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Kinetic and Crystallographic Studies on Glyceraldehyde-3-Phosphate Dehydrogenase from Trypanosoma cruzi in Complex with Iodoacetate

GUIDO, Rafael Victorio Carvalho; BALLIANO, Tatiane L.; ANDRICOPULO, Adriano Defini; OLIVA, Glaucius
Fonte: BENTHAM SCIENCE PUBL LTD Publicador: BENTHAM SCIENCE PUBL LTD
Tipo: Artigo de Revista Científica
ENG
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126.32%
Kinetic and crystallographic studies on the formation of the complex between iodoacetate and the enzyme glyceraldehyde-3-phosphate dehydrogenase from Trypanosoma cruzi were conducted in order to investigate the mechanistic and structural basis underlying enzyme inactivation. The crystallographic complex reveal important structural features useful for the design of novel inhibitors.; Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP); FAPESP (The State of Sao Paulo Research Foundation); CNPq (The National Council for Scientific and Technological Development), Brazil; Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Expression, purification and kinetic characterization of His-tagged glyceraldehyde-3-phosphate dehydrogenase from Trypanosoma cruzi

CHELESKI, Juliana; FREITAS, Renato F.; WIGGERS, Helton Jose; ROCHA, Josmar R.; ARAUJO, Ana Paula Ulian de; MONTANARI, Carlos A.
Fonte: ACADEMIC PRESS INC ELSEVIER SCIENCE Publicador: ACADEMIC PRESS INC ELSEVIER SCIENCE
Tipo: Artigo de Revista Científica
ENG
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126.41%
Trypanosomes are flagellated protozoa responsible for serious parasitic diseases that have been classified by the World Health Organization as tropical sicknesses of major importance. One important drug target receiving considerable attention is the enzyme glyceraldehyde-3-phosphate dehydrogenase from the protozoan parasite Trypanosoma cruzi, the causative agent of Chagas disease (T. cruzi Glyceraldehyde-3-phosphate dehydrogenase (TcGAPDH); EC 1.2.1.12). TcGAPDH is a key enzyme in the glycolytic pathway of T. cruzi and catalyzes the oxidative phosphorylation of D-glyceraldehyde-3-phosphate (G3P) to 1,3-bisphosphoglycerate (1,3-BPG) coupled to the reduction of oxidized nicotinamide adenine dinucleotide, (NAD(+)) to NADH, the reduced form. Herein, we describe the cloning of the T. cruzi gene for TcGAPDH into the pET-28a(+) vector, its expression as a tagged protein in Escherichia coli, purification and kinetic characterization. The His(6)-tagged TcGAPDH was purified by affinity chromatography. Enzyme activity assays for the recombinant His(6)-TcGAPDH were carried out spectrophotometrically to determine the kinetic parameters. The apparent Michaelis-Menten constant (K(M)(app)) determined for D-glyceraldehyde-3-phosphate and NAD(+) were 352 +/- 21 and 272 +/- 25 mu M...

Characterization of Heparin-induced Glyceraldehyde-3-phosphate Dehydrogenase Early Amyloid-like Oligomers and Their Implication in alpha-Synuclein Aggregation

Torres-Bugeau, Clarisa M.; Avila, Cesar L.; Raisman-Vozari, Rita; Papy-Garcia, Dulce; Itri, Rosangela; Barbosa, Leandro Ramos Souza; Cortez, Leonardo M.; Sim, Valerie L.; Chehin, Rosana N.
Fonte: AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC; BETHESDA Publicador: AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC; BETHESDA
Tipo: Artigo de Revista Científica
ENG
Relevância na Pesquisa
86.43%
Lewy bodies and Lewy neurites, neuropathological hallmarks of several neurological diseases, are mainly made of filamentous assemblies of alpha-synuclein. However, other macromolecules including Tau, ubiquitin, glyceraldehyde-3-phosphate dehydrogenase, and glycosaminoglycans are routinely found associated with these amyloid deposits. Glyceraldehyde-3-phosphate dehydrogenase is a glycolytic enzyme that can form fibrillar aggregates in the presence of acidic membranes, but its role in Parkinson disease is still unknown. In this work, the ability of heparin to trigger the amyloid aggregation of this protein at physiological conditions of pH and temperature is demonstrated by infrared and fluorescence spectroscopy, dynamic light scattering, small angle x-ray scattering, circular dichroism, and fluorescence microscopy. Aggregation proceeds through the formation of short rod-like oligomers, which elongates in one dimension. Heparan sulfate was also capable of inducing glyceraldehyde-3-phosphate dehydrogenase aggregation, but chondroitin sulfates A, B, and C together with dextran sulfate had a negligible effect. Aided with molecular docking simulations, a putative binding site on the protein is proposed providing a rational explanation for the structural specificity of heparin and heparan sulfate. Finally...

Identification of electronic and structural descriptors of adenosine analogues related to inhibition of leishmanial glyceraldehyde-3-phosphate dehydrogenase

Lozano, Norka B. H.; Oliveira, Rafael F.; Weber, Karen C.; Honório, Kathia Maria; Guido, Rafael Victório Carvalho; Andricopulo, Adriano Defini; Silva, Alberico Borges Ferreira da
Fonte: MDPI AG; Basel Publicador: MDPI AG; Basel
Tipo: Artigo de Revista Científica
ENG
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126.23%
Quantitative structure-activity relationship (QSAR) studies were performed in order to identify molecular features responsible for the antileishmanial activity of 61 adenosine analogues acting as inhibitors of the enzyme glyceraldehyde 3-phosphate dehydrogenase of Leishmania mexicana (LmGAPDH). Density functional theory (DFT) was employed to calculate quantum-chemical descriptors, while several structural descriptors were generated with Dragon 5.4. Variable selection was undertaken with the ordered predictor selection (OPS) algorithm, which provided a set with the most relevant descriptors to perform PLS, PCR and MLR regressions. Reliable and predictive models were obtained, as attested by their high correlation coefficients, as well as the agreement between predicted and experimental values for an external test set. Additional validationprocedures were carried out, demonstrating that robust models were developed, providing helpful tools for the optimization of the antileishmanial activity of adenosine compounds.; FAPESP; CNPq; CAPES

Acquisition of flocculation phenotype by Kluyveromyces marxianus when overexpressing GAP1 gene encoding an isoform of glyceraldehyde-3-phosphate dehydrogenase

Almeida, Catarina; Queirós, Odília; Wheals, Alan; Teixeira, J. A.; Ferreira, Pedro Moradas
Fonte: Elsevier Publicador: Elsevier
Tipo: Artigo de Revista Científica
Publicado em //2003 ENG
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126.23%
The use of flocculating yeast strains has been considered as a convenient approach to obtain high cell densities in bioreactors with increasing productivity in continuous operations. In Kluyveromyces marxianus ATTC 10022, the GAP1 gene encodes an isoform of glyceraldehyde-3-phosphate dehydrogenase–p37—that is accumulated in the cell wall and is involved in flocculation. To test the use of p37 as a tool for engineering Kluyveromyces cells to display a flocculation phenotype, K. marxianus CCT 3172 was transformed with an expression vector containing GAP1. This vector is based on the pY37 previously described, harbouring a S11 Kluyveromyces origin of replication, and the expression of GAP1 is under the control of GAL1. Kluyveromyces cells overexpressing GAP1 acquired a flocculent phenotype together with the accumulation of p37 in the cell wall. The results support the use of GAP1 gene as a molecular tool for inducing flocculation.; Fundação para a Ciência e a Tecnologia (FCT) - BD/18203/98.

Application of the mammalian glyceraldehyde-3-phosphate dehydrogenase gene for sample quality control in multiplex PCR for diagnosis of leishmaniasis

Gonçalves,SC; Régis-da-Silva,CG; Brito,MEFC; Brandão-Filho,SP; Paiva-Cavalcanti,M
Fonte: Centro de Estudos de Venenos e Animais Peçonhentos - CEVAP, Universidade Estadual Paulista - UNESP Publicador: Centro de Estudos de Venenos e Animais Peçonhentos - CEVAP, Universidade Estadual Paulista - UNESP
Tipo: Artigo de Revista Científica Formato: text/html
Publicado em 01/01/2012 EN
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Leishmaniasis is a neglected disease endemic in five continents. It is a severe disease that may lead to death, and its early detection is important to avoid severe damage to affected individuals. Molecular methods to detect Leishmania are considered alternatives to overcome the limitations presented by conventional methods. The aim of this study was to develop multiplex PCR systems able to detect small amounts of target DNA of Leishmania infantum and Leishmania braziliensis, and the gene coding for glyceraldehyde-3-phosphate dehydrogenase (G3PD) in mammals, enabling quality evaluation of the sample simultaneously with detection of the specific target. The systems created for G3PD recognition were combined with detection systems for L. infantum and L. braziliensis to compose multiplex PCR systems for visceral (mVL) and cutaneous (mACL) leishmaniasis diagnosis. The multiplex PCR systems developed were assessed in blood samples from five different species of mammal reservoirs involved in the disease cycle in Brazil, and 96 and 52 human samples from patients with suspected visceral leishmaniasis (VL) and cutaneous leishmaniasis (ACL), respectively. Three G3PD detection systems were created (G3PD1, G3PD2 and G3PD3) with different product sizes...

Characterization of two glyceraldehyde-3-phosphate dehydrogenase isoenzymes from the pentalenolactone producer Streptomyces arenae.

Maurer, K H; Pfeiffer, F; Zehender, H; Mecke, D
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /02/1983 EN
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Pentalenolactone (PL) irreversibly inactivates the enzyme glyceraldehyde-3-phosphate dehydrogenase [D-glyceraldehyde-3-phosphate:NAD+ oxidoreductase (phosphorylating)] (EC 1.2.1.12) and thus is a potent inhibitor of glycolysis in both procaryotic and eucaryotic cells. We showed that PL-producing strain Streptomyces arenae TU469 contains a PL-insensitive glyceraldehyde-3-phosphate dehydrogenase under conditions of PL production. In complex media no PL production was observed, and a PL-sensitive glyceraldehyde-3-phosphate dehydrogenase, rather than the insensitive enzyme, could be detected. The enzymes had the same substrate specificity but different catalytic and molecular properties. The apparent Km values of the PL-insensitive and PL-sensitive enzymes for glyceraldehyde-3-phosphate were 100 and 250 microM, respectively, and the PL-sensitive enzyme was strongly inhibited by PL under conditions in which the PL-insensitive enzyme was not inhibited. The physical properties of the PL-insensitive enzyme suggest that the protein is an octamer, whereas the PL-sensitive enzyme, like other glyceraldehyde-3-phosphate dehydrogenases, appears to be a tetramer.

Nonreversible d-Glyceraldehyde 3-Phosphate Dehydrogenase of Plant Tissues 1

Kelly, G. J.; Gibbs, Martin
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /08/1973 EN
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Preparations of TPN-linked nonreversible d-glyceraldehyde 3-phosphate dehydrogenase (EC 1.2.1.9), free of TPN-linked reversible d-glyceraldehyde 3-phosphate dehydrogenase, have been obtained from green shoots, etiolated shoots, and cotyledons of pea (Pisum sativum), cotyledons of peanut (Arachis hypogea), and leaves of maize (Zea mays). The properties of the enzyme were similar from each of these sources: the Km values for d-glyceraldehyde 3-phosphate and TPN were about 20 μm and 3 μm, respectively. The enzyme activity was inhibited by l-glyceraldehyde 3-phosphate, d-erythrose 4-phosphate, and phosphohydroxypyruvate. Activity was found predominantly in photosynthetic and gluconeogenic tissues of higher plants. A light-induced, phytochrome-mediated increase of enzyme activity in a photosynthetic tissue (pea shoots) was demonstrated. Appearance of enzyme activity in a gluconeogenic tissue (endosperm of castor bean, Ricinus communis) coincided with the conversion of fat to carbohydrate during germination. In photosynthetic tissue, the enzyme is located outside the chloroplast, and at in vivo levels of triose-phosphates and pyridine nucleotides, the activity is probably greater than that of DPN-linked reversible d-glyceraldehyde 3-phosphate dehydrogenase. Several possible roles for the enzyme in plant carbohydrate metabolism are considered.

Mutant analysis of glyceraldehyde 3-phosphate dehydrogenase in Escherichia coli

Hillman, Jeffrey D.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em 01/04/1979 EN
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NAD+-specific glyceraldehyde 3-phosphate dehydrogenase (EC 1.2.1.12) from Escherichia coli was purified to homogeneity by a relatively simple procedure involving affinity chromatography on agarose–hexane–NAD+ and repeated crystallization. Rabbit antiserum directed against this protein produced one precipitin line in double-diffusion studies against the pure enzyme, and two lines against crude extracts of wild-type E. coli strains. Both precipitin lines represent the interaction of antibody with determinants specific for glyceraldehyde 3-phosphate dehydrogenase. Nine independent mutants of E. coli lacking glyceraldehyde 3-phosphate dehydrogenase activity all possessed some antigenic cross-reacting material to the wild-type enzyme. The mutants could be divided into three groups on the basis of the types and amounts of precipitin lines observed in double-diffusion experiments; one group formed little cross-reacting material. The cross-reacting material in crude cell-free extracts of several of the mutant strains were also tested for alterations in their affinity for NAD+ and their phosphorylative activity. The cumulative data indicate that the protein in several of the mutant strains is severely altered, and thus that glyceraldehyde 3-phosphate dehydrogenase is unlikely to have an essential...

Aspects of the chemistry of d-glyceraldehyde 3-phosphate dehydrogenase

Trentham, D. R.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /10/1968 EN
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86.47%
Crystalline d-glyceraldehyde 3-phosphate dehydrogenase from lobster tail contains 4 moles of NAD+ bound and reacts specifically with 4 moles of iodoacetic acid/mole of tetramer. The essential thiol group of d-glyceraldehyde 3-phosphate dehydrogenase appears to react with iodoacetic acid with a rate constant for the overall process that is independent of the extent of carboxymethylation. The d-glyceraldehyde 3-phosphate dehydrogenase–NAD+ absorption band has a variable molar extinction coefficient in the presence of phosphate that may be correlated with a proton dissociation of pK 6·86. The binding of NAD+ to d-glyceraldehyde 3-phosphate dehydrogenase weakens as alkylating agents react with the enzyme, and NAD+ promotes the reactivity of the essential thiol group. It is suggested that, on binding to d-glyceraldehyde 3-phosphate dehydrogenase, NAD+ lowers the pK of the essential thiol group, resulting in a catalytic role of NAD+ in the reaction catalysed by d-glyceraldehyde 3-phosphate dehydrogenase. If this theory is correct, then it is likely that a proton will be liberated during the phosphorolysis of the acyl-enzyme rather than in the redox step.

THE HISTOCHEMICAL DEMONSTRATION OF GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE ACTIVITY

Himmelhoch, S. Ralph; Karnovsky, Morris J.
Fonte: The Rockefeller University Press Publicador: The Rockefeller University Press
Tipo: Artigo de Revista Científica
Publicado em 01/03/1961 EN
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A histochemical method for demonstration of glyceraldehyde-3-phosphate dehydrogenation by tissues is described. The method utilizes Nitro BT as an indicator, glyceraldehyde-3-phosphate obtained from hydrolysis of commercially obtainable glyceraldehyde-3-phosphate diethylacetal (monobarium salt) as substrate, and (ethylenediamine)tetraacetic acid acid disodium as an activating agent in a medium buffered to pH 7.2 by 0.2 M sodium phosphate. The heat lability, substrate and coenzyme specificity, and sulfhydryl and phosphate dependence of the tissue component catalyzing this reaction indicate that glyceraldehyde-3-phosphate dehydrogenase activity is being demonstrated. The disparity between the known pH optimum of this enzyme and that determined histochemically, and the anomalous histochemical localization to mitochondria of this enzyme which has been found in the soluble fraction by differential centrifugation, are thought to result from the diaphorase dependence of the tetrazolium methods and to emphasize the need for caution in the interpretation of histochemically determined intracellular localization of dehydrogenating enzymes. The evidence gathered by previous workers concerning the feasibility of demonstrating specific dehydrogenases with Nitro BT...

Structural characterization of transglutaminase-catalyzed cross-linking between glyceraldehyde 3-phosphate dehydrogenase and polyglutamine repeats

Ruoppolo, Margherita; Orrù, Stefania; Francese, Simona; Caputo, Ivana; Esposito, Carla
Fonte: Cold Spring Harbor Laboratory Press Publicador: Cold Spring Harbor Laboratory Press
Tipo: Artigo de Revista Científica
Publicado em /01/2003 EN
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86.39%
The accumulation of abnormal polyglutamine-containing protein aggregates within the cytosol and nuclei of affected neurons is a hallmark of the progressive neurodegenerative disorders caused by an elongated (CAG)n repeat in the genome. The polyglutamine domains are excellent substrates for the enzyme transglutaminase type 2 (tissue), resulting in the formation of cross-links with polypeptides containing lysyl groups. Enzymatic activity toward the Qn domains increases greatly upon lengthening of such Qn stretches (n > 40). Among the possible amine donors, the glycolytic enzyme glyceraldehyde-3-phosphate-dehydrogenase was shown to tightly bind several proteins involved in polyglutamine expansion diseases. Recently, the authors have shown that K191, K268, and K331, out of the 26 lysines present in glyceraldehyde-3-phosphate-dehydrogenase, are the reactive amine-donor sites forming cross-links with substance P, which bears the simplest Qn domain (n = 2). The present study reports that synthetic peptides of both pathological and nonpathological length (n = 43 and 17, respectively) form cross-links with the same K residues located in the C-terminal region of glyceraldehyde-3-phosphate-dehydrogenase. In addition, it is shown that extra K residues present in the C termini of glyceraldehyde-3-phosphate-dehydrogenase are susceptible to cross-linking in the presence of transglutaminase. The present results indicate a possible modulating effect of Qn stretches on tissue transglutaminase substrate specificity and mechanism of recognition.

Structure of Insoluble Rat Sperm Glyceraldehyde-3-phosphate Dehydrogenase (GAPDH) via Heterotetramer Formation with Escherichia coli GAPDH Reveals Target for Contraceptive Design*

Frayne, Jan; Taylor, Abby; Cameron, Gus; Hadfield, Andrea T.
Fonte: American Society for Biochemistry and Molecular Biology Publicador: American Society for Biochemistry and Molecular Biology
Tipo: Artigo de Revista Científica
EN
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86.49%
Sperm glyceraldehyde-3-phosphate dehydrogenase has been shown to be a successful target for a non-hormonal contraceptive approach, but the agents tested to date have had unacceptable side effects. Obtaining the structure of the sperm-specific isoform to allow rational inhibitor design has therefore been a goal for a number of years but has proved intractable because of the insoluble nature of both native and recombinant protein. We have obtained soluble recombinant sperm glyceraldehyde-3-phosphate dehydrogenase as a heterotetramer with the Escherichia coli glyceraldehyde-3-phosphate dehydrogenase in a ratio of 1:3 and have solved the structure of the heterotetramer which we believe represents a novel strategy for structure determination of an insoluble protein. A structure was also obtained where glyceraldehyde 3-phosphate binds in the Ps pocket in the active site of the sperm enzyme subunit in the presence of NAD. Modeling and comparison of the structures of human somatic and sperm-specific glyceraldehyde-3-phosphate dehydrogenase revealed few differences at the active site and hence rebut the long presumed structural specificity of 3-chlorolactaldehyde for the sperm isoform. The contraceptive activity of α-chlorohydrin and its apparent specificity for the sperm isoform in vivo are likely to be due to differences in metabolism to 3-chlorolactaldehyde in spermatozoa and somatic cells. However...

Planejamento de inibidores das enzimas gliceraldeído-3-fosfato desidrogenase e diidroorotato desidrogenase de Trypanosoma cruzi; Design of inhibitors of the enzymes glyceraldehyde-3-phosphate dehydrogenase and dihydroorotate dehydrogenase from Trypanosoma cruzi

Rocha, Josmar Rodrigues da
Fonte: Biblioteca Digitais de Teses e Dissertações da USP Publicador: Biblioteca Digitais de Teses e Dissertações da USP
Tipo: Tese de Doutorado Formato: application/pdf
Publicado em 15/03/2010 PT
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116.24%
A Doença de Chagas, causada pelo parasito tripanossomatídeo Trypanosoma cruzi, é endêmica e se distribuí por toda América Latina. É uma das parasitoses mais negligenciadas pela indústria farmacêutica e os únicos fármacos disponíveis para seu tratamento foram introduzidos há décadas. Infelizmente, eles são ineficientes e apresentam sérios efeitos colaterais. Esse panorama mostra a necessidade do desenvolvimento de novos fármacos para a quimioterapia contra a doença de Chagas. As enzimas pertencentes a vias metabólicas essenciais para a sobrevivência do parasito tais como a via glicolítica e a de síntese de novo de nucleotídeos de pirimidinas, têm sido propostas como alvos interessantes no planejamento novos fármacos para o tratamento da doença de Chagas. Neste trabalho, as enzimas Gliceraldeído 3-fosfato desidrogenase (TcGAPDH) e a Diidroorotato desidrogenase (TcDHODH) de Trypanosoma cruzi foram estudadas como alvos para o planejamento de inibidores enzimáticos com propriedades físico-químicas e características estruturais similares à de compostos-líderes. Para isso, foram utilizados métodos e ferramentas de Quiminformática tanto baseadas nas estruturas dos ligantes (LBVS) quanto dos receptores (SBVS). Para a identificação e seleção de potenciais inibidores da enzima GAPDH...

The development of SS'-polymethylenebis(methanethiosulphonates) as reversible cross-linking reagents for thiol groups and their use to form stable catalytically active cross-linked dimers within glyceraldehyde 3-phosphate dehydrogenase.

Bloxham, D P; Sharma, R P
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em 01/08/1979 EN
Relevância na Pesquisa
86.47%
The synthesis of a series of SS'-polymethylenebis(methanethiosulphonates) including the pentane, hexane, octane, decane and dodecane derivatives is described. These derivatives were synthesized by condensation between dibromoalkanes and potassium methanethiosulphonate in refluxing methanol and this seems an especially versatile reaction for the synthesis of asymmetric thiosulphonate derivatives. The synthesis of SS'-[1,8-3H4]-octamethylenebis(methanethiosulphonate) was also perfomed. Cross-linking was demonstrated in the four enzymes lactate dehydrogenase, phosphofructokinase, pyruvate kinase and glyceraldehyde 3-phosphate dehydrogenase. For all four enzymes cross-linking was efficiently reversed by reducing conditions in denaturing solvents. The reaction with glyceraldehyde 3-phosphate dehydrogenase was unique in that only the cross-linked dimer was produced in significant amounts (greater than 90% of total products as dimer). This reaction was followed in detail with radioactive cross-linking reagent. Inhibition of enzyme activity was extremely fast and showed an asymmetric distribution of enzyme activity on subunits. Thus complete modification of only one subunit resulted in up to 75% inhibition of enzyme activity. Reaction of glyceraldehyde 3-phosphate dehydrogenase with 1.25 mol of SS'-octamethylenebis(methanethiosulphonate) per mol of enzyme subunit produced two species of protein. The first species was obtained in 20% yield and was only partially re-activated on mild reduction with 2-mercaptoethanol. The second species was isolated in 66% yield and was completely re-activated on mild reduction. Before reduction there was 4 mol of inhibitor per tetramer for the latter species...

The glyceraldehyde-3-phosphate dehydrogenase gene of Moniliophthora perniciosa, the causal agent of witches' broom disease of Theobroma cacao

Lima,Juliana O.; Pereira,Jorge F.; Rincones,Johana; Barau,Joan G.; Araújo,Elza F.; Pereira,Gonçalo A.G.; Queiroz,Marisa V.
Fonte: Sociedade Brasileira de Genética Publicador: Sociedade Brasileira de Genética
Tipo: Artigo de Revista Científica Formato: text/html
Publicado em 01/01/2009 EN
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126.23%
This report describes the cloning, sequence and expression analysis of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene of Moniliophthora perniciosa, the most important pathogen of cocoa in Brazil. Southern blot analysis revealed the presence of a single copy of the GAPDH gene in the M. perniciosa genome (MpGAPDH). The complete MpGAPDH coding sequence contained 1,461 bp with eight introns that were conserved in the GAPDH genes of other basidiomycete species. The cis-elements in the promoter region of the MpGAPDH gene were similar to those of other basidiomycetes. Likewise, the MpGAPDH gene encoded a putative 339 amino acid protein that shared significant sequence similarity with other GAPDH proteins in fungi, plants, and metazoans. Phylogenetic analyses clustered the MPGAPDH protein with other homobasidiomycete fungi of the family Tricholomataceae. Expression analysis of the MpGAPDH gene by real-time PCR showed that this gene was more expressed (~1.3X) in the saprotrophic stage of this hemibiotrophic plant pathogen than in the biotrophic stage when grown in cacao extracts.

A Differential Redox Regulation or the Pathways Metabolizing Glyceraldehyde-3-phosphate Tunes the Production of Reducing Power in the Cytosol of Plant Cells

Piattoni, Claudia Vanesa; Guerrero, Sergio Adrian; Iglesias, Alberto Alvaro
Fonte: Molecular Diversity Preservation International Publicador: Molecular Diversity Preservation International
Tipo: info:eu-repo/semantics/article; info:ar-repo/semantics/artículo; info:eu-repo/semantics/publishedVersion Formato: application/pdf
ENG
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Adaptation to aerobic life leads organisms to sense reactive oxygen species and use the signal for coordination of the entire metabolism. Glycolysis in plants is a particular network where specific steps, like oxidation of glyceraldehydes-3-phosphate (Ga3P), are critical in order for it to function. The triose-phosphate can be converted into 3-phosphoglycerate through the phosphorylating Ga3P dehydrogenase (Ga3PDHase, EC 1.2.1.12) producing ATP and NADH, or via the non-phosphorylating enzyme (np-Ga3PDHase; EC 1.2.1.9) generating NADPH. In this work we found redox regulation to be a posttranslational mechanism allowing the fine-tuning of the triose-phosphate fate. Both enzymes were inactivated after oxidation by reactive oxygen and nitrogen species. Kinetic studies determined that Ga3PDHase is marked (63-fold) more sensitive to oxidants than np-Ga3PDHase. Thioredoxin-h reverted the oxidation of both enzymes (although with differences between them), suggesting a physiological redox regulation. The results support a metabolic scenario where the cytosolic triose-phosphate dehydrogenases are regulated under changeable redox conditions. This would allow coordinate production of NADPH or ATP through glycolysis, with oxidative signals triggering reducing power synthesis in the cytosol. The NADPH increment would favor antioxidant responses to cope with the oxidative situation...

Glyceraldehyde-3-phosphate dehydrogenase regulates endothelin-1 expression by a novel, redox-sensitive mechanism involving mRNA stability

Rodríguez-Pascual, Fernando; Redondo-Horcajo, Mariano; Magán-Marchal, Noemi; Lagares, David; Martínez-Ruiz, Antonio; Kleinert, Hartmut; Lamas Peláez, Santiago
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artículo Formato: 43048 bytes; application/pdf
ENG
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17 pages.-- PMID: 18809573 [PubMed].-- Printed version published on Dec 2008.; The regulation of the synthesis of the endothelial-derived vasoconstrictor endothelin-1 (ET-1) is a complex process encompassing transcriptional as well as mRNA stability mechanisms. We have described recently the existence of a mechanism for the control of ET-1 expression based on the mRNA-destabilizing capacity of specific cytosolic proteins through interaction with AU-rich elements (AREs) present in the 3'-UTR of the gene. We now identify glyceraldehyde-3'-phosphate dehydrogenase (GAPDH) as a protein which binds to the AREs and is responsible for its destabilization. Oxidant stress alters the binding of GAPDH to the mRNA and its capacity to modulate ET-1 expression, a phenomenon occurring through specific S-glutathionylation of the catalytically active residue Cys 152. Finally we provide data consistent with a role for GAPDH in mRNA unwinding, yielding this molecule more prone to degradation. By contrast, S-thiolated GAPDH appears unable to modify mRNA unwinding, thus facilitating enhanced stability. Taken together, these results describe a novel, redox-based mechanism regulating mRNA stability and add a new facet to the panoply of GAPDH cellular homeostatic actions.; Peer reviewed

Widespread occurrence of non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase among gram-positive bacteria; Amplia distribución de la gliceraldehído-3-fosfato deshidrogenasa no-fosforilante entre las bacterias gram-positivas

Iddar, Abdelghani; Valverde, Federico; Assobhei, Omar; Serrano, Aurelio; Soukri, Abdelaziz
Fonte: Sociedad Española de Microbiología Publicador: Sociedad Española de Microbiología
Tipo: Artículo
ENG
Relevância na Pesquisa
126.24%
The non-phosphorylating glyceraldehyde 3-phosphate dehydrogenase (GAPDHN, NADP+ -specific, EC 1.2.1.9) is present in green eukaryotes and some Streptococcus strains. The present report describes the results of activity and immunoblot analyses, which were used to generate the first survey of bacterial GAPDHN distribution in a number of Bacillus, Streptococcus and Clostridium strains. Putative gapN genes were identified after PCR amplification of partial 700-bp sequences using degenerate primers constructed from highly conserved protein regions. Alignment of the amino acid sequences of these fragments with those of known sequences from other eukaryotic and prokaryotic GAPDHNs, demonstrated the presence of conserved residues involved in catalytic activity that are not conserved in aldehyde dehydrogenases, a protein family closely linked to GAPDHNs. The results confirm that the basic structural features of the members of the GAPDHN family have been conserved throughout evolution and that no identity exists with phosphorylating GAPDHs. Furthermore, phylogenetic trees generated from multiple sequence alignments suggested a close relationship between plant and bacterial GAPDHN families.; La gliceraldehído-3-fosfato deshidrogenasa no-fosforilante (GAPDHN...

Widespread occurrence of non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase among gram-positive bacteria

Iddar,Abdelghani; Valverde,Federico; Assobhei,Omar; Serrano,Aurelio; Soukri,Abdelaziz
Fonte: International Microbiology Publicador: International Microbiology
Tipo: info:eu-repo/semantics/article; journal article; info:eu-repo/semantics/publishedVersion Formato: text/html; application/pdf
Publicado em 01/12/2005 ENG
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126.23%
The non-phosphorylating glyceraldehyde 3-phosphate dehydrogenase (GAPDHN, NADP+-specific, EC 1.2.1.9) is present in green eukaryotes and some Streptococcus strains. The present report describes the results of activity and immunoblot analyses, which were used to generate the first survey of bacterial GAPDHN distribution in a number of Bacillus, Streptococcus and Clostridium strains. Putative gapN genes were identified after PCR amplification of partial 700-bp sequences using degenerate primers constructed from highly conserved protein regions. Alignment of the amino acid sequences of these fragments with those of known sequences from other eukaryotic and prokaryotic GAPDHNs, demonstrated the presence of conserved residues involved in catalytic activity that are not conserved in aldehyde dehydrogenases, a protein family closely linked to GAPDHNs. The results confirm that the basic structural features of the members of the GAPDHN family have been conserved throughout evolution and that no identity exists with phosphorylating GAPDHs. Furthermore, phylogenetic trees generated from multiple sequence alignments suggested a close relationship between plant and bacterial GAPDHN families.