RESUMO
OBJECTIVES: To determine the efficacy of daptomycin for the treatment of penicillin- and cephalosporin-resistant pneumococcal meningitis using in vitro and in vivo methods. METHODS: In vitro killing curves were determined with clinically achievable CSF antibiotic concentrations. In a rabbit model of pneumococcal meningitis, we studied the efficacy (Δ cfu/mL) of daptomycin used at 15 and 25 mg/kg, comparing it with ceftriaxone 100 mg/kg/24 h and ceftriaxone plus vancomycin 30 mg/kg/24 h over a 26 h period against two different strains: HUB 2349 and ATCC 51916, with MICs of 2 and 32 mg/L of cefotaxime/ceftriaxone, respectively. RESULTS: The penetration of daptomycin into CSF ranged between 9% and 11%. Daptomycin therapy achieved an excellent response, being bactericidal within 2 h of antibiotic administration. Against strain HUB 2349, daptomycin at both doses was as effective as ceftriaxone plus vancomycin. Against the highly resistant strain, daptomycin 25 mg/kg was significantly better than ceftriaxone plus vancomycin at 2 and 6 h. CONCLUSIONS: Daptomycin at standard doses, and especially at high doses, may be a useful alternative for the treatment of penicillin- and cephalosporin-resistant pneumococcal meningitis.
Assuntos
Resistência às Cefalosporinas/efeitos dos fármacos , Cefalosporinas/uso terapêutico , Daptomicina/uso terapêutico , Meningite Pneumocócica/tratamento farmacológico , Animais , Cefalosporinas/farmacologia , Daptomicina/farmacologia , Feminino , Meningite Pneumocócica/líquido cefalorraquidiano , Coelhos , Resultado do TratamentoRESUMO
As the worldwide population continues to rise, so does global demand for agricultural production. This scenario of uncertain food supply is exacerbated by the high salinization of soils worldwide, a serious constraint to crop productivity. In this context, there is an increasing need for alternative sustainable crops. Halophytes are thought to be a promising alternative food source due to their natural ability to grow in saline soils and their multiple potential uses in the food industry. In this study, the protein and fatty acid content of the halophyte Halimione (Atriplex) portulacoides (L.) was studied in different saline conditions. Although more studies are needed to explore the nutritional properties of H. portulacoides, the data presented here suggest that this halophyte should be considered as a promising food crop for saline agriculture.
Assuntos
Chenopodiaceae/química , Ácidos Graxos/análise , Proteínas de Plantas/análise , Salinidade , Plantas Tolerantes a Sal/química , Solo/químicaRESUMO
Lipid biosynthesis in developing sunflower (Helianthus annuus L.) seeds requires reducing power. One of the main sources of cellular NADPH is the oxidative pentose phosphate pathway (OPPP), generated from the oxidation of glucose-6-phosphate. This glycolytic intermediate, which can be imported to the plastid and enter in the OPPP, is the substrate and product of cytosolic phosphoglucose isomerase (cPGI, EC 5.3.1.9). In this report, we describe the cloning of a full-length cDNA encoding cPGI from developing sunflower seeds. The sequence was predicted to code for a protein of 566 residues characterised by the presence of two sugar isomerase domains. This cDNA was heterologously expressed in Escherichia coli as a His-tagged protein. The recombinant protein was purified using immobilised metal ion affinity chromatography and biochemically characterised. The enzyme had a specific activity of 1,436 micromol min(-1) mg(-1) and 1,011 micromol min(-1) mg(-1) protein when the reaction was initiated with glucose-6-phosphate and fructose-6-phosphate, respectively. Activity was not affected by erythrose-4-phosphate, but was inhibited by 6-P gluconate and glyceraldehyde-3-phosphate. A polyclonal immune serum was raised against the purified enzyme, allowing the study of protein levels during the period of active lipid synthesis in seeds. These results were compared with PGI activity profiles and mRNA expression levels obtained from Q-PCR studies. Our results point to the existence of a possible post-translational regulatory mechanism during seed development. Immunolocalisation of the protein in seed tissues further indicated that cPGI is highly expressed in the procambial ring.
Assuntos
Citosol/enzimologia , Glucose-6-Fosfato Isomerase/química , Glucose-6-Fosfato Isomerase/metabolismo , Helianthus/enzimologia , Helianthus/metabolismo , Sementes/enzimologia , Sementes/metabolismo , Sequência de Aminoácidos , Eletroforese em Gel de Poliacrilamida , Glucose-6-Fosfato/metabolismo , Glucose-6-Fosfato Isomerase/genética , Gliceraldeído 3-Fosfato/metabolismo , Helianthus/genética , Immunoblotting , Imuno-Histoquímica , Dados de Sequência Molecular , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Reação em Cadeia da Polimerase , RNA Mensageiro/genética , Sementes/genética , Homologia de Sequência de AminoácidosRESUMO
Koelreuteria paniculata is a deciduous tree, popular in temperate regions for its ornamental value, which accumulates unusual cyanolipids in its seeds. The seed oil of this plant is rich in the unusual cis-11-eicosenoic fatty acid (20:1, or gondoic acid), a monounsaturated oil of interest to the oleochemical industry. In higher plants, de novo fatty acid biosynthesis takes place in the plastids, a process that is terminated by hydrolysis of the thioester bond between the acyl moiety and the ACP by acyl-ACP thioesterases. The specificity of acyl-ACP thioesterases is fundamental in controlling the fatty acid composition of seed oil. To determine the mechanisms involved in fatty acid biosynthesis in K. paniculata seeds, we isolated, cloned and sequenced two cDNAs encoding acyl-ACP thioesterases in this plant, KpFatA and KpFatB. Both of them were expressed heterologously in Escherichia coli and characterized with different acyl-ACP substrates. The K. paniculata FatB2 displayed unusual substrate specificity, so that unlike most FatB2 type enzymes, it displayed preference for oleoyl-ACP instead of palmitoyl-ACP. This specificity was consistent with the changes in E. coli and N. benthamiana fatty acid composition following heterologous expression of this enzyme. KpFatB also showed certain genetic divergence relative to other FatB-type thioesterases and when modelled, its structure revealed differences at the active site. Together, these results suggest that this thioesterase could be a new class of FatB not described previously.
RESUMO
Meropenem is a broad-spectrum carbapenem antibiotic that is highly active against the pathogens causing meningitis. The aims of this study was to determine the efficacies of meropenem alone and combined with rifampin against two Streptococcus pneumoniae strains with different susceptibility to beta-lactams using the guinea pig meningitis model and compare them with the standard ceftriaxone plus vancomycin therapy. All treatments except rifampin were bactericidal from 6 h. The addition of rifampin did not improve the activity of meropenem alone. Our results provide good evidence of the efficacy of meropenem in the treatment of penicillin- and cephalosporin-susceptible and -resistant pneumococcal meningitis similar to that of ceftriaxone plus vancomycin, suggesting that meropenem might be a good option in the management of this infection.
Assuntos
Antibacterianos/uso terapêutico , Meningite Pneumocócica/tratamento farmacológico , Rifampina/uso terapêutico , Streptococcus pneumoniae/efeitos dos fármacos , Tienamicinas/uso terapêutico , Animais , Ceftriaxona/uso terapêutico , Contagem de Colônia Microbiana , Modelos Animais de Doenças , Quimioterapia Combinada , Feminino , Cobaias , Humanos , Meropeném , Viabilidade Microbiana/efeitos dos fármacos , Fatores de Tempo , Resultado do Tratamento , Vancomicina/uso terapêuticoRESUMO
In the present study, we describe the molecular and biochemical characterization of sunflower (Helianthus annuus L.) enolase (ENO, EC 4.2.1.11) proteins, which catalyze the formation of phosphoenolpyruvate, the penultimate intermediate in the glycolytic pathway. We cloned and characterized three cDNAs encoding different ENO isoforms from developing sunflower seeds. Studies using fluorescently tagged ENOs confirmed the predicted subcellular localization of ENO isoforms: HaENO1 in the plastid while HaENO2 and HaENO3 were found in the cytosol. The cDNAs were used to express the corresponding 6(His)-tagged proteins in Escherichia coli. The proteins were purified to electrophoretic homogeneity, using immobilized metal ion affinity chromatography, and biochemically characterized. Recombinant HaENO1 and HaENO2, but not HaENO3 were shown to have enolase activity, in agreement with data obtained with the Arabidopsis homolog proteins. Site directed mutagenesis of several critical amino acids was used to attempt to recover enolase activity in recombinant HaENO3, resulting in very small increases that were not additive. A kinetic characterization of the two active isoforms showed that pH had similar effect on their velocity, that they had similar affinity for 2-phosphoglycerate, but that the kcat/Km of the plastidial enzyme was higher than that of the cytosolic isoform. Even though HaENO2 was always the most highly expressed transcript, the levels of expression of the three ENO genes were remarkably distinct in all the vegetative and reproductive tissues studied. This indicates that in seeds the conversion of 2-phosphoglycerate to phosphoenolpyruvate takes place through the cytosolic and the plastidial pathways therefore both routes could contribute to the supply of carbon for lipid synthesis. The identity of the main source of carbon during the period of stored products synthesis is discussed.
Assuntos
Helianthus/enzimologia , Fosfopiruvato Hidratase/metabolismo , Sementes/crescimento & desenvolvimento , Citosol/enzimologia , Glucose-6-Fosfato/metabolismo , Helianthus/genética , Helianthus/crescimento & desenvolvimento , Helianthus/metabolismo , Metabolismo dos Lipídeos , Fosfoenolpiruvato/metabolismo , Fosfopiruvato Hidratase/genética , Fosfopiruvato Hidratase/fisiologia , Filogenia , Plastídeos/enzimologia , Conformação Proteica , Reação em Cadeia da Polimerase em Tempo Real , Sementes/enzimologia , Sementes/metabolismo , Alinhamento de Sequência , Análise de Sequência de DNA , TranscriptomaRESUMO
BACKGROUND/PURPOSE: To measure the inflammatory response in terms of tumor necrosis factor-alpha (TNF-α) levels in cerebrospinal fluid (CSF), using bacteriolytic versus nonbacteriolytic antibiotic therapy and adjunctive treatment with dexamethasone in an experimental rabbit model of pneumococcal meningitis. METHODS: In a rabbit model of pneumococcal meningitis, we tested CSF TNF-α levels in several samples from rabbits infected with the HUB 2349 strain and treated with ceftriaxone 100 mg/kg/d, ceftriaxone plus vancomycin 30 mg/kg/d, or daptomycin at 15 mg/kg or 25 mg/kg. Daptomycin schedules were compared with the same doses in combination with dexamethasone at 0.125 mg/kg every 12 hours over a 26-hour period. RESULTS: The ceftriaxone group had the highest levels of TNF-α. TNF-α levels were significantly higher after ceftriaxone administration than in both daptomycin groups. The high-dose daptomycin group presented the lowest inflammatory levels in CSF samples. Adjunctive treatment with dexamethasone in this group modulated the inflammatory response, bringing down CSF TNF-α levels. CONCLUSION: CSF TNF-α levels were significantly lower in rabbits treated with daptomycin than in rabbits treated with ceftriaxone. Daptomycin avoided the inflammatory peak after administration observed in ceftriaxone-treated rabbits. The use of daptomycin plus dexamethasone achieved a significantly larger reduction in CSF TNF-α levels.
Assuntos
Antibacterianos/administração & dosagem , Líquido Cefalorraquidiano/química , Meningite Pneumocócica/microbiologia , Meningite Pneumocócica/patologia , Streptococcus pneumoniae/efeitos dos fármacos , Fator de Necrose Tumoral alfa/líquido cefalorraquidiano , Resistência beta-Lactâmica , Animais , Antibacterianos/farmacologia , Anti-Inflamatórios/administração & dosagem , Cefalosporinas/farmacologia , Dexametasona/administração & dosagem , Modelos Animais de Doenças , Feminino , Meningite Pneumocócica/tratamento farmacológico , Penicilinas/farmacologia , CoelhosRESUMO
Fatty acid desaturases (FAD) play an important role in plant lipid metabolism and they can be found in several subcellular compartments such as the plastids and endoplasmic reticulum. Lipids are critical components of the cell membrane and, as a consequence, they are fundamental for the proper growth and development of all living organisms. We have used sequences from the conserved regions of known omega-3-desaturases to design degenerated oligonucleotides and clone a cDNA encoding a plastidial omega-3 desaturase from sunflower (HaFAD7). From its presumed full-length sequence, we predict that Hafad7 encodes a protein of 443 amino acids with a molecular mass of 50.8 kDa, and that it contains a putative chloroplast transit peptide of 51 amino acids. The predicted hydrophobicity of the protein identifies four potential membrane-spanning regions and, according to the TargetP algorithm, the protein should be targeted to the plastid/chloroplast membrane. RT-PCR analysis of its expression shows the transcript is preferentially expressed in photosynthetically active tissues. Heterologous expression of this protein in the unicellular cyanobacterium Synechocystis sp. PCC 6803 confirmed that the protein produced from this cDNA has omega-3 desaturase activity.
Assuntos
Cianobactérias/metabolismo , Ácidos Graxos Dessaturases/metabolismo , Helianthus/enzimologia , Plastídeos/enzimologia , Synechocystis/genética , Synechocystis/metabolismo , Sequência de Aminoácidos , Clonagem Molecular , Cianobactérias/genética , DNA Complementar , Ácidos Graxos Dessaturases/genética , Regulação Enzimológica da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Helianthus/genética , Dados de Sequência Molecular , FilogeniaRESUMO
This study aimed to determine the effect of dexamethasone in combination with low-dose or high-dose daptomycin for the treatment of penicillin- and cephalosporin-resistant pneumococcal meningitis. Efficacy (ΔCFU/mL) and cerebrospinal fluid (CSF) levels of daptomycin at 15mg/kg and 25mg/kg were studied in a rabbit model of pneumococcal meningitis, comparing them with the same doses in combination with dexamethasone at 0.125mg/kg every 12h over a 26-h period against two different Streptococcus pneumoniae strains, HUB 2349 and ATCC 51916 with daptomycin minimum inhibitory concentrations (MICs) of 0.09mg/L and 0.19mg/L, respectively. Daptomycin levels in CSF were lower when dexamethasone was given concurrently. Against strain HUB 2349, therapeutic failure occurred with daptomycin 15mg/kg+dexamethasone; daptomycin 25mg/kg+dexamethasone was better at reducing bacterial counts than the lower dose throughout treatment. Against the highly cephalosporin-resistant ATCC 51916 strain, daptomycin 15mg/kg+dexamethasone achieved a lower bacterial decrease than daptomycin 15mg/kg alone, and therapeutic failure at 24h occurred in the daptomycin 15mg/kg+dexamethasone group. Addition of dexamethasone to a 25mg/kg daptomycin dose did not affect the efficacy of daptomycin: it remained bactericidal throughout treatment. In conclusion, against the studied strains, low-dose (15mg/kg/day) daptomycin is affected by concomitant use of dexamethasone: CSF levels are reduced and its bacterial efficacy is affected. At a higher daptomycin dose (25mg/kg/day), however, the use of dexamethasone does not alter efficacy; the combination appears to be a good choice for the treatment of pneumococcal meningitis.
Assuntos
Antibacterianos/uso terapêutico , Daptomicina/uso terapêutico , Dexametasona/uso terapêutico , Meningite Pneumocócica/tratamento farmacológico , Animais , Antibacterianos/farmacocinética , Antibacterianos/farmacologia , Carga Bacteriana , Líquido Cefalorraquidiano/química , Daptomicina/farmacocinética , Daptomicina/farmacologia , Dexametasona/farmacologia , Farmacorresistência Bacteriana , Quimioterapia Combinada , Testes de Sensibilidade Microbiana , Coelhos , Streptococcus pneumoniae , Resultado do TratamentoRESUMO
Variations of the yeast free amino acid pool under different culture conditions were studied in two Saccharomyces strains, the laboratory haploid strain S288C and the industrial fermentative yeast IFI256. The internal amino acid pool of both strains was measured when grown in laboratory (minimal and complete) versus semiindustrial (molasses with or without added biotin and/or diammonium phosphate) media, in fermentable (glucose, fructose, sucrose) versus respirable (glycerol) carbon sources, in different temperatures (22, 30, and 37 degrees C), pHs (2.0-4.75), and growth rates (0.018-0.24 h-1) in continuous culture, and at different phases of the growth curve in batch culture (lag, exponential, early and late stationary). Results indicated that environmental conditions, particularly the presence of amino acids in the media, enormously influenced the intracellular amino acid concentration. Higher values were detected in molasses than in laboratory media and in fermentable carbon sources (glucose, fructose, sucrose) than in glycerol. Variations in the amino acid pool along the growth curve were greater at 37 degrees C than at other temperatures; in all cases, the highest values were measured at the beginning of the exponential phase. In continuous culture and at different growth rates, intracellular free amino acid concentrations increased by 3-10-fold when the growth rate was lower than 0.05 h-1, representing 20-35% of the total (free plus protein) amino acid content and indicating that amino acid yield was a partly growth-linked parameter.
Assuntos
Aminoácidos/metabolismo , Saccharomyces cerevisiae/metabolismo , Meios de Cultura , Fermentação/fisiologia , Proteínas Fúngicas/metabolismo , Glucose/metabolismo , Concentração de Íons de Hidrogênio , Saccharomyces cerevisiae/crescimento & desenvolvimento , TemperaturaRESUMO
Sunflower mutant lines with high saturated fatty acid content (palmitic or stearic) in the oil have a completely different set of triacylglycerols (TAG), some of which were not found in standard sunflowers. For optimum seed germination, all of these new TAG species must be effectively catabolized. The behavior of the TAG composition during germination in cotyledons of all these mutant lines showed two different phases: an initial phase (between 0 and 2 days after sowing) with a higher catalytic activity and a preference for TAG containing at least two oleic acid molecules and a second phase with lower TAG degradation rate and a low preference for TAG containing two saturated fatty acids usually accompanied by linoleic acid. Despite the elevated content of saturated fatty acids in some TAG species, the total TAG degradation rate and germination process were similar in these lines, suggesting that sunflower seed lipases do not show a marked preference for any TAG species.
Assuntos
Ácidos Graxos/análise , Helianthus/genética , Óleos de Plantas/análise , Sementes/fisiologia , Triglicerídeos/metabolismo , Cromatografia Gasosa , Germinação , Helianthus/metabolismo , Humanos , MutaçãoRESUMO
The triacylglycerol (TAG) composition of oils from new high-saturated sunflower lines has been studied by means of GLC. The TAG profiles have been compared with the TAG reconstruction made after lipase hydrolysis (according to the 2-random 1,3-random theory). New TAG species with asclepic (cis,Delta11-octadecenoic acid, isomer of oleic acid), araquidic, or behenic acids have been synthesized and identified in oils from mutant lines. The TAG molecular species that contain asclepic acid instead of oleic acid have a longer retention time. Because each mutant oil has a specific TAG GLC pattern, this method could be used for a more precise validation of oil type than current fatty acid methyl ester analysis. The comparison of the results obtained by GLC with the reconstruction after pancreatic lipase hydrolysis shows, in general, a good agreement between both methods. However, results shown in this paper show that this is not always the case. TAG species containing two molecules of linoleic acid show a higher presence of palmitic or stearic acid than could be expected from a random distribution. The abundance of SLL increased in proportion to the stearic acid content of the oil, and the amount of TAG species with three unsaturated fatty acids (LLL or OLL) was therefore reduced.
Assuntos
Ácidos Graxos/análise , Helianthus/genética , Óleos de Plantas/análise , Sementes/química , Triglicerídeos/análise , Cromatografia Gasosa , Helianthus/química , Humanos , Mutação , Triglicerídeos/isolamento & purificaçãoRESUMO
Modifications of the fatty acid composition of plant vegetative tissues produce deficient plant growth. To determine the expression of the seed high-saturated sunflower (Helianthus annuus L.) mutant character during the vegetative cycle, five sunflower mutant lines (three high-stearic and two high-palmitic) have been studied during their germination and vegetative cycle. No significant variations with regard to the control lines were observed in the mutant vegetative tissue lipids; however, during seed germination important differences between lines were found. Although in the early steps of germination the palmitic and stearic acid levels in the respective mutants seedling cotyledons continued being higher than those of the control lines, they decreased and reached values similar to the controls, except in CAS-3. Variations in the cotyledon palmitic acid content with regard to the control line were also observed in high-stearic mutants, suggesting the expression of a modified acyl-ACP thioesterase or recycling of seed fatty acids during seedling development.
Assuntos
Ácidos Graxos/química , Helianthus/química , Lipídeos/análise , Mutação , Helianthus/genéticaRESUMO
The seed lipids from five sunflower mutants, two with high palmitic acid contents, one of them in high oleic background, and three with high stearic acid contents, have been characterized. All lipid classes of these mutant seeds have increased saturated fatty acid content although triacylglycerols had the highest levels. The increase in saturated fatty acids was mainly at the expense of oleic acid while linoleic acid levels remained unchanged. No difference between mutants and standard sunflower lines used as controls was found in minor fatty acids: linolenic, arachidic, and behenic. In the high-palmitic mutants palmitoleic acid (16:1n-7) and some palmitolinoleic acid (16:2n-7, 16:2n-4) also appeared. Phosphatidylinositol, the lipid with the highest palmitic acid content in controls, also had the highest content of palmitic or stearic acids, depending on the mutant type, suggesting that saturated fatty acids are needed for its physiological function. Positional analysis showed that mutant oils have very low content of saturated fatty acids in the sn-2 position of triacylglycerols, between the content of olive oil and cocoa butter.
Assuntos
Ácidos Graxos/análise , Helianthus/química , Helianthus/genética , Lipídeos/química , Sementes/química , Mutação , Ácido Palmítico/análise , Sementes/genética , Ácidos Esteáricos/análise , Triglicerídeos/análiseRESUMO
The chemical composition (total oil content, fatty acids, triacylglycerols (TAGs) and polar compounds) of six walnuts (Juglans regia L.) cultivars (Lauzeronne, Franquette, Hartley, Local pt, Local gd and Parisienne) collected from Mateur (north of Tunisia) was evaluated. The major fatty acids found in the walnut oils are linoleic acid (60.42-65.77%), oleic acid (13.21-19.94%) and linolenic acid (7.61-13%). The TAG species were mainly composed of trilinolein (LLL), dilinoleoyl-linolenoyl-glycerol, dilinoleoyl-oleoyl-glycerol and palmitoyl-dilinoleoyl-glycerol classes. The results revealed that Local pt variety has the highest level of oil (62.56%), linoleic acid (65.77%) and LLL (33.48%). Significant differences among oil samples were observed, therefore showing a great variability in the oil composition among cultivars.
Assuntos
Ácidos Graxos/análise , Juglans/química , Triglicerídeos/análise , Juglans/genética , TunísiaRESUMO
Three cDNAs encoding different phosphoglycerate kinase (PGK, EC 2.7.2.3) isoforms, two cytosolic (HacPGK1 and HacPGK2) and one plastidic (HapPGK), were cloned and characterized from developing sunflower (Helianthus annuus L.) seeds. The expression profiles of these genes showed differences in heterotrophic tissues, such as developing seeds and roots, where HacPGK1 was predominant, while HapPGK was highly expressed in photosynthetic tissues. The cDNAs were expressed in Escherichia coli, and the corresponding proteins purified to electrophoretic homogeneity, using immobilized metal ion affinity chromatography, and biochemically characterized. Despite the high level of identity between sequences, the HacPGK1 isoform showed strong differences in terms of specific activity, temperature stability and pH sensitivity in comparison to HacPGK2 and HapPGK. A polyclonal immune serum was raised against the purified HacPGK1 isoform, which showed cross-immunoreactivity with the other PGK isoforms. This serum allowed the localization of high expression levels of PGK isozymes in embryo tissues.
Assuntos
Helianthus/enzimologia , Helianthus/crescimento & desenvolvimento , Fosfoglicerato Quinase/genética , Fosfoglicerato Quinase/metabolismo , Sementes/enzimologia , Sementes/crescimento & desenvolvimento , Sequência de Aminoácidos , Clonagem Molecular , Citosol/enzimologia , DNA Complementar/genética , Estabilidade Enzimática , Evolução Molecular , Regulação da Expressão Gênica de Plantas , Helianthus/citologia , Helianthus/genética , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Cinética , Modelos Moleculares , Dados de Sequência Molecular , Fosfoglicerato Quinase/química , Filogenia , Plastídeos/enzimologia , Estrutura Terciária de Proteína , Transporte Proteico , Sementes/citologia , Sementes/genética , Análise de Sequência de DNA , TemperaturaRESUMO
A full-length hexokinase cDNA, HaHXK1, was cloned and characterized from Helianthus annuus L. developing seeds. Based on its sequence and phylogenetic relationships, HaHXK1 is a membrane-associated (type-B) hexokinase. The predicted structural model resembles known hexokinase structures, folding into two domains of unequal size: a large and a small one separated by a deep cleft containing the residues involved in the enzyme active site. A truncated version, without the 24 N-terminal residues, was heterologously expressed in Escherichia coli, purified to electrophoretic homogeneity using immobilized metal ion affinity chromatography and biochemically characterized. The purified enzyme behaved as a monomer on size exclusion chromatography and had a specific activity of 19.3 µmol/min/mg protein, the highest specific activity ever reported for a plant hexokinase. The enzyme had higher affinity for glucose and mannose relative to fructose, but the enzymatic efficiency was higher with glucose. Recombinant HaHXK1 was inhibited by ADP and was insensitive either to glucose-6-phosphate or to trehalose-6-phosphate. Its expression profile showed higher levels in heterotrophic tissues, developing seeds and roots, than in photosynthetic ones. A time course of HXK activity and expression in seeds showed that the highest HXK levels are found at the early stages of reserve compounds, lipids and proteins accumulation.
Assuntos
Helianthus/enzimologia , Hexoquinase/metabolismo , Proteínas de Armazenamento de Sementes/metabolismo , Sementes/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , DNA Complementar/genética , Escherichia coli/enzimologia , Escherichia coli/genética , Regulação Enzimológica da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Helianthus/química , Helianthus/genética , Helianthus/metabolismo , Hexoquinase/química , Hexoquinase/genética , Hexoquinase/isolamento & purificação , Concentração de Íons de Hidrogênio , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas de Armazenamento de Sementes/química , Proteínas de Armazenamento de Sementes/genética , Sementes/enzimologia , Sementes/genética , Alinhamento de Sequência , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , TemperaturaRESUMO
D type phospholipases (PLD) are enzymes that hydrolyze the head group of phospholipids to produce phosphatidic acid. This activity is ubiquitous in plant tissues, and has been isolated and characterized from different species and organs. Several families of these proteins have been described in plants on the basis of their gene sequences (PLD alpha, beta, gamma, delta, zeta and epsilon). They have been shown to be involved in many metabolic events, such as response to abiotic stress, signal transduction, and membrane lipid turnover and degradation. In the present study, PLD activity was measured in the soluble fractions isolated from different organs of this plant. A PLD of alpha type was cloned from leaf cDNA that was responsible for most of this activity. The gene encoding this 810 aa protein was heterologously expressed in E. coli. This protein was not lethal for the eukaryotic host, although it altered its phospholipid profile. PLDalpha was purified to almost homogeneity by His-tag affinity chromatography, displaying an optimum pH of 6.5 and strong dependence on the presence of Ca(2+) and SDS in the assay medium. The enzyme was active towards phosphatidylcholine, Phosphatidylethanolamine and phosphatidylglycerol. Furthermore, the HaPLDalpha gene was found to be expressed at high levels in leaf and stem tissues.