RESUMO
Trypanosoma cruzi, the causative agent of Chagas' disease, belongs to the Trypanosomatidae family. The parasite undergoes multiple morphological and metabolic changes during its life cycle, in which it can use both glucose and amino acids as carbon and energy sources. The glycolytic pathway is peculiar in that its first six or seven steps are compartmentalized in glycosomes, and has a two-branched auxiliary glycosomal system functioning beyond the intermediate phosphoenolpyruvate (PEP) that is also used in the cytosol as substrate by pyruvate kinase. The pyruvate phosphate dikinase (PPDK) is the first enzyme of one branch, converting PEP, PPi, and AMP into pyruvate, Pi, and ATP. Here we present a kinetic study of PPDK from T. cruzi that reveals its hysteretic behavior. The length of the lag phase, and therefore the time for reaching higher specific activity values is affected by the concentration of the enzyme, the presence of hydrogen ions and the concentrations of the enzyme's substrates. Additionally, the formation of a more active PPDK with more complex structure is promoted by it substrates and the cation ammonium, indicating that this enzyme equilibrates between the monomeric (less active) and a more complex (more active) form depending on the medium. These results confirm the hysteretic behavior of PPDK and are suggestive for its functioning as a regulatory mechanism of this auxiliary pathway. Such a regulation could serve to distribute the glycolytic flux over the two auxiliary branches as a response to the different environments that the parasite encounters during its life cycle.
Assuntos
Doença de Chagas/parasitologia , Piruvato Ortofosfato Diquinase/metabolismo , Trypanosoma cruzi/enzimologia , Monofosfato de Adenosina/metabolismo , Difosfatos/metabolismo , Glucose/metabolismo , Glicólise , Concentração de Íons de Hidrogênio , Cinética , Microcorpos/enzimologia , Fosfoenolpiruvato/metabolismo , Piruvato Ortofosfato Diquinase/química , Piruvatos/metabolismo , Proteínas Recombinantes/metabolismoRESUMO
Trypanosoma evansi is a monomorphic protist that can infect horses and other animal species of economic importance for man. Like the bloodstream form of the closely related species Trypanosoma brucei, T. evansi depends exclusively on glycolysis for its free-energy generation. In T. evansi as in other kinetoplastid organisms, the enzymes of the major part of the glycolytic pathway are present within organelles called glycosomes, which are authentic but specialized peroxisomes. Since T. evansi does not undergo stage-dependent differentiations, it occurs only as bloodstream forms, it has been assumed that the metabolic pattern of this parasite is identical to that of the bloodstream form of T. brucei. However, we report here the presence of two additional enzymes, phosphoenolpyruvate carboxykinase and PPi-dependent pyruvate phosphate dikinase in T. evansi glycosomes. Their colocalization with glycolytic enzymes within the glycosomes of this parasite has not been reported before. Both enzymes can make use of PEP for contributing to the production of ATP within the organelles. The activity of these enzymes in T. evansi glycosomes drastically changes the model assumed for the oxidation of glucose by this parasite.
Assuntos
Fosfoenolpiruvato Carboxiquinase (ATP)/metabolismo , Piruvato Ortofosfato Diquinase/metabolismo , Trypanosoma/enzimologia , Animais , Digitonina/farmacologia , Glucosefosfato Desidrogenase/isolamento & purificação , Glucosefosfato Desidrogenase/metabolismo , Glicólise , Hexoquinase/isolamento & purificação , Hexoquinase/metabolismo , Cavalos , Indicadores e Reagentes/farmacologia , Malato Desidrogenase/isolamento & purificação , Malato Desidrogenase/metabolismo , Camundongos , Microcorpos/enzimologia , Microscopia de Fluorescência , Permeabilidade/efeitos dos fármacos , Fosfoenolpiruvato Carboxiquinase (ATP)/genética , Fosfoenolpiruvato Carboxiquinase (ATP)/isolamento & purificação , Fosfoglicerato Quinase/isolamento & purificação , Fosfoglicerato Quinase/metabolismo , Fosfopiruvato Hidratase/isolamento & purificação , Fosfopiruvato Hidratase/metabolismo , Piruvato Ortofosfato Diquinase/isolamento & purificação , Coelhos , Ratos , Ratos Wistar , Trypanosoma/efeitos dos fármacosRESUMO
Gluconeogenesis is an active pathway in Leishmania amastigotes and is essential for their survival within the mammalian cells. However, our knowledge about this pathway in trypanosomatids is very limited. We investigated the role of glycerol kinase (GK), phosphoenolpyruvate carboxykinase (PEPCK), and pyruvate phosphate dikinase (PPDK) in gluconeogenesis by generating the respective Leishmania mexicana Δgk, Δpepck, and Δppdk null mutants. Our results demonstrated that indeed GK, PEPCK, and PPDK are key players in the gluconeogenesis pathway in Leishmania, although stage-specific differences in their contribution to this pathway were found. GK participates in the entry of glycerol in promastigotes and amastigotes; PEPCK participates in the entry of aspartate in promastigotes, and PPDK is involved in the entry of alanine in amastigotes. Furthermore, the majority of alanine enters into the pathway via decarboxylation of pyruvate in promastigotes, whereas pathway redundancy is suggested for the entry of aspartate in amastigotes. Interestingly, we also found that l-lactate, an abundant glucogenic precursor in mammals, was used by Leishmania amastigotes to synthesize mannogen, entering the pathway through PPDK. On the basis of these new results, we propose a revision in the current model of gluconeogenesis in Leishmania, emphasizing the differences between amastigotes and promastigotes. This work underlines the importance of studying the trypanosomatid intracellular life cycle stages to gain a better understanding of the pathologies caused in humans.
Assuntos
Gluconeogênese , Glicerol Quinase/metabolismo , Leishmania mexicana/metabolismo , Fosfoenolpiruvato Carboxilase/metabolismo , Proteínas de Protozoários/metabolismo , Piruvato Ortofosfato Diquinase/metabolismo , Vias Biossintéticas/efeitos dos fármacos , Southern Blotting , Western Blotting , DNA de Protozoário/genética , Glucose/metabolismo , Glucose/farmacologia , Glicerol Quinase/genética , Humanos , Leishmania mexicana/genética , Leishmania mexicana/crescimento & desenvolvimento , Estágios do Ciclo de Vida , Mutação , Fosfoenolpiruvato Carboxilase/genética , Proteínas de Protozoários/genética , Piruvato Ortofosfato Diquinase/genéticaRESUMO
Calmodulin (CaM) is the primary sensor for calcium in the cell. It modulates various functions by activating CaM-binding proteins (CaMBPs). This study examined the calcium/CaM-dependent system in the ancient eukaryote Giardia intestinalis. A specific antibody against the parasite's CaM was developed; this protein's expression and location during different stages of the parasite's life cycle were analyzed. The results showed that it is a housekeeping protein which is possibly involved in the parasite's motility. No CaMBP has been identified in G. intestinalis to date. Pull-down assays were used for isolating proteins which specifically bind to CaM in a calcium-dependent way. Three of them were identified through mass spectrometry; they were GASP180, α-tubulin, and pyruvate phosphate dikinase (PPDK).The first two are cytoskeleton proteins, and the last one is an essential enzyme for glycolysis. The presence of binding sites was analyzed through bioinformatics in each protein sequence. This is the first report of a CaMBP in this organism; it is considered to be a very interesting differentiation model, indicating that CaM is involved at least in two vital processes: G. intestinalis motility and energetic metabolism.
Assuntos
Proteínas de Ligação a Calmodulina/metabolismo , Calmodulina/metabolismo , Giardia lamblia/crescimento & desenvolvimento , Proteínas de Protozoários/metabolismo , Trofozoítos/metabolismo , Cálcio/metabolismo , Calmodulina/genética , Técnicas de Cultura de Células , Diferenciação Celular , Movimento Celular , Biologia Computacional , Giardia lamblia/metabolismo , Filogenia , Processamento de Proteína Pós-Traducional , Piruvato Ortofosfato Diquinase/metabolismo , Tubulina (Proteína)RESUMO
Pyruvate phosphate dikinase (PPDK) was recently reported in trypanosomatids, but its metabolic function is not yet known. The present work deals with the cellular localization and the function of the Trypanosoma cruzi enzyme. First, we show by digitonin titration and cell fractionation that the enzyme was essentially present in the glycosome matrix of the epimastigote form. Second, we address the issue of the direction of the reaction inside the glycosome for one part, our bibliographic survey evidenced a quite exergonic DeltaGo' (at least -5.2 kcal/mol at neutral pH and physiologic ionic strength); for another part, no pyrophosphatase (PPase) could be detected in fractions corresponding to the glycosomes; therefore, glycosomal PPDK likely works in the direction of pyruvate production. Third, we address the issue of the origin of the glycosomal pyrophosphate (PPi): several synthetic pathways known to produce PPi are already considered to be glycosomal. This work also indicates the presence of an NADP(+)-dependent beta-oxidation of palmitoyl-CoA in the glycosome. Several pyruvate-consuming activities, in particular alanine dehydrogenase (ADH) and pyruvate carboxylase (PC), were detected in the glycosomal fraction. PPDK appears therefore as a central enzyme in the metabolism of the glycosome of T. cruzi by providing a link between glycolysis, fatty acid oxidation and biosynthetic PPi-producing pathways. Indeed, PPDK seems to replace pyrophosphatase in its classical thermodynamic role of displacing the equilibrium of PPi-producing reactions, as well as in its role of eliminating the toxic PPi.
Assuntos
Difosfatos/metabolismo , Microcorpos/metabolismo , Piruvato Ortofosfato Diquinase/metabolismo , Trypanosoma cruzi/metabolismo , Alanina Desidrogenase , Aminoácido Oxirredutases/química , Animais , Western Blotting , Carbonatos/química , Detergentes/farmacologia , Digitonina/química , Eletroforese em Gel de Poliacrilamida , Ácidos Graxos/metabolismo , Glicólise , Concentração de Íons de Hidrogênio , Íons , Modelos Biológicos , NADP/química , Octoxinol , Oxigênio/metabolismo , Polietilenoglicóis/farmacologia , Piruvato Carboxilase/química , Piruvatos/química , Frações Subcelulares/metabolismo , TermodinâmicaRESUMO
The C(4) succulent plant Portulaca oleracea shifts its photosynthetic metabolism to crassulacean acid metabolism (CAM) after 23 d of withholding water. This is accounted by diurnal acid fluctuation, net nocturnal but not day CO(2) uptake and drastic changes in phosphoenolpyruvate carboxylase (PEPC) kinetic and regulatory properties [Lara et al. (2003) Photosynth: Res. 77: 241]. The goal of the present work was to characterize the CAM activity in leaves of P. oleracea during water stress through the study of enzymes involved in carbon fixation and carbohydrate metabolism. After drought stress, a general decrease in the photosynthetic metabolism, as accounted by the decrease in the net CO(2) fixation and in the activity of enzymes such as ribulose-1,5-bisphosphate carboxylase/oxygenase, PEPC, pyruvate orthophosphate dikinase, phosphoenolpyruvate carboxykinase and NAD-malic enzyme was observed. We also found changes in the day/night activities and level of immunoreactive protein of some of these enzymes which were correlated to night CO(2) fixation, as occurs under CAM metabolism. Based on the results obtained, including those from in situ immunolocalization studies, we propose a scheme for the possible CO(2) fixation pathways used by P. oleracea under conditions of sufficient and limiting water supply.
Assuntos
Metabolismo dos Carboidratos , Carbono/metabolismo , Fotossíntese/fisiologia , Portulaca/enzimologia , Ritmo Circadiano/fisiologia , Escuridão , Desidratação , Desastres , Regulação para Baixo/fisiologia , Malato Desidrogenase/metabolismo , Fosfoenolpiruvato Carboxiquinase (ATP)/metabolismo , Folhas de Planta/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Piruvato Ortofosfato Diquinase/metabolismo , Ribulose-Bifosfato Carboxilase/metabolismoRESUMO
We report the kinetic characterization of a previously unidentified pyruvate kinase (PK) activity in extracts from Entamoeba histolytica trophozoites. This activity was about 74% of the activity of pyruvate phosphate dikinase. EhPK differed from most PKs in that its pH optimum was 5.5-6.5 and was inhibited by high PEP concentrations (1-5mM); these are concentrations at which PK is usually assayed. The optimal temperature was above 40 degrees C with negligible activity below 20 degrees C. EhPK exhibited hyperbolic kinetics with respect to both PEP (K(m) = 0.018 mM) and ADP (K(m) = 1.05 mM). However, it exhibited a sigmoidal behavior with respect to PEP at sub-saturating ADP concentrations. EhPK did not require monovalent cations for activity. Fructose-1,6 bisphosphate was a potent non-essential activator; it increased the affinity for ADP without modification of the V(max) or the affinity for PEP. Phosphate, citrate, malate, and alpha-ketoglutarate significantly inhibited EhPK activity. A putative EhPK gene fragment found in EhDNA was analyzed. The data indicate that E. histolytica trophozoites contain an active PK, which might contribute to the generation of glycolytic ATP for parasite survival.
Assuntos
Entamoeba histolytica/enzimologia , Piruvato Quinase/metabolismo , Difosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Animais , Clonagem Molecular , Cricetinae , Eletroforese em Gel de Poliacrilamida , Entamoeba histolytica/genética , Concentração de Íons de Hidrogênio , Cinética , Abscesso Hepático Amebiano/parasitologia , Dados de Sequência Molecular , Fosfoenolpiruvato/metabolismo , Piruvato Quinase/química , Piruvato Quinase/genética , Piruvato Ortofosfato Diquinase/metabolismo , Coelhos , Alinhamento de Sequência , TemperaturaRESUMO
This work studies damage to rat liver mitochondrial protein, lipid, and DNA caused by electronically excited states generated by cytochrome c-catalyzed diphenylacetaldehyde enol oxidation to triplet benzophenone. The extension of lipid peroxidation was estimated by production of thiobarbituric acid-reactive substances and by formation of Schiff bases with membrane proteins, evaluated by SDS-polyacrylamide gel electrophoresis. Concomitant with DPAA-driven mitochondrial permeabilization, extensive mtDNA fragmentation occurred and DNA adducts with aldehydes-products of fatty acid oxidation-were observed. The degree of lipid peroxidation and mtDNA alterations were significantly decreased by butylated hydroxytoluene, a potent peroxidation chain breaker. The lipid peroxidation process was also partially inhibited by the bioflavonoid rutin and urate totally prevented the mitochondrial transmembrane potential collapse. In all cases, the mitochondrial damage was dependent on the presence of phosphate ions, a putative bifunctional catalyst of carbonyl enolization. These data are consistent with the notion that triplet ketones may act like alkoxyl radicals as deleterious reactive oxygen species on biologic structures. Involvement of singlet dioxygen formed by triplet-triplet energy transfer from benzophenone in the model reaction with DPAA/cytochrome c in the presence of DCP liposomes was suggested by quenching of the accompanying chemiluminescence upon addition of histidine and lycopene.
Assuntos
Aldeídos/farmacologia , Dano ao DNA , DNA Mitocondrial/metabolismo , Mitocôndrias Hepáticas/efeitos dos fármacos , Fosfolipídeos/metabolismo , Animais , Benzofenonas/farmacologia , Grupo dos Citocromos c/metabolismo , Adutos de DNA/análise , Histidina/farmacologia , Lipossomos/química , Potenciais da Membrana/efeitos dos fármacos , Proteínas de Membrana/análise , Piruvato Ortofosfato Diquinase/metabolismo , Ratos , Ratos Wistar , Substâncias Reativas com Ácido Tiobarbitúrico/análiseRESUMO
The parasite Entamoeba histolytica is an organism whose main energetic source comes from glycolysis. It has the singularity that several of its glycolytic enzymes use pyrophosphate as an alternative phosphate donor. Thus, pyruvate phosphate dikinase (PPDK), an inorganic pyrophosphate (PPi)-dependent enzyme, substitutes pyruvate kinase present in humans. We previously cloned and sequenced the gene that codifies for PPDK in E. histolytica. We now report its expression in a bacterial system and its purification to 98% homogeneity. We determined its K(m) for phosphoenolpyruvate, AMP and PPi (21, < 5 and 100 microM, respectively). Unlike PPDK from maize and bacteria and pyruvate kinase from other cells, EhPPDk is dependent on divalent cations but does not require monovalent cations for activity. The enzyme has an optimum pH of 6.0, it is labile to low temperatures and has a tetrameric structure. Since EhPPDK is a PPi-dependent enzyme, we also tested the effect of some pyrophosphate analogs as inhibitors of activity. Studies on the function and structure of this enzyme may be important for therapeutic research in several parasitic diseases, since it has no counterpart in humans.