RESUMO
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
Hexokinase from Leishmania mexicana was purified to homogeneity from a glycosome-enriched fraction obtained after a differential centrifugation of promastigote form. The kinetic properties of the pure enzyme were determined and the Km values for glucose (Km = 66 microM) and ATP (Km = 303 muM) were comparable to those from hexokinase of Trypanosoma cruzi. L. mexicana hexokinase was able to use fructose (Km = 142 microM), which reflects the condition found in the insect host. In contrast with hexokinases from other trypanosomatids, the enzyme exhibited a moderate sensitivity to inhibition by glucose 6-phosphate. This inhibition was competitive with respect to both ATP and glucose, indicating that an allosteric site for glucose 6-phosphate does not exist in this enzyme. The enzyme was also inhibited by inorganic pyrophosphate, the inhibition being higher than that observed for T. cruzi enzyme. As expected, the enzyme was localized, by immunofluorescence analysis, in glycosomes and is present in both promastigotes and true amastigotes obtained from hamster lesion. Hexokinase specific activity increased with the aging of promastigote culture, and this increment was related to glucose consumption. However, the level of the hexokinase protein remains constant as determined by Western blotting. Several hypotheses are discussed to explain this result.
Assuntos
Hexoquinase/isolamento & purificação , Hexoquinase/metabolismo , Leishmania mexicana/enzimologia , Animais , Difosfatos/metabolismo , Hexoquinase/antagonistas & inibidores , Hexoquinase/químicaRESUMO
In mammals, hexokinase (HK) is strategically located at the outer membrane of mitochondria bound to the porin protein. The mitochondrial HK is a crucial modulator of apoptosis and reactive oxygen species generation. In plants, these properties related to HK are unknown. In order to better understand the physiological role of non-cytosolic hexokinase (NC-HK) in plants, we developed a purification strategy here described. Crude extract of 400 g of maize roots (230 mg protein) contained a specific activity of 0.042 micromol G6P min(-1) mg PTN(-1). After solubilization with detergent two fractions were obtained by DEAE column chromatography, NC-HK 1 (specific activity = 3.6 micromol G6P min(-1) mg PTN(-1) and protein recovered = 0.7 mg) and NC-HK 2. A major purification (yield = 500-fold) was obtained after passage of NC-HK 1 through the hydrophobic phenyl-Sepharose column. The total amount of protein and activity recovered were 0.04 and 18%, respectively. The NC-HK 1 binds to the hydrophobic phenyl-Sepharose matrix, as observed for rat brain HK. Mild chymotrypsin digestion did not affect adsorption of NC-HK 1 to the hydrophobic column as it does for rat HK I. In contrast to mammal mitochondrial HK, glucose-6-phosphate, clotrimazole or thiopental did not dissociate NC-HK from maize (Zea mays) or rice (Oryza sativa) mitochondrial membranes. These data show that the interaction between maize or rice NC-HK to mitochondria differs from that reported in mammals, where the mitochondrial enzyme can be displaced by modulators or pharmacological agents known to interfere with the enzyme binding properties with the mitochondrial porin protein.
Assuntos
Hexoquinase/isolamento & purificação , Mitocôndrias/enzimologia , Raízes de Plantas/enzimologia , Zea mays/enzimologia , Animais , Encéfalo/enzimologia , Cromatografia DEAE-Celulose , Hexoquinase/metabolismo , Oryza/enzimologia , Ratos , SolubilidadeRESUMO
In mammals, hexokinase (HK) is strategically located at the outer membrane of mitochondria bound to the porin protein. The mitochondrial HK is a crucial modulator of apoptosis and reactive oxygen species generation. In plants, these properties related to HK are unknown. In order to better understand the physiological role of non-cytosolic hexokinase (NC-HK) in plants, we developed a purification strategy here described. Crude extract of 400 g of maize roots (230 mg protein) contained a specific activity of 0.042 æmol G6P min-1 mg PTN-1. After solubilization with detergent two fractions were obtained by DEAE column chromatography, NC-HK 1 (specific activity = 3.6 æmol G6P min-1 mg PTN-1 and protein recovered = 0.7 mg) and NC-HK 2. A major purification (yield = 500-fold) was obtained after passage of NC-HK 1 through the hydrophobic phenyl-Sepharose column. The total amount of protein and activity recovered were 0.04 and 18 percent, respectively. The NC-HK 1 binds to the hydrophobic phenyl-Sepharose matrix, as observed for rat brain HK. Mild chymotrypsin digestion did not affect adsorption of NC-HK 1 to the hydrophobic column as it does for rat HK I. In contrast to mammal mitochondrial HK, glucose-6-phosphate, clotrimazole or thiopental did not dissociate NC-HK from maize (Zea mays) or rice (Oryza sativa) mitochondrial membranes. These data show that the interaction between maize or rice NC-HK to mitochondria differs from that reported in mammals, where the mitochondrial enzyme can be displaced by modulators or pharmacological agents known to interfere with the enzyme binding properties with the mitochondrial porin protein.
Assuntos
Animais , Ratos , Hexoquinase/isolamento & purificação , Hexoquinase/metabolismo , Mitocôndrias/enzimologia , Raízes de Plantas/enzimologia , Zea mays/enzimologia , Encéfalo/enzimologia , Cromatografia DEAE-Celulose , Oryza , SolubilidadeRESUMO
This study dealt with the partition behavior and partial purification of hexokinase (HK) from baker's yeast by liquid-liquid extraction using aqueous two-phase polyethylene glycol (PEG)/citrate systems. First, we investigated the effect of agitation type (vortex and 8 rpm rotation) on the stability of the system, and then the effects of sodium citrate concentration, PEG concentration, and molar mass of PEG on the partition coefficient of this enzyme by using a 25 factorial experimental design. The results of this factorial experiment showed the possibility of a partial purification of HK by using two extraction steps, since the enzyme preferentially migrated to the top phase and the total proteins (mainly contaminants) remained in the bottom phase. The purification factor (PurTOP) of the enzyme in the top phase was 1.87, and the partition coefficient of the total proteins (KProt) was 0.47.
Assuntos
Hexoquinase/isolamento & purificação , Hexoquinase/metabolismo , Saccharomyces cerevisiae/enzimologia , Citratos , Estabilidade Enzimática , Indicadores e Reagentes , Cinética , Peso Molecular , Polietilenoglicóis , Citrato de Sódio , ÁguaRESUMO
Hexokinase (HK) and glucose 6-phosphate dehydrogenase (G6PDH) are important enzymes used in biochemical studies and in analytical methods. The stability of the enzymes can be affected by several variables, pH being one of them. The effect of pH on the stability of HK and G6PDH was evaluated in this work. Baker's yeast cells were suspended in 50 mM Tris-HCl buffer (pH 7.5) containing 5.0 mM MgCl2, and submitted to disruption by agitation with glass beads and in the presence of protease inhibitors. The cell-free extract was obtained by centrifugation (2880g; 10 min), followed by dilution into the buffers: 0.1 M acetate-acetic acid (pH: 4.0, 4.5, 5.0, or 5.5), 0.1 M phosphate buffer (pH: 6.0, 6.5, or 7.0), and 0.1 M Tris-HCl buffer (pH: 7.5, 8.0, 8.5, 9.0 or 9.5). The residual activity of HK and G6PDH, expressed as micromol of NADPH formed per min, were measured through a period of buffer-enzyme contact from 0 to 51 h at 4 degrees C. It was observed that up to 4 h both enzymes were stable in all buffers used. However, after 51 h HK was stable at pH 6.0 and 7.5, whereas G6PDH was stable at pH 7.0, 9.5, and between 4.5 and 5.5.
Assuntos
Glucosefosfato Desidrogenase/química , Hexoquinase/química , Concentração de Íons de Hidrogênio , Estabilidade Enzimática , Glucosefosfato Desidrogenase/isolamento & purificação , Glucosefosfato Desidrogenase/metabolismo , Hexoquinase/isolamento & purificação , Hexoquinase/metabolismo , Cinética , Saccharomyces cerevisiae/enzimologia , Fatores de TempoRESUMO
This study describes the effect of some saturated and unsaturated free fatty acids and acyl-CoA thioesters on Trypanosoma cruzi glucose 6-phosphate dehydrogenase and hexokinase activities. Glucose 6-phosphate dehydrogenase was sensitive to the destabilizing effect provoked by free fatty acids, while hexokinase remained unaltered. Glucose 6-phosphate dehydrogenase inhibition by free fatty acids was dependent on acid concentration and chain length. Both enzymes were inhibited when they were incubated with acyl-CoA thioesters. The acyl-CoA thioesters inhibited glucose 6-phosphate dehydrogenase at a lower concentration than the free fatty acids; the ligands glucose 6-phosphate and NADP+ afforded protection. The inhibition of hexokinase by acyl-CoAs was not reverted when the enzyme was incubated with ATP. The type of inhibition found with acyl-CoAs in relation to glucose 6-phosphate dehydrogenase and hexokinase suggests that this type inhibition may produce an in vivo modulation of these enzymatic activities.
Assuntos
Acil Coenzima A/farmacologia , Glucosefosfato Desidrogenase/antagonistas & inibidores , Hexoquinase/antagonistas & inibidores , Trypanosoma cruzi/enzimologia , Trifosfato de Adenosina/metabolismo , Animais , Inibidores Enzimáticos/farmacologia , Ácidos Graxos não Esterificados/farmacologia , Ácidos Graxos Insaturados/farmacologia , Glucose-6-Fosfato/metabolismo , Glucosefosfato Desidrogenase/isolamento & purificação , Glucosefosfato Desidrogenase/metabolismo , Hexoquinase/isolamento & purificação , Hexoquinase/metabolismo , Cinética , NADP/metabolismo , Trypanosoma cruzi/crescimento & desenvolvimento , Trypanosoma cruzi/metabolismoRESUMO
Hexokinase is the prime enzyme of the Embden-Meyerhof pathway and is responsible for the first stage of energy conversion. It catalyzes the transfer of a phosphate to glucose to form glucose-6-phosphate. Yeast hexokinase PII is also known to play an important role in glucose signal transduction. Crystals of yeast hexokinase isoforms PI and PII were obtained by vapour-diffusion techniques using the hanging-drop method. Isoform PI crystals belong to the space group P2(1)2(1)2(1), with unit-cell parameters a = 62.12, b = 78.87, c = 144.74 A. Unit-cell parameters for isoform PII crystals are a = b = 142.81, c = 58.46 A and the space group is I4. Synchrotron diffraction data have been collected to 2.2 A resolution from the isoform PII crystal, whereas isoform PI diffracted to 3.1 A.
Assuntos
Hexoquinase/química , Hexoquinase/isolamento & purificação , Isoenzimas/química , Isoenzimas/isolamento & purificação , Saccharomyces cerevisiae/enzimologia , Cristalização , Cristalografia por Raios XRESUMO
Buffalo erythrocytes contain one isozyme of hexokinase that apparently lacks microheterogeneity as shown by chromatographic properties. A single protein band was detected by means of Western blotting using an antibody raised in rabbits against homogeneous rat brain hexokinase I. The native protein has a molecular weight of 200,000 +/- 2880 by gel filtration. Partial purification of erythrocyte hexokinase by a combination of several procedures, including affinity chromatography, which was previously applied successfully to the purification of other mammalian type I hexokinases, produced a partially purified enzyme that showed several contaminants after SDS-polyacrylamide gel electrophoresis. The affinity of buffalo erythrocyte hexokinase for glucose (K(m) = 0.012 +/- 0.001 mM) is lower than most other mammal hexokinases type I. It phosphorylates other sugars, with considerably higher K(m) values. This isozyme is able to use MgATP but does not use MgGTP, MgCTP or MgUTP. We used inhibition patterns, obtained with products to elucidate enzyme sequential mechanisms. Our results are clearly in agreement with a random sequential mechanism and in disagreement with an ordered sequential mechanism with either glucose or ATP as the obligatory first substrates. The ADP inhibition was of mixed type with both ATP and glucose as substrates.
Assuntos
Búfalos , Eritrócitos/enzimologia , Hexoquinase/isolamento & purificação , Hexoquinase/fisiologia , Animais , Eletroforese/métodos , Hexoquinase/antagonistas & inibidores , Concentração de Íons de Hidrogênio , Ponto Isoelétrico , Cinética , Peso Molecular , Coelhos , Ratos , Especificidade por Substrato , Distribuição TecidualRESUMO
Kinetic and structural studies have been carried out of two isoenzymes of hexokinase from the rat, hexokinase II and glucokinase. Although both enzymes are monomeric, hexokinase II has a molecular weight double that of glucokinase and resembles a dimer of glucokinase. The co-operativity of glucokinase, which is not observed for hexokinase II, appears to be kinetic in origin rather than the consequence of ineractions between distinct glucose-binding sites.