Assuntos
Arabidopsis/genética , Genes de Plantas , Canais Iônicos/genética , Receptores de Glutamato/genética , Terminologia como Assunto , Arabidopsis/metabolismo , Canais Iônicos/química , Canais Iônicos/classificação , Canais Iônicos/metabolismo , Filogenia , Plantas/genética , Receptores de Glutamato/química , Receptores de Glutamato/classificação , Receptores de Glutamato/metabolismoRESUMO
The action of gamma-aminobutyrate (GABA) as an intercellular signaling molecule has been intensively studied, but the role of this amino acid metabolite in intracellular metabolism is poorly understood. In this work, we identify a Saccharomyces cerevisiae homologue of the GABA-producing enzyme glutamate decarboxylase (GAD) that is required for normal oxidative stress tolerance. A high copy number plasmid bearing the glutamate decarboxylase gene (GAD1) increases resistance to two different oxidants, H(2)O(2) and diamide, in cells that contain an intact glutamate catabolic pathway. Structural similarity of the S. cerevisiae GAD to previously studied plant enzymes was demonstrated by the cross-reaction of the yeast enzyme to a antiserum directed against the plant GAD. The yeast GAD also bound to calmodulin as did the plant enzyme, suggesting a conservation of calcium regulation of this protein. Loss of either gene encoding the downstream steps in the conversion of glutamate to succinate reduced oxidative stress tolerance in normal cells and was epistatic to high copy number GAD1. The gene encoding succinate semialdehyde dehydrogenase (UGA5) was identified and found to be induced by H(2)O(2) exposure. Together, these data strongly suggest that increases in activity of the glutamate catabolic pathway can act to buffer redox changes in the cell.
Assuntos
Regulação Enzimológica da Expressão Gênica , Glutamato Descarboxilase/genética , Glutamato Descarboxilase/metabolismo , Estresse Oxidativo , Saccharomyces cerevisiae/enzimologia , Sequência de Aminoácidos , Calmodulina/metabolismo , Clonagem Molecular , Diamida/farmacologia , Resistência Microbiana a Medicamentos , Dosagem de Genes , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Genes Fúngicos/genética , Glutamato Descarboxilase/química , Ácido Glutâmico/metabolismo , Peróxido de Hidrogênio/farmacologia , Hidroxibutirato Desidrogenase/genética , Hidroxibutirato Desidrogenase/metabolismo , Dados de Sequência Molecular , Oxidantes/farmacologia , Ligação Proteica , Proteínas Recombinantes de Fusão , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Ácido gama-Aminobutírico/metabolismo , Ácido gama-Aminobutírico/farmacologiaRESUMO
Two distinct cDNA clones encoding for the glutamate decarboxylase (GAD) isoenzymes GAD1 and GAD2 from Arabidopsis (L.) Heynh. were characterized. The open reading frames for GAD1 and GAD2 were expressed in Escherichia coli and the recombinant proteins were purified by affinity chromatography. Analysis of the recombinant proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot analysis suggest that GAD1 and GAD2 encode for 58- and 56-kD peptides, respectively. The enzymatic activities of the pure recombinant GAD1 and GAD2 proteins were stimulated 35- and 13-fold, respectively, by Ca2+/calmodulin but not by Ca2+ or calmodulin alone. Southern-blot analysis of genomic DNA suggests that there is only one copy of each gene in Arabidopsis. The GAD1 transcript and a corresponding 58-kD peptide were detected in roots only. Conversely, the GAD2 transcript and a corresponding 56-kD peptide were detected in all organs tested. The specific activity, GAD2 transcript, and 56-kD peptide increased in leaves of plants treated with 10 mM NH4Cl, 5 mM NH4NO3, 5 mM glutamic acid, or 5 mM glutamine as the sole nitrogen source compared with samples from plants treated with 10 mM KNO3. The results from these experiments suggest that in leaves GAD activity is partially controlled by gene expression or RNA stability. Results from preliminary analyses of different tissues imply that these tendencies were not the same in flower stalks and flowers, suggesting that other factors may control GAD activity in these organs. The results from this investigation demonstrate that GAD activity in leaves is altered by different nitrogen treatments, suggesting that GAD2 may play a unique role in nitrogen metabolism.
Assuntos
Arabidopsis/genética , Glutamato Descarboxilase/genética , Isoenzimas/genética , Sequência de Aminoácidos , Arabidopsis/enzimologia , Sequência de Bases , Southern Blotting , Cálcio/metabolismo , Calmodulina/metabolismo , Clonagem Molecular , DNA Complementar , Escherichia coli/genética , Glutamato Descarboxilase/metabolismo , Isoenzimas/metabolismo , Dados de Sequência Molecular , Nitrogênio/metabolismo , Ligação Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homologia de Sequência de AminoácidosRESUMO
Screening of a cDNA library from soybean (Glycine max (L.) Merr. cv. Century) with probes based upon cytosolic ascorbate peroxidase (APx; EC 1.11.1.11) genes identified two full-length clones (SOYAPx1, SOYAPx2) apparently encoding for different soybean leaf cytosolic APxs. The deduced amino acid sequences of the two APx cDNA products differed in 13 of the 250 amino acids. The SOYAPx1 cDNA was identical to the cytosolic APx cDNA previously found in soybean root nodules. Escherichia coli expression systems were developed using both soybean APx cDNAs. Recombinant SOYAPx1 and SOYAPx2 were then utilized to characterize the enzymatic properties of the two APx cDNA products.
Assuntos
Escherichia coli/metabolismo , Glycine max/enzimologia , Peroxidases/genética , Sequência de Aminoácidos , Ascorbato Peroxidases , Sequência de Bases , Citosol/enzimologia , DNA Complementar , DNA de Plantas , Eletroforese em Gel de Poliacrilamida , Expressão Gênica , Dados de Sequência Molecular , Peroxidases/isolamento & purificação , Peroxidases/metabolismo , Folhas de Planta , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Glycine max/genéticaRESUMO
Two distinct cDNA clones encoding NAD(H)-dependent glutamate dehydrogenase (NAD[H]-GDH) in Arabidopsis thaliana were identified and sequenced. The genes corresponding to these cDNA clones were designated GDH1 and GDH2. Analysis of the deduced amino acid sequences suggest that both gene products contain putative mitochondrial transit polypeptides and NAD(H)- and alpha-ketoglutarate-binding domains. Subcellular fractionation confirmed the mitochondrial location of the NAD(H)-GDH isoenzymes. In addition, a putative EF-hand loop, shown to be associated with Ca2+ binding, was identified in the GDH2 gene product but not in the GDH1 gene product. GDH1 encodes a 43.0-kD polypeptide, designated alpha, and GDH2 encodes a 42.5-kD polypeptide, designated beta. The two subunits combine in different ratios to form seven NAD(H)-GDH isoenzymes. The slowest-migrating isoenzyme in a native gel, GDH1, is a homohexamer composed of alpha subunits, and the fastest-migrating isoenzyme, GDH7, is a homohexamer composed of beta subunits. GDH isoenzymes 2 through 6 are heterohexamers composed of different ratios of alpha and beta subunits. NAD(H)-GDH isoenzyme patterns varied among different plant organs and in leaves of plants irrigated with different nitrogen sources or subjected to darkness for 4 d. Conversely, there were little or no measurable changes in isoenzyme patterns in roots of plants treated with different nitrogen sources. In most instances, changes in isoenzyme patterns were correlated with relative differences in the level of alpha and beta subunits. Likewise, the relative difference in the level of alpha or beta subunits was correlated with changes in the level of GDH1 or GDH2 transcript detected in each sample, suggesting that NAD(H)-GDH activity is controlled at least in part at the transcriptional level.
Assuntos
Arabidopsis/enzimologia , Arabidopsis/genética , Genes de Plantas , Glutamato Desidrogenase/biossíntese , Glutamato Desidrogenase/genética , Sequência de Aminoácidos , Sequência de Bases , Cloroplastos/enzimologia , Clonagem Molecular , Escuridão , Glutamato Desidrogenase/química , Isoenzimas/biossíntese , Isoenzimas/química , Isoenzimas/genética , Cinética , Luz , Microcorpos/enzimologia , Mitocôndrias/enzimologia , Dados de Sequência Molecular , Folhas de Planta , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Homologia de Sequência de AminoácidosRESUMO
Proteins in extracts from cotyledons, hypocotyls, and roots of 5-d-old, dark-grown soybean (Glycine max L. Merr. cv Williams) seedlings were separated by polyacrylamide gel electrophoresis. Three isoforms of glutamate dehydrogenase (GDH) were resolved and visualized in gels stained for GDH activity. Two isoforms with high electrophoretic mobility, GDH1 and GDH2, were in protein extracts from cotyledons and a third isoform with the lowest electrophoretic mobility, GDH3, was identified in protein extracts from root and hypocotyls. Subcellular fractionation of dark-grown soybean tissues demonstrated that GDH3 was associated with intact mitochondria. GDH3 was purified to homogeneity, as determined by native and sodium dodecyl sulfate-polyacrylamide gels. The isoenzyme was composed of a single 42-kD subunit. The pH optima for the reductive amination and the oxidative deamination reactions were 8.0 and 9.3, respectively. At any given pH, GDH activity was 12- to 50-fold higher in the direction of reductive amination than in the direction of the oxidative deamination reaction. GDH3 had a cofactor preference for NAD(H) over NADP(H). The apparent Michaelis constant values for [alpha]-ketoglutarate, ammonium, and NADH at pH 8.0 were 3.6, 35.5, and 0.07 mM, respectively. The apparent Michaelis constant values for glutamate and NAD were 15.8 and 0.10 mM at pH 9.3, respectively. To our knowledge, this is the first biochemical and physical characterization of a purified mitochondrial NAD(H)-dependent GDH isoenzyme from soybean.
RESUMO
A full-length cDNA clone encoding aspartate aminotransferase (AAT) has been identified from a carrot root cDNA library. Degenerate oligo primers were synthesized from the known amino acid sequence of AAT form I from carrot (Daucus carota L. cv Danvers). These primers were utilized in a polymerase chain reaction to amplify a portion of a carrot AAT gene from first strand cDNA synthesized from poly(A)(+) RNA isolated from 5-d-old cell suspension cultures. The resulting 750-bp fragment was cloned, mapped, and sequenced. The cloned fragment, mpAAT1, was used as a probe to identify a full-length cDNA clone in a library constructed from poly(A)(+) RNA isolated from carrot roots. A 1.52-kb full-length clone, AAT7, was isolated and sequenced. AAT7 has 54% nucleotide identity with both the mouse cytoplasmic and mitochondrial AAT genes. The deduced amino acid sequence has 52 and 53% identity with the deduced amino acid sequences of mouse cytoplasmic and mitochondrial AAT genes, respectively. Further analysis of the sequence data suggests that AAT7 encodes a cytoplasmic form of carrot AAT; the evidence includes the (a) absence of a transit or signal sequence, (b) lack of "m-residues," or invariant mitochondrial residues, in the carrot AAT sequence, and (c) high degree of sequence similarity with the amino acid sequence previously obtained for form I of carrot, a cytoplasmic isoenzyme. High- and low-stringency hybridizations to Southern blots of carrot nuclear DNA with AAT7 show that AAT is part of a small multigene family. Northern blot analysis of AAT7 suggests that AAT is expressed throughout cell culture up to 7 d and is highly expressed in roots but not in leaves.
RESUMO
Several isoenzymic forms of aspartate aminotransferase (AAT) have been identified in protein extracts from carrot (Daucus carota) cell suspension cultures. The cellular location of the major form (form I) of AAT in carrot suspension cultures was determined by heat inactivation, subcellular fractionation, and amino acid sequence analysis. In mammalian systems, there are two forms of AAT, a heat-stable cytoplasmic form and a heat-labile form in the mitochondria. The thermostability of three isoenzymes of carrot AAT was examined, and the results showed that form I was more thermostable than forms II or III. Organelles were separated in sucrose gradients by isopynic centrifugation. Activity for form I was identified in the soluble fractions and not in fractions containing peroxisomes, proplastids, or mitochondria. Form I was purified to homogeneity and endoproteolytically cleaved, and the peptide fragments were separated by reverse phase chromatography. Analysis of the sequence data from two of the polypeptides showed that the amino acid identity of form I is more conserved to the animal cytoplasmic AAT than to animal mitochondrial AAT sequences. These data strongly suggest that form I of AAT from carrot is the cytoplasmic isoenzyme. Additionally, a rapid purification scheme for form I of AAT from carrot is presented using selective heat denaturation and anion-exchange chromatography.
RESUMO
Three aspartate aminotransferase isoenzymes were identified from extracts of carrot (Daucus carota L.) cell suspension cultures. These isoenzymes were separated by DEAE chromatography and were analyzed on native gradient polyacrylamide gels. The relative molecular weights of the isoenzymes were 111,000 +/- 5000, 105,000 +/- 5000, and 94,000 +/- 4000 daltons; they were designated forms I, II, and III, respectively. Form I, the predominant form, has been purified to apparent homogeneity (>300-fold) using immunoaffinity chromatography with rabbit anti-pig AAT antibodies. Form I has a subunit size of 43,000 M(r), as determined on sodium dodecyl sulfate polyacrylamide gel electrophoresis. Isoelectric focusing (IEF)-PAGE has resolved three bands at a pl of approximately 5.2. Form I may be composed of subunits of similar molecular weight and different charges, and the three bands with AAT activity on the IEF-PAGE gel are a combination of hetero- and homodimers. Form I has a broad pH optimum of 7.5 to 10.0. K(m) values of 23.6, 2.8, 0.05, and 0.22 millimolar were obtained for glutamate, aspartate, oxaloacetate, and alpha-ketoglutarate, respectively. The mode of action is a ping-pong-bi-bi mechanism.
RESUMO
Multiple forms of homoserine dehydrogenase (HSDH) from carrot (Daucus carota L.) have been identified. One form of HSDH (T-form) has a relative molecular weight of 240,000 and is strongly inhibited by threonine. Another form (K-form) has a relative molecular weight of 180,000 and is insensitive to inhibition by threonine. The interconversion of these two forms is dependent upon the presence or absence of threonine and potassium. Polyacrylamide electrophoretic gels stained for HSDH activity and protein, paralleled with Western blot analysis, verified the interconversion of the T- and K-forms in 5 millimolar threonine and 100 millimolar potassium, respectively. Carrot HSDH also aggregates to form higher molecular weight complexes of 240,000 up to 720,000 M(r.) Polyclonal antibody from mouse was raised against the T-form (240,000 M(r)) of carrot HSDH. Specificity of the mouse antisera to carrot HSDH was verified by immunoprecipitation and Western blot analysis. The T-form, K-form, and all of the higher molecular aggregates of carrot HSDH cross-reacted with the anti-HSDH antiserum. The antiserum also cross-reacted with soybean HSDH, but did not cross-react with either of the two HSDH forms found in Escherichia coli. A model for the in vivo regulation of threonine biosynthesis in the chloroplast is presented. The model is based on the interconversion of the HSDH forms by potassium and threonine.
RESUMO
Introns in the cytochrome oxidase subunit II (COXII) gene of plant mitochondrial DNA (mtDNA) have been observed only in monocots. The COXII genes in dicots investigated to date do not contain introns. This is the first report of an intron in the COXII gene of a dicot. The presence of an intron in the carrot COXII intron was verified by restriction mapping and hybridization using specific maize and wheat COXII probes. Regions of the carrot COXII intron are homologous to the maize COXII intron and homologous to the wheat COXII intron-insert as demonstrated by hybridization. Homology of these regions was confirmed by sequencing portions of the gene. A comparison of the restriction map of the carrot COXII gene with the restriction maps of the COXII genes from pea, Oenothera, maize, wheat, and rice revealed that the carrot map coincides with the rice restriction map.