleled by accumulation of glucose-6-phosphate (G6P), suggesting a metabolic flux shift toward the pentose phosphate shunt. Upon zwf overexpression, bacterial cells also show increased levels of antioxidant molecules (NADPH, GSH), better-protected oxidation-sensitive enzymes and decreased amounts of oxidized proteins and membrane lipids. These results suggest that by increasing NADPH content, G6PDH plays an important role in E. coli survival under tellurite stress.
Assuntos
Proteínas de Escherichia coli/metabolismo , Escherichia coli/efeitos dos fármacos , Escherichia coli/enzimologia , Glucosefosfato Desidrogenase/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Telúrio/farmacologia , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Glucose-6-Fosfato/metabolismo , Glucosefosfato Desidrogenase/genética , NADP/metabolismo , Espécies Reativas de Oxigênio/metabolismoRESUMO
Because of its severe side effects and variable efficacy, the current treatment for Chagas disease is unsatisfactory. Natural compounds are good alternative chemotherapeutic agents for the treatment of this infection. Recently, our group reported the antiproliferative activity and morphological alterations in epimastigotes and intracellular amastigotes of Trypanosoma cruzi treated with eupomatenoid-5, a neolignan isolated from leaves of Piper regnellii var. pallescens. Here, we demonstrate that eupomatenoid-5 exhibited activity against trypomastigotes, the infective form of T. cruzi (EC50 40.5 µM), leading to ultrastructural alteration and lipoperoxidation in the cell membrane. Additionally, eupomatenoid-5 induced depolarization of the mitochondrial membrane, lipoperoxidation and increased G6PD activity in epimastigotes of T. cruzi. These findings support the possibility that different mechanisms may be targeted, according to the form of the parasite, and that the plasma membrane and mitochondria are the structures that are most affected in trypomastigotes and epimastigotes, respectively. Thus, the trypanocidal action of eupomatenoid-5 may be associated with mitochondrial dysfunction and oxidative damage, which can trigger destructive effects on biological molecules of T. cruzi, leading to parasite death.
Assuntos
Benzofuranos/farmacologia , Mitocôndrias/metabolismo , Fenóis/farmacologia , Piper/química , Extratos Vegetais/química , Tripanossomicidas/farmacologia , Trypanosoma cruzi/efeitos dos fármacos , Benzofuranos/química , Benzofuranos/isolamento & purificação , Doença de Chagas/tratamento farmacológico , Doença de Chagas/parasitologia , Glucose-6-Fosfato/metabolismo , Humanos , Peróxido de Hidrogênio/metabolismo , Lignanas/química , Lignanas/isolamento & purificação , Lignanas/farmacologia , Peroxidação de Lipídeos/efeitos dos fármacos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Membranas Mitocondriais/efeitos dos fármacos , Membranas Mitocondriais/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Fenóis/química , Fenóis/isolamento & purificação , Fosfogluconato Desidrogenase/efeitos dos fármacos , Fosfogluconato Desidrogenase/metabolismo , Folhas de Planta/química , Tripanossomicidas/química , Tripanossomicidas/isolamento & purificação , Trypanosoma cruzi/metabolismo , Trypanosoma cruzi/ultraestruturaRESUMO
Kluyveromyces lactis Lac12 permease mediates lactose and low-affinity galactose transports. In this study we investigated the effects of carbon sources on internalization of Lac12 using a LAC12-GFP fusion construct. When galactose- or lactose-grown cells are shifted to a fresh sugar medium, Lac12-GFP is removed from the plasma membrane and is localized intracellularly. Surprisingly, either galactose or lactose in the new media caused the internalization, and cells responded differently to these two sugars. Our results reveal that this process is dependent on sugar species and also sugar concentration. Lac12-GFP internalization causes reduction of [C(14) ]lactose uptake rates and also occurs in a Klsnf1 mutant strain; it is therefore independent of KlSnf1 activity. We suggest that glucose-6-phosphate is the intracellular signal, as internalization was induced by 2-deoxyglucose, and inhibition of phosphoglucomutase by lithium prevented galactose- but not lactose- or glucose-induced internalization. Lac12-GFP internalization was not triggered by 6-deoxyglucose, and was irreversible in the absence of protein synthesis.