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Sample records for 3-phosphate 4-nitrophenyl phosphate

  1. A chromogenic substrate for phosphatidylinositol-specific phospholipase C: 4-nitrophenyl myo-inositol-1-phosphate.

    PubMed

    Shashidhar, M S; Volwerk, J J; Griffith, O H; Keana, J F

    1991-12-01

    A chromogenic water-soluble substrate for phosphatidylinositol-specific phospholipase C was synthesized starting from myo-inositol employing isopropylidene and 4-methoxytetrahydropyranyl protecting groups. In this analogue of phosphatidylinositol, 4-nitrophenol replaces the diacylglycerol moiety, resulting in synthetic, racemic 4-nitrophenyl myo-inositol-1-phosphate. Using this synthetic substrate a rapid, convenient and sensitive spectrophotometric assay for the phosphatidylinositol-specific phospholipase C from Bacillus cereus was developed. Initial rates of the cleavage of the nitrophenol substrate were linear with time and the amount of enzyme used. At pH 7.0, specific activities for the B. cereus enzyme were 77 and 150 mumol substrate cleaved min-1 (mg protein)-1 at substrate concentrations of 1 and 2 mM, respectively. Under these conditions, less than 50 ng quantities of enzyme were easily detected. The chromogenic substrate was stable during long term storage (6 months) as a solid at -20 degrees C.

  2. 4-Nitrophenol in 4-nitrophenyl phosphate, a substrate for alkaline phosphatase, as measured by paired-ion high-performance liquid chromatography.

    PubMed

    Culbreth, P H; Duncan, I W; Burtis, C A

    1977-12-01

    We used paired-ion high-performance liquid chromatography to determine the 4-nitrophenol content of 4-nitrophenyl phosphate, a substrate for alkaline phosphatase analysis. This was done on a reversed-phase column with a mobile phase of methanol/water, 45/55 by vol, containing 3 ml of tetrabutylammonium phosphate reagent per 200 ml of solvent. At a flow rate of 1 ml/min, 4-nitrophenol was eluted at 9 min and monitored at 404 nm; 4-nitrophenyl phosphate was eluted at 5 min and could be monitored at 311 nm. Samples of 4-nitrophenyl phosphate obtained from several sources contained 0.3 to 7.8 mole of 4-nitrophenol per mole of 4-nitrophenyl phosphate.

  3. l-Glyceraldehyde 3-Phosphate, a Bactericidal Agent

    PubMed Central

    Tang, Chu-Tay; Engel, Robert; Tropp, Burton E.

    1977-01-01

    At a concentration of 2.5 mM, dl-glyceraldehyde 3-phosphate has a bactericidal effect upon Escherichia coli. The glycerol 3-phosphate transport system is required for the entry of the biologically active l-enantiomer. l-Glyceraldehyde must be phosphorylated by the cell to exert its full effect upon growth. The addition of dl-glyceraldehyde 3-phosphate to a culture of E. coli caused no preferential inhibition of the accumulation of deoxyribonucleic acid, ribonucleic acid, or phosphoglycerides, although protein accumulation was less affected. Studies with mutant strains ruled out catabolic glycerol 3-phosphate dehydrogenase, anabolic nicotinamide adenine dinucleotide (phosphate):sn-glycerol 3-phosphate oxidoreductase, and fructose 1,6-diphosphate aldolase as the primary sites of action. l-Glyceraldehyde 3-phosphate is a competitive inhibitor of sn-glycerol 3-phosphate in the reactions catalyzed by acyl coenzyme A:sn-glycerol 3-phosphate acyltransferase (Ki of 1.8 mM) and cytidine 5′-diphosphate-diglyceride:sn-glycerol 3-phosphate phosphatidyltransferase (Ki of 2.7 mM). A Km mutant for the former enzyme was susceptible to the inhibitor. l-Glyceraldehyde 3-phosphate does not affect acyl coenzyme A:lysophosphatidate acyltransferase activity. In vivo, phosphatidylethanolamine and phosphatidylglycerol accumulation are inhibited to the same extent by the addition of dl-glyceraldehyde 3-phosphate to a culture of E. coli. PMID:319747

  4. Determination of acid phosphatase in biological fluids using a new substrate, 2,6-dichloro-4-nitrophenyl phosphate.

    PubMed

    Teshima, S; Hayashi, Y; Ando, M

    1987-09-30

    A new substrate, 2,6-dichloro-4-nitrophenyl phosphate (DCNP-P), is used for the determination of acid phosphatase (EC 3.1.3.2) in serum and urine. It was hydrolyzed by acid phosphatase to 2,6-dichloro-4-nitrophenol (DCNP) and phosphoric acid. At a pH of 4.5-6.0, the absorption of DCNP liberated by acid phosphatase was much higher than that of p-nitrophenol, which is commonly used as an aglycone in the acid phosphatase assay. By using DCNP-P as a substrate for acid phosphatase activity, determinations can be made without the colour reaction which requires the addition of an alkaline solution, and can be determined by the rate assay that does not require measurement of sample blanks in serum or urine. This method using DCNP-P is highly sensitive and is the most suitable for the rate assay of acid phosphatase activity in biological fluids.

  5. Iodination of glyceraldehyde 3-phosphate dehydrogenase

    PubMed Central

    Thomas, Jean O.; Harris, J. Ieuan

    1970-01-01

    1. A high degree of homology in the positions of tyrosine residues in glyceraldehyde 3-phosphate dehydrogenase from lobster and pig muscle, and from yeast, prompted an examination of the reactivity of tyrosine residues in the enzyme. 2. Iodination of the enzyme from lobster muscle with low concentrations of potassium tri-[125I]-iodide led to the identification of tyrosine residues of differing reactivity. Tyrosine-46 appeared to be the most reactive in the native enzyme. 3. When the monocarboxymethylated enzyme was briefly treated with small amounts of iodine, iodination could be confined almost entirely to tyrosine-46 in the lobster enzyme; tyrosine-39 or tyrosine-42, or both, were also beginning to react. 4. These three tyrosine residues were also those that reacted most readily in the carboxymethylated pig and yeast enzymes. 5. The difficulties in attaining specific reaction of the native enzyme are considered. 6. The differences between our results and those of other workers are discussed. ImagesPLATE 1PLATE 2 PMID:5530750

  6. Two potential fish glycerol-3-phosphate phosphatases.

    PubMed

    Raymond, James A

    2015-06-01

    Winter-acclimated rainbow smelt (Osmerus mordax Mitchill) produce high levels of glycerol as an antifreeze. A common pathway to glycerol involves the enzyme glycerol-3-phosphate phosphatase (GPP), but no GPP has yet been identified in fish or any other animal. Here, two phosphatases assembled from existing EST libraries (from winter-acclimated smelt and cold-acclimated smelt hepatocytes) were found to resemble a glycerol-associated phosphatase from a glycerol-producing alga, Dunaliella salina, and a recently discovered GPP from a bacterium, Mycobacterium tuberculosis. Recombinant proteins were generated and were found to have GPP activity on the order of a few μMol Pi/mg enzyme/min. The two enzymes have acidic pH optima (~5.5) similar to that previously determined for GPP activity in liver tissue, with about 1/3 of their peak activities at neutral pH. The two enzymes appear to account for the GPP activity of smelt liver, but due to their reduced activities at neutral pH, their contributions to glycerol production in vivo remain unclear. Similar enzymes may be active in a glycerol-producing insect, Dendroctonus ponderosae.

  7. Structure of RNA 3'-phosphate cyclase bound to substrate RNA.

    PubMed

    Desai, Kevin K; Bingman, Craig A; Cheng, Chin L; Phillips, George N; Raines, Ronald T

    2014-10-01

    RNA 3'-phosphate cyclase (RtcA) catalyzes the ATP-dependent cyclization of a 3'-phosphate to form a 2',3'-cyclic phosphate at RNA termini. Cyclization proceeds through RtcA-AMP and RNA(3')pp(5')A covalent intermediates, which are analogous to intermediates formed during catalysis by the tRNA ligase RtcB. Here we present a crystal structure of Pyrococcus horikoshii RtcA in complex with a 3'-phosphate terminated RNA and adenosine in the AMP-binding pocket. Our data reveal that RtcA recognizes substrate RNA by ensuring that the terminal 3'-phosphate makes a large contribution to RNA binding. Furthermore, the RNA 3'-phosphate is poised for in-line attack on the P-N bond that links the phosphorous atom of AMP to N(ε) of His307. Thus, we provide the first insights into RNA 3'-phosphate termini recognition and the mechanism of 3'-phosphate activation by an Rtc enzyme. © 2014 Desai et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

  8. Polymetallic complexes in microemulsions for the hydrolysis of 4-nitrophenyl phosphate: a bio-mimetic model for decontamination of organophosphates in the environment.

    PubMed

    Tafesse, Fikru; Deppa, Ntsapokazi C

    2004-06-01

    Reactions of several metal ions and polymetallic complexes toward hydrolysis of 4-nitrophenyl phosphate (NPP) were investigated at 10(-3)M concentration in oil-in-water microemulsions and aqueous media. The reactions were monitored by measuring the absorbance of the nitrophenolate ion produced in the reaction aliquots with time. The order of effectiveness of the metal ions and polymetallic complexes at neutral pH toward hydrolysis of NPP was found to be Turnbull's Blue>magnetite>Fe(III)>Fe(II)>Co(II)>Ca(II)>Cu(II). The possible application of these systems for sites contaminated with chemical warfare agents such as organophosphates is discussed.

  9. Metabolism of L-glyceraldehyde 3-phosphate in Escherichia coli

    SciTech Connect

    Kalyananda, M.K.G.S.

    1985-01-01

    E. coli is able to incorporate L-glyceraldehyde and L-glyceraldehyde 3-phosphate into phospholipids, L-(3-/sup 3/H)Glyceraldehyde was synthesized and the purity and the chemical identity of the product were checked by paper chromatography. L-(3-/sup 3/H)Glyceraldehyde 3-phosphate was synthesized from L-(3-/sup 3/H)glyceraldehyde in a reaction catalyzed by glycerokinase. E. coli extract contains a new enzyme activity which catalyzes an NADPH dependent reduction of L-glyceraldehyde 3-phosphate into sn-glycerol 3-phosphate. A procedure, specifically suitable for assaying the reductase activity in the crude extract, was developed. A more convenient spectrophotometric assay method was employed for the purified enzyme. At moderate concentrations sulfhydryl group inhibitors had no effect on the enzyme activity of L-GAP reductase. At 100..mu..M concentration Zn/sup +2/ inhibited the enzyme activity by about 30% while Mn/sup +2/ elevated the activity by about the same margin. Mg/sup +2/, Ca/sup +2/ and Fe/sup +2/ were without effect at this concentration. L-Glyceraldehyde 3-phosphate is known to be bactericidal at 1.25 ..mu..M concentration and the D-enantiomer is without effect. Furthermore, methylglyoxal is known to be bactericidal at or above 0.5 mM concentration. Strains of E. coli resistant to 1 mM methylglyoxal were isolated. The cell extract prepared from the mutant possessed increased capacity to transform methylglyoxal into D-lactate via a glutathione dependent reaction. These mutants were less sensitive to 2.5 mM DL-GAP suggesting that conversion of L-glyceraldehyde 3-phosphate into methylglyoxal may at least partly be responsible for the bactericidal activity of L-GAP.

  10. Quantitative cytochemical measurement of glyceraldehyde 3-phosphate dehydrogenase activity.

    PubMed

    Henderson, B

    1976-08-25

    A system has been developed for the quantitative measurment of glyceraldehyde 3-phosphate dehydrogenase activity in tissue sections. An obstacle to the histochemical study of this enzyme has been the fact that the substrate, gylceraldehyde 3-phosphate, is very unstable. In the present system a stable compound, fructose 1, 6-diphosphate, is used as the primary substrate and the demonsatration of the glyceraldehyde 3-phosphate dehydrogenase activity depends on the conversion of this compound into the specific substrate by the aldolase present in the tissue. The characteristics of the dehydrogenase activity resulting from the addition of fructose 1, 6-diphosphate, resemble closely the known properties of purified glyceraldehyde 3-phosphate dehydrogenase. Use of polyvinyl alcohol in the reaction medium prevents release of enzymes from the sections, as occurs in aqueous media. Although in this study intrinsic aldolase activity was found to be adequate for the rapid conversion of fructose 1, 6-diphosphate into the specific substrate for the dehydrogenase, the use of exogenous aldolase may be of particular advantage in assessing the intergrity of the Embden-Meyerhof pathway.

  11. Toxic Neuronal Death by Glyeraldehyde-3-Phosphate Dehydrogenase and Mitochondria

    DTIC Science & Technology

    2003-08-01

    Neuroreport, 10(5), 1149-1153. Sioud, M., & Jespersen, L. (1996). Enhancement of hammerhead ribozyme catalysis by glyceraldehyde-3-phosphate dehydrogenase...1996) Enhancemen t of hammerhead r ibozyme cata lysis by glycera ldehyde-3- phospha te dehydrogenase. J Mol Biol 257:775–789. Sirover MA (1997) Role of

  12. Temperature dependence of the absorbance of alkaline solutions of 4-nitrophenyl phosphate--a potential source of error in the measurement of alkaline phosphatase activity.

    PubMed

    Burtis, C A; Seibert, L E; Baird, M A; Sampson, E J

    1977-09-01

    The absorbance of an alkaline solution of 4-nitrophenyl phosphate is a function of temperature. Quantitative evaluation of this phenomenon indicates that it (a) depends on the concentration of the compound and is independent of source, buffer concentration, and pH above 9.0; (b) is reversible; (c) is not a result of alkaline hydrolysis or 4-nitrophenol contamination; and (d) correlates with a temperature-induced shift of its absorbance spectrum. The phenomenon may represent a potential analytical problem in methods for alkaline phosphatase in which this compound is the substrate. If thermal equilibrium is not reached and maintained during an alkaline phosphatase assay, the thermochromic response will be included in the measured rate. The magnitude of this error depends on the thermal response and control characteristics of each particular instrument and the reaction conditions under which such an analysis is performed.

  13. Uptake of glycerol 3-phosphate and some of its analogs by the hexose phosphate transport system of Escherichia coli.

    PubMed Central

    Guth, A; Engel, R; Tropp, B E

    1980-01-01

    The hexose phosphate transport system transported glycerol 3-phosphate and its analogs 3,4-dihydroxybutyl-1-phosphonate, glyceraldehyde 3-phosphate, and 3-hydroxy-4-oxobutyl-1-phosphonate. PMID:6995450

  14. sn-Glycerol-3-phosphate transport in Salmonella typhimurium.

    PubMed Central

    Hengge, R; Larson, T J; Boos, W

    1983-01-01

    Salmonella typhimurium contains a transport system for sn-glycerol-3-phosphate that is inducible by growth on glycerol and sn-glycerol-3-phosphate. In fully induced cells, the system exhibited an apparent Km of 50 microM and a Vmax of 2.2 nmol/min . 10(8) cells. The corresponding system in Escherichia coli exhibits, under comparable conditions, a Km of 14 microM and a Vmax of 2.2 nmol/min . 10(8) cells. Transport-defective mutants were isolated by selecting for resistance against the antibiotic fosfomycin. They mapped in glpT at 47 min in the S. typhimurium linkage map, 37% cotransducible with gyrA. In addition to the glpT-dependent system, S. typhimurium LT2 contains, like E. coli, a second, ugp-dependent transport system for sn-glycerol-3-phosphate that was derepressed by phosphate starvation. A S. typhimurium DNA bank containing EcoRI restriction fragments in phage lambda gt7 was used to clone the glpT gene in E. coli. Lysogens that were fully active in the transport of sn-glycerol-3-phosphate with a Km of 33 microM and a Vmax of 2.0 nmol/min . 10(8) cells were isolated in a delta glpT mutant of E. coli. The EcoRI fragment harboring glpT was 3.5 kilobases long and carried only part of glpQ, a gene distal to glpT but on the same operon. The fragment was subcloned in multicopy plasmid pACYC184. Strains carrying this hybrid plasmid produced large amounts of cytoplasmic membrane protein with an apparent molecular weight of 33,000, which was identified as the sn-glycerol-3-phosphate permease. Its properties were similar to the corresponding E. coli permease. The presence of the multicopy glpT hybrid plasmid had a strong influence on the synthesis or assembly of other cell envelope proteins of E. coli. For instance, the periplasmic ribose-binding protein was nearly absent. On the other hand, the quantity of an unidentified E. coli outer membrane protein usually present only in small amounts increased. Images PMID:6408060

  15. Novel Inhibitors of Mitochondrial sn-Glycerol 3-phosphate Dehydrogenase

    PubMed Central

    Orr, Adam L.; Ashok, Deepthi; Sarantos, Melissa R.; Ng, Ryan; Shi, Tong; Gerencser, Akos A.; Hughes, Robert E.; Brand, Martin D.

    2014-01-01

    Mitochondrial sn-glycerol 3-phosphate dehydrogenase (mGPDH) is a ubiquinone-linked enzyme in the mitochondrial inner membrane best characterized as part of the glycerol phosphate shuttle that transfers reducing equivalents from cytosolic NADH into the mitochondrial electron transport chain. Despite the widespread expression of mGPDH and the availability of mGPDH-null mice, the physiological role of this enzyme remains poorly defined in many tissues, likely because of compensatory pathways for cytosolic regeneration of NAD+ and mechanisms for glycerol phosphate metabolism. Here we describe a novel class of cell-permeant small-molecule inhibitors of mGPDH (iGP) discovered through small-molecule screening. Structure-activity analysis identified a core benzimidazole-phenyl-succinamide structure as being essential to inhibition of mGPDH while modifications to the benzimidazole ring system modulated both potency and off-target effects. Live-cell imaging provided evidence that iGPs penetrate cellular membranes. Two compounds (iGP-1 and iGP-5) were characterized further to determine potency and selectivity and found to be mixed inhibitors with IC50 and Ki values between ∼1–15 µM. These novel mGPDH inhibitors are unique tools to investigate the role of glycerol 3-phosphate metabolism in both isolated and intact systems. PMID:24587137

  16. Model of early self-replication based on covalent complementarity for a copolymer of glycerate-3-phosphate and glycerol-3-phosphate

    NASA Technical Reports Server (NTRS)

    Weber, Arthur L.

    1989-01-01

    Glyceraldehyde-3-phosphate acts as the substrate in a model of early self-replication of a phosphodiester copolymer of glycerate-3-phosphate and glycerol-3-phosphate. This model of self-replication is based on covalent complementarity in which information transfer is mediated by a single covalent bond, in contrast to multiple weak interactions that establish complementarity in nucleic acid replication. This replication model is connected to contemporary biochemistry through its use of glyceraldehyde-3-phosphate, a central metabolite of glycolysis and photosynthesis.

  17. Green tea catechins: inhibitors of glycerol-3-phosphate dehydrogenase.

    PubMed

    Kao, Chung-Cheng; Wu, Bo-Tsung; Tsuei, Yi-Wei; Shih, Li-Jane; Kuo, Yu-Liang; Kao, Yung-Hsi

    2010-05-01

    Green tea catechins, especially (-)-epigallocatechin-3-gallate (EGCG), are known to regulate obesity and fat accumulation. We performed a kinetic analysis in a cell-free system to determine the mode of inhibition of glycerol-3-phosphate dehydrogenase (GPDH; EC 1.1.1.8) by EGCG. GPDH catalyzes the beta-nicotinamide adenine dinucleotide (NADH)-dependent reduction of dihydroxyacetone phosphate (DHAP) to yield glycerol-3-phosphate, which serves as one of the major precursors of triacylglycerols. We found that EGCG dose-dependently inhibited GPDH activity at a concentration of approximately 20 muM for 50 % inhibition. The IC (50) values of other green tea catechins, such as (-)-epicatechin, (-)-epicatechin-3-gallate, and (-)-epigallocatechin, were all above 100 microM. This suggests a catechin type-dependent effect. Based on double-reciprocal plots of the kinetic data, EGCG was a noncompetitive inhibitor of the GPDH substrates, NADH and DHAP, with respective inhibition constants (Ki) of 18 and 31 microM. Results of this study possibly support previous studies that EGCG mediates fat content. Georg Thieme Verlag KG Stuttgart. New York.

  18. Metabolism of L-glyceraldehyde 3-phosphate in Escherichia coli

    SciTech Connect

    Kalyananda, M.K.G.S.; Engel, R.; Tropp, B.E.

    1987-06-01

    When either /sup 3/H-labeled L-glyceraldehyde or /sup 3/H-labeled L-glyceraldehyde 3-phosphate (GAP) was added to cultures of Escherichia coli, the phosphoglycerides were labeled. More than 81% of the label appeared in the backbone of the phosphoglycerides. Chromatographic analyses of the labeled phosphoglycerides revealed that the label was normally distributed into phosphatidylethanolamine, phosphatidylglycerol, and cardiolipin. These results suggest that L-glyceraldehyde is phosphorylated and the resultant L-GAP is converted into sn-glycerol 3-phosphate (G3P) before being incorporated into the bacterial phosphoglycerides. Cell-free bacterial extracts catalyzed an NADPH-dependent reduction of L-GAP to sn-G3P. The partially purified enzyme was specific for L-GAP and recognized neither D-GAP nor dihydroxyacetone phosphate as a substrate. NADH could not replace NADPH as a coenzyme. The L-GAP:NADPH oxidoreductase had an apparent K/sub m/ of 28 and 35 ..mu..M for L-GAP and NADPH, respectively. The enzyme was insensitive to sulfhydryl reagents and had a pH optimum of approximately 6.6. The phosphonic acid analog of GAP, 3-hydroxy-4-oxobutyl-1-phosphonate, was a substrate for the reductase, with an apparent K/sub m/ of 280 ..mu..M.

  19. Toxic Neuronal Death by Glyceraldehyde-3-Phosphate Dehydrongenase and Mitochondria

    DTIC Science & Technology

    2001-08-01

    Parkinson’s Disease (PD) and after a number of forms of toxic exposure. If unique elements in the signaling pathways for the PD or toxic apoptosis can be identified and their apoptosis signaling impeded, neuronal loss may be slowed or reduced in the conditions. The research proposed in this grant was designed to examine the role of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in apoptotic neuronal signaling. Recent studies in postmortem brain have implicated GAPDH apoptosis signaling in Parkinson’s disease (PD). Propargylamines, with

  20. Catalytically active monomers of E. coli glyceraldehyde-3-phosphate dehydrogenase.

    PubMed

    Levashov, P A; Muronetz, V I; Klyachko, N L; Nagradova, N K

    1998-04-01

    Monomeric forms of E. coli glyceraldehyde-3-phosphate dehydrogenase have been prepared using two different experimental approaches: (1) covalent immobilization of a tetramer on a solid support via a single subunit with subsequent dissociation of non-covalently bound subunits in the presence of urea, and (2) entrapment of monomeric species into reversed micelles of Aerosol OT in octane. Isolated monomers were shown to be catalytically active, exhibiting KM values close to the parameters characteristic of the tetrameric forms. Like tetramers, isolated monomers did not use NADP7 as a coenzyme.

  1. Enzyme-enzyme interaction in the chloroplast: glyceraldehyde-3-phosphate dehydrogenase, triose phosphate isomerase and aldolase.

    PubMed

    Anderson, L E; Goldhaber-Gordon, I M; Li, D; Tang, X Y; Xiang, M; Prakash, N

    1995-01-01

    Apparent physical interaction between pea chloroplast (Pisum sativum L.) glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.13) and aldolase (EC 4.1.2.13) is seen in phase-partitioning, fluorescent-anisotropy and isoelectric-focusing experiments. Similarly, results obtained in phase-partitioning and isoelectric-focusing experiments indicate physical interaction between aldolase and triose-phosphate isomerase (EC 5.3.1.1). Kinetic experiments suggest that both aldolase-bound glyceraldehyde-3-phosphate can act as substrate for glyceraldehyde-3-phosphate dehydrogenase. These results are consistent with the notion that there is interaction between these three enzymes both during photosynthetic CO2 fixation and during glycolysis in the chloroplast.

  2. Dihydropyrazine-induced inactivation of glyceraldehyde-3-phosphate dehydrogenase.

    PubMed

    Takechi, Shinji; Nakahara, Kazuhide; Yamaguchi, Tadatoshi

    2010-01-01

    Dihydropyrazine (DHP), which is produced during the Maillard reaction, generates radicals that not only cause breakage of chromosomal DNA leading to mutagenic lesions but also induce oxidative damage to cellular proteins. In the present study, we show that three DHP derivatives, which generated superoxide anions, caused inhibition of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). SH-compounds, such as cysteine, dithiothreitol (DTT), 2-mercaptoethanol, 2-mercaptoethylamine, and N-acetyl-cysteine, suppressed the inhibition of GAPDH by DHP in vitro, although the effect of DHP on GAPDH was not reversed by DTT. In addition, DHP-exposed Escherichia coli showed almost unaffected growth on plates containing a rich medium, but poor growth on plates containing M9 synthetic medium with glucose as the sole carbon source. Furthermore, DHP-exposed E. coli exhibited reduced GAPDH activity. These findings indicate that DHP disturbs the glycolytic pathway by inhibiting GAPDH activity.

  3. Phosphate closes the solution structure of the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) from Mycobacterium tuberculosis.

    PubMed

    Borges, Júlio C; Pereira, José H; Vasconcelos, Igor B; dos Santos, Giovanni C; Olivieri, Johnny R; Ramos, Carlos H I; Palma, Mário S; Basso, Luiz A; Santos, Diógenes S; de Azevedo, Walter F

    2006-08-15

    The 5-enolpyruvylshikimate-3-phosphate synthase catalyses the sixth step of the shikimate pathway that is responsible for synthesizing aromatic compounds and is absent in mammals, which makes it a potential target for drugs development against microbial diseases. Here, we report the phosphate binding effects at the structure of the 5-enolpyruvylshikimate-3-phosphate synthase from Mycobacterium tuberculosis. This enzyme is formed by two similar domains that close on each other induced by ligand binding, showing the occurrence of a large conformation change. We have monitored the phosphate binding effects using analytical ultracentrifugation, small angle X-ray scattering and, circular dichroism techniques. The low resolution results showed that the enzyme in the presence of phosphate clearly presented a more compact structure. Thermal-induced unfolding experiments followed by circular dichroism suggested that phosphate rigidified the enzyme. Summarizing, these data suggested that the phosphate itself is able to induce conformational change resulting in the closure movement in the M. tuberculosis 5-enolpyruvylshikimate-3-phosphate synthase.

  4. Glycerol-3-phosphate acyltransferase 2 is essential for normal spermatogenesis.

    PubMed

    Garcia-Fabiani, Maria B; Montanaro, Mauro A; Stringa, Pablo; Lacunza, Ezequiel; Cattaneo, Elizabeth R; Santana, Marianela; Pellon-Maison, Magali; Gonzalez-Baro, Maria R

    2017-08-30

    Glycerol-3-phosphate acyltransferases (GPATs) catalyze the first and rate-limiting step in the de novo glycerolipid synthesis. The GPAT2 isoform differs from the other isoforms because its expression is restricted to male germ cells and cancer cells. It has been recently reported that GPAT2 expression in mouse testis fluctuates during sexual maturation and that it is regulated by epigenetic mechanisms in combination with vitamin A derivatives. Despite progress made in this field, information about GPAT2 role in the developing male germ cells remains unclear. The aim of the present study was to confirm the hypothesis that GPAT2 is required for the normal physiology of testes and male germ cell maturation. The gene was silenced in vivo by inoculating lentiviral particles carrying the sequence of a short-hairpin RNA targeting Gpat2 mRNA into mouse testis. Histological and gene expression analysis showed impaired spermatogenesis and arrest at the pachytene stage. Defects in reproductive fitness were also observed, and the analysis of apoptosis-related gene expression demonstrated the activation of apoptosis in Gpat2-silenced germ cells. These findings indicate that GPAT2 protein is necessary for the normal development of male gonocytes, and that its absence triggers apoptotic mechanisms, thereby decreasing the number of dividing germ cells. © 2017 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.

  5. Mechanism of glyceraldehyde-3-phosphate dehydrogenase inactivation by tyrosine nitration

    PubMed Central

    Palamalai, Vikram; Miyagi, Masaru

    2010-01-01

    Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a multifaceted protein that is involved in numerous processes including glycolysis, translational silencing, transcriptional regulation of specific genes, and acting as a nitric oxide sensor. The precise mechanism on how GAPDH is targeted to these different roles is unclear but believed to involve specific posttranslational modification to the protein. Numerous studies have demonstrated that GAPDH is a target for tyrosine nitration. However, the site of modification and the molecular consequence have not been defined. Rabbit GAPDH with a reversibly protected catalytic cysteine was nitrated in vitro with tetranitromethane, resulting in complete loss of GAPDH catalytic activity. Nitration was estimated as 0.32 mol of nitrotyrosine residue per mole of GAPDH. Mass spectrometry analysis of nitrated GAPDH indicated that Tyr311 and Tyr317 were the sole sites of nitration. The X-ray crystal structure revealed that the distances between Tyr311 and Tyr317 and the cofactor nicotinamide adenine dinucleotide (NAD+) were less than 7.2 and 3.7 Å, respectively, implying that nitration of these two residues may affect NAD+ binding. This possibility was assessed using an NAD+ binding assay, which showed that nitrated GAPDH was incapable of binding NAD+. Thus, these results strongly suggest that Tyr311 and Tyr317 nitration prohibits NAD+ binding, leading to the loss of catalytic activity. PMID:20014444

  6. Transport of 3,4-dihydroxybutyl-1-phosphonate, an analogue of sn-glycerol 3-phosphate.

    PubMed Central

    Leifer, Z; Engel, R; Tropp, B E

    1977-01-01

    3,4-Dihydroxybutyl-1-phosphonate (DHBP), an analogue of glycerol 3-phosphate, is actively transported by the sn-glycerol 3-phosphate transport system of Escherichia coli strain 8. The Km for the transport of DHBP is 200 microM. PMID:400804

  7. Direct nonchromatographic assay for 1-acyl-sn-glycerol-3-phosphate acyltransferase

    SciTech Connect

    Rajasekharan, R.; Ray, T.K.; Cronan, J.E. Jr.

    1988-09-01

    1-Acyl-sn-glycerol-3-phosphate acyltransferase (also called lysophosphatidic acid acyltransferase) which catalyzes the acylation of 1-acyl-sn-glycerol-3-phosphate to phosphatidic acid is generally assayed by the use of a radioactive substrate followed by a time-consuming chromatographic separation of substrate and product. We report a direct and highly sensitive nonchromatographic assay for this enzyme based on the ability of Escherichia coli alkaline phosphatase to dephosphorylate 1-acyl-sn-glycerol-3-phosphate but not phosphatidic acid. This selective hydrolysis coupled with the use of /sup 32/P-labeled 1-acyl-sn-glycerol-3-phosphate as substrate permits measurement of the product, /sup 32/P-labeled phosphatidic acid by solvent extraction or precipitation. We also report a series of enzymatic reactions for the efficient conversion of /sup 32/Pi to /sup 32/P-labeled 1-acyl-sn-glycerol-3-phosphate.

  8. Characterization of mitochondrial glycerol-3-phosphate acyltransferase in notothenioid fishes.

    PubMed

    Keenan, Kelly A; Grove, Theresa J; Oldham, Corey A; O'Brien, Kristin M

    2017-02-01

    Hearts of Antarctic icefishes (suborder Notothenioidei, family Channichthyidae) have higher densities of mitochondria, and mitochondria have higher densities of phospholipids, compared to red-blooded notothenioids. Glycerol-3-phosphate acyltransferase (GPAT) catalyzes the rate-limiting step in glycerolipid biosynthesis. There are four isoforms of GPAT in vertebrates; GPAT1 and GPAT2 are localized to the outer mitochondrial membrane, whereas GPAT3 and GPAT4 are localized to the endoplasmic reticulum membrane. We hypothesized that transcript levels of GPAT1 and/or GPAT2 would mirror densities of mitochondrial phospholipids and be higher in the icefish Chaenocephalus aceratus compared to the red-blooded species Notothenia coriiceps. Transcript levels of GPAT1 were quantified in heart ventricles and liver using qRT-PCR. Additionally, GPAT1 cDNA was sequenced in the Antarctic notothenioids, C. aceratus and N. coriiceps, and in the sub-Antarctic notothenioid, Eleginops maclovinus, to identify amino acid substitutions that may maintain GPAT1 function at cold temperature. Transcript levels of GPAT1 were higher in liver compared to heart ventricles but were not significantly different between the two species. In contrast, transcripts of GPAT2 were only detected in ventricle where they were 6.6-fold higher in C. aceratus compared to N. coriiceps. These data suggest GPAT1 may be more important for synthesizing triacylglycerol, whereas GPAT2 may regulate synthesis of phospholipids. GPAT1 amino acid sequences are highly conserved among the three notothenioids with 97.9-98.7% identity. Four amino acid substitutions within the cytosolic region of Antarctic notothenioid GPAT1 may maintain conformational changes necessary for binding and catalysis at cold temperature.

  9. Testis-specific glyceraldehyde-3-phosphate dehydrogenase: origin and evolution.

    PubMed

    Kuravsky, Mikhail L; Aleshin, Vladimir V; Frishman, Dmitrij; Muronetz, Vladimir I

    2011-06-10

    Glyceraldehyde-3-phosphate dehydrogenase (GAPD) catalyses one of the glycolytic reactions and is also involved in a number of non-glycolytic processes, such as endocytosis, DNA excision repair, and induction of apoptosis. Mammals are known to possess two homologous GAPD isoenzymes: GAPD-1, a well-studied protein found in all somatic cells, and GAPD-2, which is expressed solely in testis. GAPD-2 supplies energy required for the movement of spermatozoa and is tightly bound to the sperm tail cytoskeleton by the additional N-terminal proline-rich domain absent in GAPD-1. In this study we investigate the evolutionary history of GAPD and gain some insights into specialization of GAPD-2 as a testis-specific protein. A dataset of GAPD sequences was assembled from public databases and used for phylogeny reconstruction by means of the Bayesian method. Since resolution in some clades of the obtained tree was too low, syntenic analysis was carried out to define the evolutionary history of GAPD more precisely. The performed selection tests showed that selective pressure varies across lineages and isoenzymes, as well as across different regions of the same sequences. The obtained results suggest that GAPD-1 and GAPD-2 emerged after duplication during the early evolution of chordates. GAPD-2 was subsequently lost by most lineages except lizards, mammals, as well as cartilaginous and bony fishes. In reptilians and mammals, GAPD-2 specialized to a testis-specific protein and acquired the novel N-terminal proline-rich domain anchoring the protein in the sperm tail cytoskeleton. This domain is likely to have originated by exonization of a microsatellite genomic region. Recognition of the proline-rich domain by cytoskeletal proteins seems to be unspecific. Besides testis, GAPD-2 of lizards was also found in some regenerating tissues, but it lacks the proline-rich domain due to tissue-specific alternative splicing.

  10. THE HEME BINDING PROPERTIES OF GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE

    PubMed Central

    Hannibal, Luciana; Collins, Daniel; Brassard, Julie; Chakravarti, Ritu; Vempati, Rajesh; Dorlet, Pierre; Santolini, Jérôme; Dawson, John H.; Stuehr, Dennis J.

    2012-01-01

    Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a glycolytic enzyme that also functions in transcriptional regulation, oxidative stress, vesicular trafficking, and apoptosis. Because GAPDH is required for cellular heme insertion into inducible nitric oxide synthase (Chakravarti et al, PNAS 2010, 107(42):18004-9), we extensively characterized the heme binding properties of GAPDH. Substoichiometric amounts of ferric heme bound to GAPDH (1 heme per GAPDH tetramer) to form a low-spin complex with UV-visible maxima at 362, 418 and 537 nm, and when reduced to ferrous gave maxima at 424, 527 and 559 nm. Ferric heme association and dissociation rate constants at 10 °C were kon =17,800 M−1s−1 and koff1 = 7.0 × 10−3 s−1; koff2 = 3.3 × 10−4 s−1 respectively, giving approximate affinities of 19–390 nM. Ferrous heme bound more poorly to GAPDH and dissociated with a koff = 4.2 × 10−3 s−1. Magnetic circular dichroism (MCD), resonance Raman (rR) and EPR spectroscopic data on the ferric, ferrous, and ferrous-CO complexes of GAPDH showed that the heme is bis-ligated with His as the proximal ligand. The distal ligand in ferric complex was not displaced by CN− or N3− but in ferrous complex was displaceable by CO at a rate of 1.75 s−1 (for [CO]>0.2 mM). Studies with heme analogs revealed selectivity toward the coordinating metal and porphyrin ring structure. GAPDH-heme was isolated from bacteria induced to express rabbit GAPDH in the presence of δ-amino levulinic acid. Our finding of heme binding to GAPDH expands the protein’s potential roles. The strength, selectivity, reversibility, and redox sensitivity of heme binding to GAPDH is consistent with it performing heme sensing or heme chaperone-like functions in cells. PMID:22957700

  11. Buformin suppresses the expression of glyceraldehyde 3-phosphate dehydrogenase.

    PubMed

    Yano, Akiko; Kubota, Masafumi; Iguchi, Kazuhiro; Usui, Shigeyuki; Hirano, Kazuyuki

    2006-05-01

    The biguanides metformin and buformin, which are clinically used for diabetes mellitus, are known to improve resistance to insulin in patients. Biguanides were reported to cause lactic acidosis as a side effect. Since the mechanism of the side effect still remains obscure, we have examined genes whose expression changes by treating HepG2 cells with buformin in order to elucidate the mechanisms of the side effect. A subtraction cDNA library was constructed by the method of suppressive subtractive hybridization and the screening of the library was performed with cDNA probes prepared from HepG2 cells treated with or without buformin for 12 h. The expression of the gene and the protein obtained by the screening was monitored by real-time RT-PCR with specific primers and Western blotting with specific antibody. The amounts of ATP and NAD+ were determined with luciferase and alcohol dehydrogenase, respectively. We found that expression of the glyceraldehyde 3-phosphate dehydrogenase (GAPD) gene was suppressed by treating HepG2 cells with 0.25 mM buformin for 12 h as a result of the library screening. The decrease in the expression depended on the treatment period. The amount of GAPD protein also decreased simultaneously with the suppression of the gene expression by the treatment with buformin. The amount of ATP and NAD+ in the HepG2 cells treated with buformin decreased to 10 and 20% of the control, respectively. These observations imply that the biguanide causes deactivation of the glycolytic pathway and subsequently the accumulation of pyruvate and NADH and a decrease in NAD+. Therefore, the reaction equilibrium catalyzed by lactate dehydrogenase leans towards lactate production and this may result in lactic acidosis.

  12. Periplasmic protein related to the sn-glycerol-3-phosphate transport system of Escherichia coli.

    PubMed Central

    Silhavy, T J; Hartig-Beecken, I; Boos, W

    1976-01-01

    Two-dimensional gel electrophoresis of shock fluids of Escherichia coli K-12 revealed the presence of a periplasmic protein related to sn-glycerol-3-phosphate transport (GLPT) that is under the regulation of glpR, the regulatory gene of the glp regulon. Mutants selected for their resistance to phosphonomycin and found to be defective in sn-glycerol-3-phosphate transport either did not produce GLPT or produced it in reduced amounts. Other mutations exhibited no apparent effect of GLPT. Transductions of glpT+ nalA phage P1 into these mutants and selection for growth on sn-glycerol-3-phosphate revealed a 50% cotransduction frequency to nalA. Reversion of mutants taht did not produce GLPT to growth on sn-glycerol-3-phosphate resulted in strains that produce GLPT. This suggests a close relationship of GLPT to the glpT gene and to sn-glycerol-3-phosphate transport. Attempts to demonstrate binding activity of GLPT in crude shock fluid towards sn-glycerol-3-phosphate have failed so far. However, all shock fluids, independent of their GLPT content, exhibited an enzymatic activity that hydrolyzes under the conditions of the binding assay, 30 to 60% of the sn-glycerol-3-phosphate to glycerol and inorganic orthophosphate. Images PMID:770459

  13. 40 CFR 174.523 - CP4 Enolpyruvylshikimate-3-phosphate (CP4 EPSPS) synthase in all plants; exemption from the...

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 40 Protection of Environment 24 2014-07-01 2014-07-01 false CP4 Enolpyruvylshikimate-3-phosphate... CP4 Enolpyruvylshikimate-3-phosphate (CP4 EPSPS) synthase in all plants; exemption from the requirement of a tolerance. Residues of the CP4 Enolpyruvylshikimate-3-phosphate (CP4 EPSPS) synthase enzyme...

  14. 40 CFR 174.523 - CP4 Enolpyruvylshikimate-3-phosphate (CP4 EPSPS) synthase in all plants; exemption from the...

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 40 Protection of Environment 25 2012-07-01 2012-07-01 false CP4 Enolpyruvylshikimate-3-phosphate... CP4 Enolpyruvylshikimate-3-phosphate (CP4 EPSPS) synthase in all plants; exemption from the requirement of a tolerance. Residues of the CP4 Enolpyruvylshikimate-3-phosphate (CP4 EPSPS) synthase enzyme...

  15. 40 CFR 174.523 - CP4 Enolpyruvylshikimate-3-phosphate (CP4 EPSPS) synthase in all plants; exemption from the...

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 40 Protection of Environment 25 2013-07-01 2013-07-01 false CP4 Enolpyruvylshikimate-3-phosphate... CP4 Enolpyruvylshikimate-3-phosphate (CP4 EPSPS) synthase in all plants; exemption from the requirement of a tolerance. Residues of the CP4 Enolpyruvylshikimate-3-phosphate (CP4 EPSPS) synthase enzyme...

  16. 40 CFR 174.523 - CP4 Enolpyruvylshikimate-3-phosphate (CP4 EPSPS) synthase in all plants; exemption from the...

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 40 Protection of Environment 23 2010-07-01 2010-07-01 false CP4 Enolpyruvylshikimate-3-phosphate... CP4 Enolpyruvylshikimate-3-phosphate (CP4 EPSPS) synthase in all plants; exemption from the requirement of a tolerance. Residues of the CP4 Enolpyruvylshikimate-3-phosphate (CP4 EPSPS) synthase enzyme...

  17. 40 CFR 174.523 - CP4 Enolpyruvylshikimate-3-phosphate (CP4 EPSPS) synthase in all plants; exemption from the...

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 40 Protection of Environment 24 2011-07-01 2011-07-01 false CP4 Enolpyruvylshikimate-3-phosphate... CP4 Enolpyruvylshikimate-3-phosphate (CP4 EPSPS) synthase in all plants; exemption from the requirement of a tolerance. Residues of the CP4 Enolpyruvylshikimate-3-phosphate (CP4 EPSPS) synthase enzyme...

  18. Atomic-level characterization of transport cycle thermodynamics in the glycerol-3-phosphate:phosphate antiporter

    NASA Astrophysics Data System (ADS)

    Moradi, Mahmoud; Enkavi, Giray; Tajkhorshid, Emad

    2015-09-01

    Membrane transporters actively translocate their substrate by undergoing large-scale structural transitions between inward- (IF) and outward-facing (OF) states (`alternating-access' mechanism). Despite extensive structural studies, atomic-level mechanistic details of such structural transitions, and as importantly, their coupling to chemical events supplying the energy, remain amongst the most elusive aspects of the function of these proteins. Here we present a quantitative, atomic-level description of the functional thermodynamic cycle for the glycerol-3-phosphate:phosphate antiporter GlpT by using a novel approach in reconstructing the free energy landscape governing the IF<-->OF transition along a cyclic transition pathway involving both apo and substrate-bound states. Our results provide a fully atomic description of the complete transport process, offering a structural model for the alternating-access mechanism and substantiating the close coupling between global structural transitions and local chemical events.

  19. Atomic-level characterization of transport cycle thermodynamics in the glycerol-3-phosphate:phosphate antiporter

    PubMed Central

    Moradi, Mahmoud; Enkavi, Giray; Tajkhorshid, Emad

    2015-01-01

    Membrane transporters actively translocate their substrate by undergoing large-scale structural transitions between inward- (IF) and outward-facing (OF) states (‘alternating-access' mechanism). Despite extensive structural studies, atomic-level mechanistic details of such structural transitions, and as importantly, their coupling to chemical events supplying the energy, remain amongst the most elusive aspects of the function of these proteins. Here we present a quantitative, atomic-level description of the functional thermodynamic cycle for the glycerol-3-phosphate:phosphate antiporter GlpT by using a novel approach in reconstructing the free energy landscape governing the IF↔OF transition along a cyclic transition pathway involving both apo and substrate-bound states. Our results provide a fully atomic description of the complete transport process, offering a structural model for the alternating-access mechanism and substantiating the close coupling between global structural transitions and local chemical events. PMID:26417850

  20. Synthesis of sn-glycerol 3-phosphate, a key precursor of membrane lipids, in Bacillus subtilis.

    PubMed Central

    Morbidoni, H R; de Mendoza, D; Cronan, J E

    1995-01-01

    The Bacillus subtilis gpsA gene was cloned by complementation of an Escherichia coli gpsA strain auxotrophic for sn-glycerol 3-phosphate. The gene was sequenced and found to encode an NAD(P)H-dependent dihydroxyacetone phosphate reductase with a deduced molecular mass of 39.5 kDa. The deduced amino acid sequence showed strong conservation with that of the E. coli homolog and to other procaryotic and eucaryotic dihydroxyacetone phosphate reductases. The physical location of gpsA on the B. subtilis chromosome was at about 200 degrees. Disruption of the chromosomal gpsA gene yielded B. subtilis strains auxotrophic for glycerol, indicating that the gpsA gene product is responsible for synthesis of the sn-glycerol 3-phosphate required for phospholipid synthesis. We also found that transformation of the classical B. subtilis glycerol auxotrophs with a gpsA-containing genomic fragment yielded transformants that grew in the absence of glycerol. In agreement with prior work, our attempts to determine the reductase activity in B. subtilis extracts were unsuccessful. However, expression of the B. subtilis gpsA gene in E. coli gave reductase activity that was only slightly inhibited by sn-glycerol 3-phosphate. Since the E. coli GpsA dihydroxyacetone phosphate reductase is very sensitive to allosteric inhibition by sn-glycerol 3-phosphate, these results indicate that the B. subtilis gpsA-encoded reductase differs from that of E. coli. It seems that B. subtilis regulates sn-glycerol 3-phosphate synthesis at the level of gene expression rather than through the E. coli mechanism of strong allosteric inhibition of an enzyme produced in excess. PMID:7592341

  1. Mapping of a genetic locus that affects glycerol 3-phosphate transport in Bacillus subtilis.

    PubMed Central

    Lindgren, V

    1978-01-01

    Two types of fosfomycin-resistant mutants of Bacillus subtilis were isolated. Mutants of the first type (GlpT mutants) were resistant to at least 200 microgram of fosfomycin per ml and failed to take up exogenous glycerol 3-phosphate. Mutants of the second type were resistant to lower concentrations of fosfomycin and transported glycerol-3-phosphate as efficiently as wild-type bacteria. The glpT mutations, but not the mutations in the second type of fosfomycin-resistant mutants, map in the cysA-aroI region of the B. subtilis chromosome. PMID:415047

  2. Structural Basis of Substrate Selectivity in the Glycerol-3-Phosphate: Phosphate Antiporter GlpT

    PubMed Central

    Law, Christopher J.; Enkavi, Giray; Wang, Da-Neng; Tajkhorshid, Emad

    2009-01-01

    Abstract Major facilitators represent the largest superfamily of secondary active transporter proteins and catalyze the transport of an enormous variety of small solute molecules across biological membranes. However, individual superfamily members, although they may be architecturally similar, exhibit strict specificity toward the substrates they transport. The structural basis of this specificity is poorly understood. A member of the major facilitator superfamily is the glycerol-3-phosphate (G3P) transporter (GlpT) from the Escherichia coli inner membrane. GlpT is an antiporter that transports G3P into the cell in exchange for inorganic phosphate (Pi). By combining large-scale molecular-dynamics simulations, mutagenesis, substrate-binding affinity, and transport activity assays on GlpT, we were able to identify key amino acid residues that confer substrate specificity upon this protein. Our studies suggest that only a few amino acid residues that line the transporter lumen act as specificity determinants. Whereas R45, K80, H165, and, to a lesser extent Y38, Y42, and Y76 contribute to recognition of both free Pi and the phosphate moiety of G3P, the residues N162, Y266, and Y393 function in recognition of only the glycerol moiety of G3P. It is the latter interactions that give the transporter a higher affinity to G3P over Pi. PMID:19720022

  3. A second transport system for sn-glycerol-3-phosphate in Escherichia coli.

    PubMed Central

    Argast, M; Ludtke, D; Silhavy, T J; Boos, W

    1978-01-01

    Strains containing phage Mucts inserted into glpT were isolated as fosfomycin-resistant clones. These mutants did not transport sn-glycerol-3-phosphate, and they lacked GLPT, a protein previously shown to be a product of the glpT operon. By plating these mutants on sn-glycerol-3-phosphate at 43 degrees C, we isolated revertants that regained the capacity to grow on G3P. Most of these revertants did not map in glpT and did not regain GLPT. These revertants exhibited a highly efficient uptake system for sn-glycerol-3-phosphate within an apparent Km of 5 micron. In addition, three new proteins (GP 1, 2, and 3) appeared in the periplasm of these revertants. None of these proteins were antigentically related to GLPT. However, like GLPT, GP1 exhibits abnormal behavior on sodium dodecyl sulfate-polyacrylamide gels. GP 2 is an efficient binding protein. The new uptake system showed different characteristics than the system that is coded for by the glpT operon. It was inhibited neither by phosphate nor fosfomycin. So far, none of the systems that transport organic acids in Escherichia coli could be implicated in the new sn-glycerol-3-phosphate uptake activity. The mutation ugp+, which was responsible for the appearance of the new transport system and the appearance of GP 1, 2, and 3 in the periplasm was cotransducible with araD by phage P1 transduction and was recessive in merodiploids. Images PMID:363686

  4. Chemical and enzymatic methodologies for the synthesis of enantiomerically pure glyceraldehyde 3-phosphates.

    PubMed

    Gauss, Dominik; Schoenenberger, Bernhard; Wohlgemuth, Roland

    2014-05-07

    Glyceraldehyde 3-phosphates are important intermediates of many central metabolic pathways in a large number of living organisms. d-Glyceraldehyde 3-phosphate (d-GAP) is a key intermediate during glycolysis and can as well be found in a variety of other metabolic pathways. The opposite enantiomer, l-glyceraldehyde 3-phosphate (l-GAP), has been found in a few exciting new pathways. Here, improved syntheses of enantiomerically pure glyceraldehyde 3-phosphates are reported. While d-GAP was synthesized by periodate cleavage of d-fructose 6-phosphate, l-GAP was obtained by enzymatic phosphorylation of l-glyceraldehyde. (1)H- and (31)P NMR spectroscopy was applied in order to examine pH-dependent behavior of GAP over time and to identify potential degradation products. It was found that GAP is stable in acidic aqueous solution below pH 4. At pH 7, methylglyoxal is formed, whereas under alkaline conditions, the formation of lactic acid could be observed.

  5. Glycerol-3-phosphate is a critical mobile inducer of systemic immunity in plants

    USDA-ARS?s Scientific Manuscript database

    Glycerol-3-phosphate (G3P) is an important metabolite that contributes to the growth and disease-related physiologies of prokaryotes, plants, animals and humans alike. Here we show that G3P serves as the inducer of an important form of broad-spectrum immunity in plants, termed systemic acquired resi...

  6. EXPRESSION OF THE SPERMATOGENIC CELL-SPECIFIC GLYCERALDEHYDE 3-PHOSPHATE DEHYDROGENASE (GAPDS) IN RAT TESTIS

    EPA Science Inventory

    The spermatogenic cell-specific variant of glyceraldehyde 3-phosphate dehydrogenase (GAPDS) has been cloned from a rat testis cDNA library and its pattern of expression determined. A 1417 nucleotide cDNA has been found to encode an enzyme with substantial homology to mouse GAPDS...

  7. EXPRESSION OF THE SPERMATOGENIC CELL-SPECIFIC GLYCERALDEHYDE 3-PHOSPHATE DEHYDROGENASE (GAPDS) IN RAT TESTIS

    EPA Science Inventory

    The spermatogenic cell-specific variant of glyceraldehyde 3-phosphate dehydrogenase (GAPDS) has been cloned from a rat testis cDNA library and its pattern of expression determined. A 1417 nucleotide cDNA has been found to encode an enzyme with substantial homology to mouse GAPDS...

  8. Redundant Systems of Phosphatidic Acid Biosynthesis via Acylation of Glycerol-3-Phosphate or Dihydroxyacetone Phosphate in the Yeast Saccharomyces cerevisiae

    PubMed Central

    Athenstaedt, Karin; Weys, Sabine; Paltauf, Fritz; Daum, Günther

    1999-01-01

    In the yeast Saccharomyces cerevisiae lipid particles harbor two acyltransferases, Gat1p and Slc1p, which catalyze subsequent steps of acylation required for the formation of phosphatidic acid. Both enzymes are also components of the endoplasmic reticulum, but this compartment contains additional acyltransferase(s) involved in the biosynthesis of phosphatidic acid (K. Athenstaedt and G. Daum, J. Bacteriol. 179:7611–7616, 1997). Using the gat1 mutant strain TTA1, we show here that Gat1p present in both subcellular fractions accepts glycerol-3-phosphate and dihydroxyacetone phosphate as a substrate. Similarly, the additional acyltransferase(s) present in the endoplasmic reticulum can acylate both precursors. In contrast, yeast mitochondria harbor an enzyme(s) that significantly prefers dihydroxyacetone phosphate as a substrate for acylation, suggesting that at least one additional independent acyltransferase is present in this organelle. Surprisingly, enzymatic activity of 1-acyldihydroxyacetone phosphate reductase, which is required for the conversion of 1-acyldihydroxyacetone phosphate to 1-acylglycerol-3-phosphate (lysophosphatidic acid), is detectable only in lipid particles and the endoplasmic reticulum and not in mitochondria. In vivo labeling of wild-type cells with [2-3H, U-14C]glycerol revealed that both glycerol-3-phosphate and dihydroxyacetone phosphate can be incorporated as a backbone of glycerolipids. In the gat1 mutant and the 1-acylglycerol-3-phosphate acyltransferase slc1 mutant, the dihydroxyacetone phosphate pathway of phosphatidic acid biosynthesis is slightly preferred as compared to the wild type. Thus, mutations of the major acyltransferases Gat1p and Slc1p lead to an increased contribution of mitochondrial acyltransferase(s) to glycerolipid synthesis due to their substrate preference for dihydroxyacetone phosphate. PMID:10049376

  9. Structure of glycerol-3-phosphate dehydrogenase, an essential monotopic membrane enzyme involved in respiration and metabolism

    SciTech Connect

    Yeh, Joanne I.; Chinte, Unmesh; Du, Shoucheng

    2008-04-02

    Sn-glycerol-3-phosphate dehydrogenase (GlpD) is an essential membrane enzyme, functioning at the central junction of respiration, glycolysis, and phospholipid biosynthesis. Its critical role is indicated by the multitiered regulatory mechanisms that stringently controls its expression and function. Once expressed, GlpD activity is regulated through lipid-enzyme interactions in Escherichia coli. Here, we report seven previously undescribed structures of the fully active E. coli GlpD, up to 1.75 {angstrom} resolution. In addition to elucidating the structure of the native enzyme, we have determined the structures of GlpD complexed with substrate analogues phosphoenolpyruvate, glyceric acid 2-phosphate, glyceraldehyde-3-phosphate, and product, dihydroxyacetone phosphate. These structural results reveal conformational states of the enzyme, delineating the residues involved in substrate binding and catalysis at the glycerol-3-phosphate site. Two probable mechanisms for catalyzing the dehydrogenation of glycerol-3-phosphate are envisioned, based on the conformational states of the complexes. To further correlate catalytic dehydrogenation to respiration, we have additionally determined the structures of GlpD bound with ubiquinone analogues menadione and 2-n-heptyl-4-hydroxyquinoline N-oxide, identifying a hydrophobic plateau that is likely the ubiquinone-binding site. These structures illuminate probable mechanisms of catalysis and suggest how GlpD shuttles electrons into the respiratory pathway. Glycerol metabolism has been implicated in insulin signaling and perturbations in glycerol uptake and catabolism are linked to obesity in humans. Homologs of GlpD are found in practically all organisms, from prokaryotes to humans, with >45% consensus protein sequences, signifying that these structural results on the prokaryotic enzyme may be readily applied to the eukaryotic GlpD enzymes.

  10. Glycerolipid biosynthesis in Saccharomyces cerevisiae: sn-glycerol-3-phosphate and dihydroxyacetone phosphate acyltransferase activities.

    PubMed Central

    Schlossman, D M; Bell, R M

    1978-01-01

    Yeast acyl-coenzyme A:dihydroxyacetone-phosphate O-acyltransferase (DHAP acyltransferase; EC 2.3.1.42) was investigated to (i) determine whether its activity and that of acyl-coenzyme A:sn-glycerol-3-phosphate O-acyltransferase (glycerol-P acyltransferase; EC 2.3.1.15) represent dual catalytic functions of a single membranous enzyme, (ii) estimate the relative contributions of the glycerol-P and DHAP pathways for yeast glycerolipid synthesis, and (iii) evaluate the suitability of yeast for future genetic investigations of the eucaryotic glycerol-P and DHAP acyltransferase activities. The membranous DHAP acyltransferase activity showed an apparent Km of 0.79 mM for DHAP, with a Vmax of 5.3 nmol/min per mg, whereas the glycerol-P acyltransferase activity showed an apparent Km of 0.05 mM for glycerol-P, with a Vmax of 3.4 nmol/min per mg. Glycerol-P was a competitive inhibitor (Ki, 0.07 mM) of the DHAP acyltransferase activity, and DHAP was a competitive inhibitor (Ki, 0.91 mM) of the glycerol-P acyltransferase activity. The two acyltransferase activities exhibited marked similarities in their pH dependence, acyl-coenzyme A chain length preference and substrate concentration dependencies, thermolability, and patterns of inactivation by N-ethylmaleimide, trypsin, and detergents. Thus, the data strongly suggest that yeast glycerol-P and DHAP acyltransferase activities represent dual catalytic functions of a single membrane-bound enzyme. Furthermore, since no acyl-DHAP oxidoreductase activity could be detected in yeast membranes, the DHAP pathway for glycerolipid synthesis may not operate in yeast. PMID:25265

  11. L-glycerol-3-phosphate dehydrogenase from the insect Ceratitis capitata. Purfication, physicochemical and enzymic properties.

    PubMed

    Fernández-Sousa, J M; Gavilanes, J G; Municio, A M; Pérez-Aranda, A

    1977-03-15

    Soluble L-glycerol-3-phosphate dehydrogenase (sn-glycerol-3-phosphate: NAD+ 2-oxidoreductase, EC 1.1.1.8) from the mediterranean fruit fly Ceratitis capitata has been purified 130-fold with an overall yield of about 40%. The final preparation had a specific activity of about 200 mumol NADH/min/mg protein. The enzyme preparation has been shown to be homogeneous throughout disc gel electrophoresis, dodecyl sulphate gel electrophoresis, isoelectric focusing and ultracentrifugation. The Km values for dihydroxyacetone phosphate, NADH, L-glycerol-3-phosphate and NAD+ were respectively 0.33, 0.018, 0.74 and 0.26 mM. L-glycerol-3-phosphate dehydrogenase from the insect had a maximal activity around pH 6.6 for the oxidation of NADH and pH 10.0 for the reduction of NAD+. It was stable from pH 6.0 to pH 9.0 at 20 degrees C for 1 h and remained active after incubating at 30 degrees C for 30 min at pH 6.6. The enzyme was completely inactivated by incubating at 60 degrees C for 5 min. Enzyme stability versus ionic strength as well as the dependence of the reaction velocity on temperature are also reported. The active enzyme was found to have a minimum molecular weight of approx. 63 000. Molecular weight determinations by sodium dodecyl sulphate gel electrophoresis gave subunit weights of 33 500. The isoelectric point of the protein was determined by electrofocusing and found to be 5.75 +/- 0.05. The extinction coefficient at 278 nm was calculated by dry weight measurements to be E1cm 1mg/ml = 0.42 +/- 0.1. Sedimentation velocity studies on ultracentrifuge indicated a dependence of the sedimentation coefficient on the enzyme concentration. The amino acid composition of the enzyme was determined. The protein has no free N-terminal residue and the digestion with carboxypeptidases gave the C-terminal sequence: -ala-gly-ser. All these data are discussed in relation to the properties of the enzyme from other sources.

  12. Characterization of an Arabidopsis thaliana mutant lacking a cytosolic non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase.

    PubMed

    Rius, Sebastián P; Casati, Paula; Iglesias, Alberto A; Gomez-Casati, Diego F

    2006-08-01

    Non-phosphorylating glyceraldehyde- 3-phosphate dehydrogenase (NP-GAPDH) is a conserved cytosolic protein found in higher plants. In photosynthetic cells, the enzyme is involved in a shuttle transfer mechanism to export NADPH from the chloroplast to the cytosol. To investigate the role of this enzyme in plant tissues, we characterized a mutant from Arabidopsis thaliana having an insertion at the NP-GAPDH gene locus. The homozygous mutant was determined to be null respect to NP-GAPDH, as it exhibited undetectable levels of both transcription of NP-GAPDH mRNA, protein expression and enzyme activity. Transcriptome analysis demonstrated that the insertion mutant plant shows altered expression of several enzymes involved in carbohydrate metabolism. Significantly, cytosolic phosphorylating (NAD-dependent) glyceraldehyde-3-phosphate dehydrogenase mRNA levels are induced in the mutant, which correlates with an increase in enzyme activity. mRNA levels and enzymatic activity of glucose-6-phosphate dehydrogenase were also elevated, correlating with an increase in NADPH concentration. Moreover, increased ROS levels were measured in the mutant plants. Down-regulation of several glycolytic and photosynthetic genes suggests that NP-GAPDH is important for the efficiency of both metabolic processes. The results presented demonstrate that NP-GAPDH has a relevant role in plant growth and development.

  13. An EPSP synthase inhibitor joining shikimate 3-phosphate with glyphosate: synthesis and ligand binding studies.

    PubMed

    Marzabadi, M R; Gruys, K J; Pansegrau, P D; Walker, M C; Yuen, H K; Sikorski, J A

    1996-04-02

    A novel EPSP synthase inhibitor 4 has been designed and synthesized to probe the configurational details of glyphosate recognition in its herbicidal ternary complex with enzyme and shikimate 3-phosphate (S3P). A kinetic evaluation of the new 3-dephospho analog 12, as well as calorimetric and (31)P NMR spectroscopic studies of enzyme-bound 4, now provides a more precise quantitative definition for the molecular interactions of 4 with this enzyme. The very poor binding, relative to 4, displayed by the 3-dephospho analog 12 is indicative that 4 has a specific interaction with the S3P site. A comparison of Ki(calc) for 12 versus the Ki(app) for 4 indicates that the 3-phosphate group in 4 contributes about 4.8 kcal/mol to binding. This compares well with the 5.2 kcal/mol which the 3-phosphate group in S3P contributes to binding. Isothermal titration calorimetry demonstrates that 4 binds to free enzyme with an observed Kd of 0.53 +/- 0.04 microM. As such, 4 binds only 3-fold weaker than glyphosate and about 150-fold better than N-methylglyphosate. Consequently, 4 represents the most potent N-alkylglyphosate derivative identified to date. However, the resulting thermodynamic binding parameters clearly demonstrate that the formation of EPSPS x 4 is entropy driven like S3P. The binding characteristics of 4 are fully consistent with a primary interaction localized at the S3P subsite. Furthermore, (31)P NMR studies of enzyme-bound 4 confirm the expected interaction at the shikimate 3-phosphate site. However, the chemical shift observed for the phosphonate signal of EPSPS x 4 is in the opposite direction than that observed previously when glyphosate binds with enzyme and S3P. Therefore, when 4 occupies the S3P binding site, there is incomplete overlap at the glyphosate phosphonate subsite. As a glyphosate analog inhibitor, the potency of 4 most likely arises from predominant interactions which occur outside the normal glyphosate binding site. Consequently, 4 is best described

  14. Cloning and characterization of 5-enopyruvylshikimate-3-phosphate synthase from Pantoea sp.

    PubMed

    Liu, F; Cao, Y P

    2015-12-29

    The shikimate pathway enzyme 5-enopyruvylshikimate-3-phosphate synthase (EPSPS) is the target of the broad spectrum herbicide glyphosate. A novel aroA gene encoding an EPSPS from Pantoea sp was identified and subcloned into the pET-28a vector to construct the recombinant pET-AroAPantoea sp plasmid. Amino acid sequence analysis indicated that AroAPantoea sp is a class I AroA enzyme. When expressed in Escherichia coli, it conveyed high tolerance to glyphosate. AroAPantoea sp may be used to generate transgenic glyphosate-tolerant plants.

  15. Functional characterization of 5-enopyruvylshikimate-3-phosphate synthase from Alkaliphilus metalliredigens in transgenic Arabidopsis.

    PubMed

    Xing, Xiao-Juan; Tian, Yong-Sheng; Peng, Ri-He; Xu, Jing; Zhao, Wei; Yao, Quan-Hong; Sun, Sheng

    2014-10-01

    Although a large number of AroA enzymes (EPSPS: 5-enopyruvylshikimate-3-phosphate synthase) have been identified, cloned, and tested for glyphosate resistance, only two AroA variants, derived from Agrobacterium tumefaciens strain CP4 and Zea mays, have been utilized to produce the commercial glyphosate-resistant crops. Here, we have used a PCR-based twostep DNA synthesis method to synthesize an aroA gene (aroAA. metalliredigens) from Alkaliphilus metalliredigens, encoding a new EPSPS. Furthermore, transgenic Arabidopsis with the new aroAA. metalliredigens gene was obtained to confirm the potential of the novel aroA gene in developing glyphosate-resistant crops.

  16. Relationship between a stress membrane protein of Oenococcus oeni and glyceraldehyde-3-phosphate dehydrogenases.

    PubMed

    Carreté, Ramon; Reguant, Cristina; Bordons, Albert; Constantí, Magda

    2005-10-01

    The goal of this study was to analyze how the profiles of membrane proteins of Oenococcus oeni change under particular stress conditions of wine. Sodium dodecyl sulfate polyacrylamide gel electrophoresis protein profiles of membrane fraction showed that a 40-kDa protein was overexpressed in the presence of SO2. The sequence of its N-terminal fragment showed a significant identity with glyceraldehyde-3-phosphate dehydrogenases (GAPDHs), but the protein showed no GAPDH activity. This sequence was compared with those of other GAPDHs with ClustalW alignment, and it was found to be somewhat similar to that of the cell-wall and membrane proteins of other lactic acid bacteria.

  17. Influence of heavy metals on glyceraldehyde-3-phosphate dehydrogenase interactions in Chironomus riparius larvae.

    PubMed

    Wai, Isaac; Chong, King; Ho, Wing Shing

    2013-08-01

    Some aquatic organisms can live in contaminated environment due to their adaptable defense mechanism related to their inducible detoxification and excretion. A recent study showed glyceraldehyde-3-phosphate dehydrogenase (GAPDH) can modulate different cellular activities including transcription activation and detoxification. In the present study, the authors report on experiments to test the GAPDH activity of Chironomus riparius toward heavy metals. Glyceraldehyde-3-phosphate dehydrogenase was isolated and purified from C. riparius. The kinetics of the enzyme was measured. The results showed that GAPDH was inhibited by heavy metals including Co(2+) , Cu(2+) , Fe(2+) , Ni(2+) , Pb(2+) , but was activated by zinc ions. The kinetics study of the enzyme showed maximum initial velocity (Vmax) of GAPDH increased by 50%. In addition, the substrate and cofactor affinity increased in the presence of zinc. The GAPDH from C. riparius had maximum activities at pH 8.5 and 37 °C. The protein sequence analysis shows that there are 2 additional cysteine and histidine residues in the conserved region of GAPDH from C. riparius, which is believed to play an important role in the interactions with heavy metals. The results suggest that exposure to zinc could modulate GAPDH, which could be related to response of antioxidant defense to other heavy metals.

  18. Cloning and characterization of glyceraldehyde-3-phosphate dehydrogenase encoding gene in Gracilaria/Gracilariopsis lemaneiformis

    NASA Astrophysics Data System (ADS)

    Xueying, Ren; Zhenghong, Sui; Xuecheng, Zhang

    2006-04-01

    Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) plays important roles in various cellular processes. A cytosolic GAPDH encoding gene ( gpd) of Gracilaria/Gracilariopsis lemaneiformis was cloned and characterized. Deduced amino acid sequence of the enzyme of G. lemaneiformis had high homology with those of seven red algae. The 5'-untranslated regions of the GAPDHs encoding genes of these red algae varied greatly. GAPDHs of these red algae shared the highly conserved glyceraldehyde 3-phosphate dehydrogenase active site ASCTTNCL. However, such active site of Cyanidium caldarium was different from those of the other six algae at the last two residues (CL to LF), thus the spatial structure of its GAPDH active center may be different from those of the other six. Phylogenetic analysis indicated that GAPDH of G. lemaneiformis might have undergone an evolution similar to those of Porphyra yezoensis, Chondrus crispus, and Gracilaria verrucosa. C. caldarium had a closer evolutionary relationship with Cyanidioschyzon merolae than with Cyanidium sp. Virtual Northern blot analysis revealed that gpd of G. lemaneiformis expressed constitutively, which suggested that it might be house-keeping and could be adapted as an inner control in gene expression analysis of G. lemaneiformis.

  19. Moonlighting glycolytic protein glyceraldehyde-3-phosphate dehydrogenase (GAPDH): an evolutionarily conserved plasminogen receptor on mammalian cells.

    PubMed

    Chauhan, Anoop Singh; Kumar, Manoj; Chaudhary, Surbhi; Patidar, Anil; Dhiman, Asmita; Sheokand, Navdeep; Malhotra, Himanshu; Raje, Chaaya Iyengar; Raje, Manoj

    2017-03-15

    Prokaryotic pathogens establish infection in mammals by capturing the proteolytic enzyme plasminogen (Plg) onto their surface to digest host extracellular matrix (ECM). One of the bacterial surface Plg receptors is the multifunctional glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH). In a defensive response, the host mounts an inflammatory response, which involves infiltration of leukocytes to sites of inflammation. This requires macrophage exit from the blood and migration across basement membranes, a phenomenon dependent on proteolytic remodeling of the ECM utilizing Plg. The ability of Plg to facilitate inflammatory cell recruitment critically depends on receptors on the surface of phagocyte cells. Utilizing a combination of biochemical, cellular, knockdown, and in vivo approaches, we demonstrated that upon inflammation, macrophages recruit GAPDH onto their surface to carry out the same task of capturing Plg to digest ECM to aid rapid phagocyte migration and combat the invading pathogens. We propose that GAPDH is an ancient, evolutionarily conserved receptor that plays a key role in the Plg-dependent regulation of macrophage recruitment in the inflammatory response to microbial aggression, thus pitting prokaryotic GAPDH against mammalian GAPDH, with both involved in a conserved role of Plg activation on the surface of their respective cells, to conflicting ends.-Chauhan, A. S., Kumar, M., Chaudhary, S., Patidar, A., Dhiman, A., Sheokand, N., Malhotra, H., Raje, C. I., Raje, M. Moonlighting glycolytic protein glyceraldehyde-3-phosphate dehydrogenase (GAPDH): an evolutionarily conserved plasminogen receptor on mammalian cells.

  20. Conformational and activity changes during guanidine denaturation of D-glyceraldehyde-3-phosphate dehydrogenase.

    PubMed

    Xie, G F; Tsou, C L

    1987-01-05

    Changes in intrinsic protein fluorescence of lobster muscle D-glyceraldehyde-3-phosphate dehydrogenase (D-glyceraldehyde-3-phosphate: NAD+ oxidoreductase (phosphorylating), EC 1.2.1.12) have been compared with inactivation of the enzyme during denaturation in guanidine solutions. The holoenzyme is completely inactivated at guanidine concentrations less than 0.5 M and this is accompanied by a red shift of the emission maximum at 335 nm and a marked decrease in intensity of the intrinsic fluorescence. At 0.5 M guanidine, the inactivation is a slow process, with a first-order rate constant of 2.4 X 10(-3) s-1. A further red shift in the emission maximum and a decrease in intensity occur at guanidine concentrations higher than 1.5 M. The emission peak at 410 nm of the fluorescent NAD derivative introduced at the active site of this enzyme (Tsou, C.L. et al. (1983) Biochem. Soc. Trans. 11, 425-429) shows both a red shift and a marked decrease in intensity at the same guanidine concentration required to bring about the inactivation and the initial changes in the intrinsic fluorescence of the holoenzyme. It appears that treatment by low guanidine concentrations leads to both complete inactivation and perturbation of the active site conformation and that a tryptophan residue is situated at or near the active site.

  1. U(VI) Sequestration in Hydroxyapatite Produced by Microbial Glycerol 3-Phosphate Metabolism▿ †

    PubMed Central

    Shelobolina, Evgenya S.; Konishi, Hiromi; Xu, Huifang; Roden, Eric E.

    2009-01-01

    Previous studies have demonstrated the potential for removal of U(VI) from solution via precipitation of U(VI)-bearing calcium-phosphate (Ca-P) minerals coupled to microbial hydrolysis of glycerol phosphate compounds. We evaluated this process in circumneutral-pH groundwater from Area 2 of the U.S. Department of Energy Field Research Center at Oak Ridge National Laboratory. Area 2 groundwater contains high concentrations of dissolved calcium (ca. 4 mM), and thus, release of phosphate during glycerol phosphate metabolism has the potential to create conditions favorable for U(VI) sequestration in Ca-P minerals. Microbial enumeration and isolation studies verified the presence of aerobic and nitrate-reducing glycerol 3-phosphate (G3P)-metabolizing microorganisms in Area 2 sediments. Coprecipitation of U(VI) with Ca-P minerals coupled to microbial G3P hydrolysis was demonstrated in artificial groundwater under aerobic and nitrate-reducing conditions. Transmission electron microscopy analysis and mineral-washing experiments demonstrated that U(VI) was incorporated into the structure of the insoluble Ca-P mineral hydroxyapatite [Ca5(PO4)3OH]. Our results support the idea that U(VI) can be effectively removed from solution in contaminated aquifers through stimulation of microbial organophosphate metabolism. PMID:19633115

  2. Isolation of a FAD-GPDH gene encoding a mitochondrial FAD-dependent glycerol-3-phosphate dehydrogenase from Dunaliella salina.

    PubMed

    Yang, Wanggui; Cao, Yi; Sun, Xiaofei; Huang, Fei; He, Qinghua; Qiao, Dairong; Bai, Linhan

    2007-06-01

    The mitochondrial FAD-dependent glycerol-3-phosphate dehydrogenase (FAD-GPDH), recently reported in plants, has been detailed in yeast and animal systems. It oxidizes glycerol-3-phosphate (G-3-P) to dihydroxyacetone phosphate (DHAP) on the outer surface of mitochondrial inner membrane. A cDNA encoding the Dunaliella salina mitochondrial glycerol-3-phosphate dehydrogenase (DsFAD-GPDH) has been cloned and sequenced. The full length cDNA is 2791 bp, with an open reading frame (ORF) encoding 650 predicted amino acids, which show strong homology to reported FAD-GPDHs and have an apparent mitochondrial targeting sequence in the N-terminal. The sequence has been submitted to the GenBank database under Accession No. DQ916107. Results of Real-Time Quantitative PCR and enzymatic assays show that expression of DsFAD-GPDH is enhanced at first by salt treatment, and repressed by oxygen deficiency and cold stress.

  3. Structure of glyceraldehyde-3-phosphate dehydrogenase from the archaeal hyperthermophile Methanocaldococcus jannaschii

    PubMed Central

    Malay, Ali D.; Bessho, Yoshitaka; Ellis, Mark J.; Antonyuk, Svetlana V.; Strange, Richard W.; Hasnain, S. Samar; Shinkai, Akeo; Padmanabhan, Balasundaram; Yokoyama, Shigeyuki

    2009-01-01

    The X-ray crystal structure of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from the hyperthermophilic archaeon Methanocaldococcus jannaschii (Mj-GAPDH) was determined to 1.81 Å resolution. The crystal belonged to space group C2221, with unit-cell parameters a = 83.4, b = 152.0, c = 118.6 Å. The structure was solved by molecular replacement and was refined to a final R factor of 17.1% (R free = 19.8%). The final structure included the cofactor NADP+ at the nucleotide-binding site and featured unoccupied inorganic and substrate phosphate-binding sites. A comparison with GAPDH structures from mesophilic sources suggested that Mj-GAPDH is stabilized by extensive electrostatic interactions between the C-terminal α-helices and various distal loop regions, which are likely to contribute to thermal stability. The key phosphate-binding residues in the active site of Mj-GAPDH are conserved in other archaeal GAPDH proteins. These residues undergo a conformational shift in response to occupancy of the inorganic phosphate site. PMID:20054117

  4. Folding domains and intramolecular ionic interactions of lysine residues in glyceraldehyde 3-phosphate dehydrogenase.

    PubMed Central

    Lambert, J M; Perham, R N

    1977-01-01

    1. Treatment with methyl acetimidate was used to probe the topography of several tetrameric glyceraldehyde 3-phosphate dehydrogenases, in particular the holoenzymes from rabbit muscle and Bacillus stearothermophilus. During the course of the reaction with the rabbit muscle enzyme, the number of amino groups fell rapidly from the starting value of 27 per subunit to a value of approx. five per subunit. This number could be lowered further to values between one and two per subunit by a second treatment with methyl acetimidate. The enzyme remained tetrameric throughout and retained 50% of its initial catalytic activity at the end of the experiment. 2. Use of methyl [1-14C]acetimidate and small-scale methods of protein chemistry showed that only one amino group per subunit, that of lysine-306, was completely unavailable for reaction with imido ester in the native enzyme. This results is consistent with the structure of the highly homologous glyceraldehyde 3-phosphate dehydrogenase of lobster muscle deduced from X-ray-crystallographic analysis, since lysine-306 can be seen to form an intrachain ion-pair with aspartic acid-241 in the hydrophobic environment of a subunit-subunit interface. 3. Several other amino groups in the rabbit muscle enzyme that reacted only slowly with the reagent were also identified chemically. These were found to be located entirely in the C-terminal half of the polypeptides chain, which comprises a folding domain associated with catalytic activity and subunit contact in the three-dimensional structure. Slow reaction of these 'surface' amino groups with methyl acetimidate is attributed to intramolecular ionic interactions of the amino groups with neighbouring side-chain carboxyl groups, a conclusion that is compatible with the reported three-dimensional structure and with the dependence of the reaction of ionic stength. 4. Very similar results were obtained with the enzymes from B. stearothermophilus and from ox muscle and ox liver, supporting

  5. Receptor protein kinase FERONIA controls leaf starch accumulation by interacting with glyceraldehyde-3-phosphate dehydrogenase.

    PubMed

    Yang, Tao; Wang, Long; Li, Chiyu; Liu, Ying; Zhu, Sirui; Qi, Yinyao; Liu, Xuanming; Lin, Qinglu; Luan, Sheng; Yu, Feng

    2015-09-11

    Cell expansion is coordinated by several cues, but available energy is the major factor determining growth. Receptor protein kinase FERONIA (FER) is a master regulator of cell expansion, but the details of its control mechanisms are not clear. Here we show that FER interacts with cytosolic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH, GAPC1 and GAPC2), that catalyzes a key reaction in glycolysis, which contributes to energy production. When there is an FER deficiency, there are corresponding decreases in the enzyme activity of GAPDH and increased amounts of starch. More importantly, gapc1/2 mutants mimic fer4 mutants. These data indicate that FER regulated starch content is an evolutionarily conserved function in plants that connects the cell expansion and energy metabolism pathways.

  6. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a specific substrate of yeast metacaspase.

    PubMed

    Silva, A; Almeida, B; Sampaio-Marques, B; Reis, M I R; Ohlmeier, S; Rodrigues, F; Vale, A do; Ludovico, P

    2011-12-01

    Yeast metacaspase (Yca1p) is required for the execution of apoptosis upon a wide range of stimuli. However, the specific degradome of this yeast protease has not been unraveled so far. By combining different methodologies described as requisites for a protein to be considered a protease substrate, such as digestome analysis, cleavage of recombinant GAPDH by metacaspase and evaluation of protein levels in vivo, we show that upon H(2)O(2)-induced apoptosis, the metabolic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a specific target of metacaspase. Nitric oxide (NO) signaling, which mediates H(2)O(2)-induced apoptosis, is required for metacaspase specific GAPDH cleavage. In conclusion, in this work we identified GAPDH as the first direct yeast metacaspase substrate described so far. Although mammalian caspases and yeast metacaspase apparently have distinct target cleavage sites, GAPDH arises as a common substrate for these proteases.

  7. Succination of proteins by fumarate: mechanism of inactivation of glyceraldehyde-3-phosphate dehydrogenase in diabetes.

    PubMed

    Blatnik, Matthew; Thorpe, Suzanne R; Baynes, John W

    2008-04-01

    S-(2-succinyl)cysteine (2SC) is a chemical modification of proteins formed by a Michael addition reaction between the Krebs cycle intermediate, fumarate, and thiol groups in protein--a process known as succination of protein. Succination causes irreversible inactivation of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in vitro. GAPDH was immunoprecipitated from muscle of diabetic rats, then analyzed by ultra-performance liquid chromatography-electrospray ionization-mass spectroscopy. Succination of GAPDH was increased in muscle of diabetic rats, and the extent of succination correlated strongly with the decrease in specific activity of the enzyme. We propose that 2SC is a biomarker of mitochondrial and oxidative stress in diabetes and that succination of GAPDH and other thiol proteins may provide the chemical link between glucotoxicity and the pathogenesis of diabetic complications.

  8. Molecular cloning and characterization of the glyceraldehyde-3-phosphate dehydrogenase gene from Penicillium expansum PE-12.

    PubMed

    Zhang, T; Qi, Z; Yu, Q S; Tang, K X

    2013-07-15

    Penicillium expansum produces large amounts of lipase, which is widely used in laundry detergent and leather industry. We isolated the glyceraldehyde-3-phosphate dehydrogenase gene (PeGPD) from P. expansum PE-12 through reverse transcriptase PCR and 5'-3' rapid amplification of cDNA ends (RACE-PCR). The gene is 1266 bp long, including an ORF of 1014 bp, encoding a polypeptide chain of 337 amino acids. A phylogenetic tree based on GPD proteins showed that P. expansum is close to Aspergillus species, but comparatively distant from P. marneffei. Southern blot results revealed a single copy of PeGPD, and expression analysis gave evidence of high expression levels. PeGPD genes have potential for genetic engineering of P. expansum for industrial lipase production.

  9. Succination of Proteins by Fumarate: Mechanism of Inactivation of Glyceraldehyde-3-Phosphate Dehydrogenase in Diabetes

    PubMed Central

    Blatnik, Matthew; Thorpe, Suzanne R.; Baynes, John W.

    2008-01-01

    S-(2-succinyl)cysteine (2SC) is a chemical modification of proteins formed by a Michael addition reaction between the Krebs cycle intermediate, fumarate, and thiol groups in protein—a process known as succination of protein. Succination causes irreversible inactivation of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in vitro. GAPDH was immunoprecipitated from muscle of diabetic rats, then analyzed by ultra-performance liquid chromatography–electrospray ionization–mass spectroscopy. Succination of GAPDH was increased in muscle of diabetic rats, and the extent of succination correlated strongly with the decrease in specific activity of the enzyme. We propose that 2SC is a biomarker of mitochondrial and oxidative stress in diabetes and that succination of GAPDH and other thiol proteins may provide the chemical link between glucotoxicity and the pathogenesis of diabetic complications. PMID:18448829

  10. Daily Variations in the Glycerol-3-Phosphate Dehydrogenase Isoforms Expression in Triatoma infestans Flight Muscles

    PubMed Central

    Stroppa, María M.; Carriazo, Carlota S.; Gerez de Burgos, Nelia M.; Garcia, Beatríz A.

    2014-01-01

    Triatoma infestans, the main vector of Chagas disease, is a blood-sucking insect. Flight dispersal of adults is the most important mechanism for reinfestation of houses after insecticide spraying. Flight muscles have two glycerol-3-phosphate dehydrogenase (GPDH) isoforms: GPDH-1 is involved in flight metabolism and GPDH-2 provides lipid precursors. In this study, we explored the profile of GPDH expression in females and males adult flight muscles under light/dark cycle, constant light, and constant dark conditions. Under constant dark conditions, GPDH-1 flight muscles of T. infestans showed a rhythmic pattern of transcription synchronous with a rhythmic profile of activity suggesting regulation by the endogenous circadian clock. Otherwise, the GPDH-2 expression analysis showed no regulation by the endogenous clock, but showed that an external factor, such as the dark/light period, was necessary for synchronization of GPDH-2 transcription and activity. PMID:24914000

  11. Sperm-Specific Glyceraldehyde-3-Phosphate Dehydrogenase - An Evolutionary Acquisition of Mammals.

    PubMed

    Muronetz, V I; Kuravsky, M L; Barinova, K V; Schmalhausen, E V

    2015-12-01

    This review is focused on the mammalian sperm-specific glyceraldehyde-3-phosphate dehydrogenase (GAPDS). GAPDS plays the major role in the production of energy required for sperm cell movement and does not perform non-glycolytic functions that are characteristic of the somatic isoenzyme of glyceraldehyde-3-phosphate dehydrogenase. The GAPDS sequence is composed of 408 amino acid residues and includes an additional N-terminal region of 72 a.a. that binds the protein to the sperm tail cytoskeleton. GAPDS is present only in the sperm cells of mammals and lizards, possibly providing them with certain evolutionary advantages in reproduction. In this review, studies concerning the problems of GAPDS isolation, its catalytic properties, and its structural features are described in detail. GAPDS is much more stable compared to the somatic isoenzyme, perhaps due to the necessity of maintaining the enzyme function in the absence of protein expression. The site-directed mutagenesis approach revealed the two GAPDS-specific proline residues, as well as three salt bridges, which seem to be the basis of the increased stability of this protein. As distinct from the somatic isoenzyme, GAPDS exhibits positive cooperativity in binding of the coenzyme NAD+. The key role in transduction of structural changes induced by NAD+ is played by the salt bridge D311-H124. Disruption of this salt bridge cancels GAPDS cooperativity and twofold increases its enzymatic activity instead. The expression of GAPDS was detected in some melanoma cells as well. Its role in the development of certain pathologies, such as cancer and neurodegenerative diseases, is discussed.

  12. Evidence for ligand-induced conformational changes in rabbit-muscle glyceraldehyde-3-phosphate dehydrogenase.

    PubMed

    Henis, Y I; Levitzki, A; Gafni, A

    1979-07-01

    The tetrameric glyceraldehyde-3-phosphate dehydrogenase from rabbit muscle binds NAD+ and some of its analogues in a negatively cooperative manner, whereas other NAD+ analogues bind non-cooperatively to this enzyme. Subsequent to alkylation of a fraction of the active sites of the enzyme with the fluorescent SH reagent N-iodoacetyl-N'-(5-sulfo-1-naphthyl)-ethylenediamine, it was found that the alkylated sites bind NAD+ and NAD+ analogues with a markedly reduced affinity as compared with non-alkylated sites. It was therefore feasible to measure the fluorescence and the circular polarization of the luminescence of the enzyme-bound alkyl groups as a function of binding of NAD+ and of NAD+ analogues to the non-alkylated sites. The changes observed indicate that ligand binding to the non-alkylated sites induces changes in the fluorescence properties of the alkyl groups bound to neighbouring subunits, most likely through the protein moiety. The nature of these changes appears to depend on the structure of the coenzyme analogue. The binding of the non-cooperative binders acetyl-pyridine--adenine dinucleotide, ATP and ADP-ribose induce different conformational changes in the neighbouring vacant subunit, as monitored by the spectroscopic properties of the bound alkyl group. These results in conjunction with other data support the view that the negative cooperativity in NAD+ binding to glyceraldehyde-3-phosphate dehydrogenase results from ligand-induced conformational changes. Furthermore, these results further support the view that subtle structural changes in the coenzyme molecule determine the nature of the conformational changes induced within the enzyme tetramer.

  13. Lysine post-translational modification of glyceraldehyde-3-phosphate dehydrogenase regulates hepatic and systemic metabolism.

    PubMed

    Bond, Simon T; Howlett, Kirsten F; Kowalski, Greg M; Mason, Shaun; Connor, Timothy; Cooper, Adrian; Streltsov, Victor; Bruce, Clinton R; Walder, Ken R; McGee, Sean L

    2017-03-03

    Reciprocal regulation of hepatic glycolysis and gluconeogenesis contributes to systemic metabolic homeostasis. Recent evidence from lower order organisms has found that reversible post-translational modification of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), particularly acetylation, contributes to the reciprocal regulation of glycolysis/gluconeogenesis. However, whether this occurs in mammalian hepatocytes in vitro or in vivo is unknown. Several proteomics studies have identified 4 lysine residues in critical regions of mammalian GAPDH that are altered by multiple post-translational modifications. In FAO hepatoma cells, mutation of all 4 lysine residues (4K-R GAPDH) to mimic their unmodified state reduced GAPDH glycolytic activity and glycolytic flux and increased gluconeogenic GAPDH activity and glucose production. Hepatic expression of 4K-R GAPDH in mice increased GAPDH gluconeogenic activity and the contribution of gluconeogenesis to endogenous glucose production in the unfed state. Consistent with the increased reliance on the energy-consuming gluconeogenic pathway, plasma free fatty acids and ketones were elevated in mice expressing 4K-R GAPDH, suggesting enhanced lipolysis and hepatic fatty acid oxidation. In normal mice, food withholding and refeeding, as well as hormonal regulators of reciprocal glycolysis/gluconeogenesis, such as insulin, glucagon, and norepinephrine, had no effect on global GAPDH acetylation. However, GAPDH acetylation was reduced in obese and type 2 diabetic db/db mice. These findings show that post-translational modification of GAPDH lysine residues regulates hepatic and systemic metabolism, revealing an unappreciated role for hepatic GAPDH in substrate selection and utilization.-Bond, S. T., Howlett, K. F., Kowalski, G. M., Mason, S., Connor, T., Cooper, A., Streltsov, V., Bruce, C. R., Walder, K. R., McGee, S. L. Lysine post-translational modification of glyceraldehyde-3-phosphate dehydrogenase regulates hepatic and systemic

  14. A Novel 5-Enolpyruvylshikimate-3-Phosphate Synthase from Rahnella aquatilis with Significantly Reduced Glyphosate Sensitivity

    PubMed Central

    Xiong, Ai-Sheng; Zhao, Wei; Fu, Xiao-Yan; Han, Hong-Juan; Chen, Chen; Jin, Xiao-Fen; Yao, Quan-Hong

    2012-01-01

    The 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS; EC 2.5.1.19) is a key enzyme in the shikimate pathway for the production of aromatic amino acids and chorismate-derived secondary metabolites in plants, fungi, and microorganisms. It is also the target of the broad-spectrum herbicide glyphosate. Natural glyphosate resistance is generally thought to occur within microorganisms in a strong selective pressure condition. Rahnella aquatilis strain GR20, an antagonist against pathogenic agrobacterial strains of grape crown gall, was isolated from the rhizosphere of grape in glyphosate-contaminated vineyards. A novel gene encoding EPSPS was identified from the isolated bacterium by complementation of an Escherichia coli auxotrophic aroA mutant. The EPSPS, named AroAR.aquatilis, was expressed and purified from E. coli, and key kinetic values were determined. The full-length enzyme exhibited higher tolerance to glyphosate than the E. coli EPSPS (AroAE.coli), while retaining high affinity for the substrate phosphoenolpyruvate. Transgenic plants of AroAR.aquatilis were also observed to be more resistant to glyphosate at a concentration of 5 mM than that of AroAE.coli. To probe the sites contributing to increased tolerance to glyphosate, mutant R.aquatilis EPSPS enzymes were produced with the c-strand of subdomain 3 and the f-strand of subdomain 5 (Thr38Lys, Arg40Val, Arg222Gln, Ser224Val, Ile225Val, and Gln226Lys) substituted by the corresponding region of the E. coli EPSPS. The mutant enzyme exhibited greater sensitivity to glyphosate than the wild type R.aquatilis EPSPS with little change of affinity for its first substrate, shikimate-3-phosphate (S3P) and phosphoenolpyruvate (PEP). The effect of the residues on subdomain 5 on glyphosate resistance was more obvious. PMID:22870190

  15. Glycerol-3-phosphate dehydrogenase 1 deficiency induces compensatory amino acid metabolism during fasting in mice.

    PubMed

    Sato, Tomoki; Yoshida, Yuma; Morita, Akihito; Mori, Nobuko; Miura, Shinji

    2016-11-01

    Glucose is used as an energy source in many organs and obtained from dietary carbohydrates. However, when the external energy supply is interrupted, e.g., during fasting, carbohydrates preserved in the liver and glycogenic precursors derived from other organs are used to maintain blood glucose levels. Glycerol and glycogenic amino acids derived from adipocytes and skeletal muscles are utilized as glycogenic precursors. Glycerol-3-phosphate dehydrogenase 1 (GPD1), an NAD(+)/NADH-dependent enzyme present in the cytosol, catalyzes the reversible conversion of glycerol-3-phosphate (G3P) to dihydroxyacetone phosphate (DHAP). Since G3P is one of the substrates utilized for gluconeogenesis in the liver, the conversion of G3P to DHAP by GPD1 is essential for maintaining blood glucose levels during fasting. We focused on GPD1 and examined its roles in gluconeogenesis during fasting. Using GPD1 null model BALB/cHeA mice (HeA mice), we measured gluconeogenesis from glycerol and the change of blood glucose levels under fasting conditions. We also measured gene expression related to gluconeogenesis in the liver and protein metabolism in skeletal muscle. BALB/cBy mice (By mice) were used as a control. The blood glucose levels in the HeA mice were lower than that in the By mice after glycerol administration. Although lack of GPD1 inhibited gluconeogenesis from glycerol, blood glucose levels in the HeA mice after 1-4h of fasting were significantly higher than that in the By mice. Muscle protein synthesis in HeA mice was significantly lower than that in the By mice. Moreover, blood alanine levels and usage of alanine for gluconeogenesis in the liver were significantly higher in the HeA mice than that in the By mice. Although these data indicate that a lack of GPD1 inhibits gluconeogenesis from glycerol, chronic GPD1 deficiency may induce an adaptation that enhances gluconeogenesis from glycogenic amino acids. Copyright © 2016 Elsevier Inc. All rights reserved.

  16. Inactivation of glyceraldehyde-3-phosphate dehydrogenase of human malignant cells by methylglyoxal.

    PubMed

    Ray, M; Basu, N; Ray, S

    1997-12-01

    The effect of methylglyoxal on the activity of glyceraldehyde-3-phosphate dehydrogenase (GA3PD) of several normal human tissues and benign and malignant tumors has been tested. Methylglyoxal inactivated GA3PD of all the malignant cells (47 samples) and the degree of inactivation was in the range of 25-90%, but it had no inhibitory effect on this enzyme from several normal cells (24 samples) and benign tumors (13 samples). When the effect of methylglyoxal on other two dehydrogenases namely glucose 6-phosphate dehydrogenase (G6PD) and L-lactic dehydrogenase (LDH) of similar cells was tested as controls it has been observed that methylglyoxal has some inactivating effect on G6PD of all the normal, benign and malignant samples tested, whereas, LDH remained completely unaffected. These studies indicate that the inactivating effect of methylglyoxal on GA3PD specifically of the malignant cells may be a common feature of all the malignant cells, and this phenomenon can be used as a simple and rapid device for the detection of malignancy.

  17. Structure and kinetic characterization of human sperm-specific glyceraldehyde-3-phosphate dehydrogenase, GAPDS.

    PubMed

    Chaikuad, Apirat; Shafqat, Naeem; Al-Mokhtar, Ruby; Cameron, Gus; Clarke, Anthony R; Brady, R Leo; Oppermann, Udo; Frayne, Jan; Yue, Wyatt W

    2011-04-15

    hGAPDS (human sperm-specific glyceraldehyde-3-phosphate dehydrogenase) is a glycolytic enzyme essential for the survival of spermatozoa, and constitutes a potential target for non-hormonal contraception. However, enzyme characterization of GAPDS has been hampered by the difficulty in producing soluble recombinant protein. In the present study, we have overexpressed in Escherichia coli a highly soluble form of hGAPDS truncated at the N-terminus (hGAPDSΔN), and crystallized the homotetrameric enzyme in two ligand complexes. The hGAPDSΔN-NAD+-phosphate structure maps the two anion-recognition sites within the catalytic pocket that correspond to the conserved Ps site and the newly recognized Pi site identified in other organisms. The hGAPDSΔN-NAD+-glycerol structure shows serendipitous binding of glycerol at the Ps and new Pi sites, demonstrating the propensity of these anion-recognition sites to bind non-physiologically relevant ligands. A comparison of kinetic profiles between hGAPDSΔN and its somatic equivalent reveals a 3-fold increase in catalytic efficiency for hGAPDSΔN. This may be attributable to subtle amino acid substitutions peripheral to the active centre that influence the charge properties and protonation states of catalytic residues. Our data therefore elucidate structural and kinetic features of hGAPDS that might provide insightful information towards inhibitor development.

  18. Effector-induced dissociation of glyceraldehyde-3-phosphate dehydrogenase discriminated by urea solvation.

    PubMed

    Ivanova, V; Krusteva, N; Atanasov, B

    1995-08-01

    The dissociation of glyceraldehyde-3-phosphate dehydrogenase (GAPD) from pig muscle in water solutions (0.1 M phosphate, pH 7) at increased urea concentrations was studied by means of frontal-gel chromatography, intrinsic (TRP) fluorescence, differential absorption spectroscopy and selective chemical modification at TRP0193. The results are in agreement with a consecutive two-step model of dissociation of the tetramer and the dimer (C*T = 0.42 M urea < C*D = 1.39 M urea). The binding effector(s) destabilizes the oligomeric structures (delta GT changes from -1.00 to -0.54 kcal/mol; delta GD from -2.30 to -1.22 kcal/mol). The introduction of the bulky Koshland-reagent group to TRP-193 at the subunit-subunit interface leads to a decrease of the stability with delta delta G approximate to 1 kcal/mol, owing to TRP-193...TYR-39...TYR-92 cluster destruction. By using lobster GAPD atomic coordinates (PDB file 1GPD) and pig muscle GAPD amino-acid sequence, a tentative molecular model was constructed and the subunit contacts in terms of the Lee-Richard static accessibilities were described. A detailed analysis of the dissociation as a transfer of the buried residues from the molecular interface to the urea solutions was performed.

  19. Transition state analysis of enolpyruvylshikimate 3-phosphate (EPSP) synthase (AroA)-catalyzed EPSP hydrolysis.

    PubMed

    Lou, Meiyan; Burger, Steven K; Gilpin, Meghann E; Gawuga, Vivian; Capretta, Alfredo; Berti, Paul J

    2012-08-08

    Proton transfer to carbon atoms is a significant catalytic challenge because of the large intrinsic energetic barrier and the frequently unfavorable thermodynamics. The main catalytic challenge for enolpyruvylshikimate 3-phosphate synthase (EPSP synthase, AroA) is protonating the methylene carbon atom of phosphoenolpyruvate, or EPSP, in the reverse reaction. We performed transition state analysis using kinetic isotope effects (KIEs) on AroA-catalyzed EPSP hydrolysis, which also begins with a methylene carbon (C3) protonation, as an analog of AroA's reverse reaction. As part of this analysis, an inorganic phosphate scavenging system was developed to remove phosphate which, though present in microscopic amounts in solution, is ubiquitous. The reaction was stepwise, with irreversible C3 protonation to form an EPSP cation intermediate; that is, an AH(‡)*AN mechanism. The large experimental 3-(14)C KIE, 1.032 ± 0.005, indicated strong coupling of C3 with the motion of the transferring proton. Calculated 3-(14)C KIEs for computational transition state models revealed that the transition state occurs early during C3-H(+) bond formation, with a C3-H(+) bond order of ≈0.24. The observed solvent deuterium KIE, 0.97 ± 0.04, was the lowest observed to date for this type of reaction, but consistent with a very early transition state. The large 2-(14)C KIE reflected an "electrostatic sandwich" formed by Asp313 and Glu341 to stabilize the positive charge at C2. In shifting the transition state earlier than the acid-catalyzed reaction, AroA effected a large Hammond shift, indicating that a significant part of AroA's catalytic strategy is to stabilize the positive charge in the EPSP cation. A computational model containing all the charged amino acid residues in the AroA active site close to the reactive center showed a similar Hammond shift relative to the small transition state models.

  20. Enhanced resistance in Theobroma cacao against oomycete and fungal pathogens by secretion of phosphatidylinositol-3-phosphate-binding proteins

    USDA-ARS?s Scientific Manuscript database

    The internalization of oomycete and fungal pathogen effectors into host plant cells has been reported to be blocked by proteins that bind to the effectors’ cell entry receptor, phosphatidylinositol-3-phosphate (PI3P). This finding suggested a novel strategy for disease control by engineering plants ...

  1. GLYCERALDEHYDE 3-PHOSPHATE DEHYDROGENASE-S, A SPERM-SPECIFIC GLYCOLYTIC ENZYME, IS REQUIRED FOR SPERM MOTILITY AND MALE FERTILITY

    EPA Science Inventory

    While glycolysis is highly conserved, it is remarkable that several novel isozymes in this central metabolic pathway are found in mammalian sperm. Glyceraldehyde 3-phosphate dehydrogenase-S (GAPDS) is the product of a mouse gene expressed only during spermatogenesis and, like it...

  2. GLYCERALDEHYDE 3-PHOSPHATE DEHYDROGENASE-S, A SPERM-SPECIFIC GLYCOLYTIC ENZYME, IS REQUIRED FOR SPERM MOTILITY AND MALE FERTILITY

    EPA Science Inventory

    While glycolysis is highly conserved, it is remarkable that several novel isozymes in this central metabolic pathway are found in mammalian sperm. Glyceraldehyde 3-phosphate dehydrogenase-S (GAPDS) is the product of a mouse gene expressed only during spermatogenesis and, like it...

  3. Isolation from Ochrobactrum anthropi of a Novel Class II 5-Enopyruvylshikimate-3-Phosphate Synthase with High Tolerance to Glyphosate▿ †

    PubMed Central

    Tian, Yong-Sheng; Xiong, Ai-Sheng; Xu, Jing; Zhao, Wei; Gao, Feng; Fu, Xiao-Yan; Xu, Hu; Zheng, Jian-Li; Peng, Ri-He; Yao, Quan-Hong

    2010-01-01

    Applying the genomic library construction process and colony screening, a novel aroA gene encoding 5-enopyruvylshikimate-3-phosphate synthase from Ochrobactrum anthropi was identified, cloned, and overexpressed, and the enzyme was purified to homogeneity. Furthermore, site-directed mutagenesis was employed to assess the role of single amino acid residues in glyphosate resistance. PMID:20601515

  4. Isolation from Ochrobactrum anthropi of a novel class II 5-enopyruvylshikimate-3-phosphate synthase with high tolerance to glyphosate.

    PubMed

    Tian, Yong-Sheng; Xiong, Ai-Sheng; Xu, Jing; Zhao, Wei; Gao, Feng; Fu, Xiao-Yan; Xu, Hu; Zheng, Jian-Li; Peng, Ri-He; Yao, Quan-Hong

    2010-09-01

    Applying the genomic library construction process and colony screening, a novel aroA gene encoding 5-enopyruvylshikimate-3-phosphate synthase from Ochrobactrum anthropi was identified, cloned, and overexpressed, and the enzyme was purified to homogeneity. Furthermore, site-directed mutagenesis was employed to assess the role of single amino acid residues in glyphosate resistance.

  5. Modeling of glycerol-3-phosphate transporter suggests a potential 'tilt' mechanism involved in its function.

    PubMed

    Tsigelny, Igor F; Greenberg, Jerry; Kouznetsova, Valentina; Nigam, Sanjay K

    2008-10-01

    Many major facilitator superfamily (MFS) transporters have similar 12-transmembrane alpha-helical topologies with two six-helix halves connected by a long loop. In humans, these transporters participate in key physiological processes and are also, as in the case of members of the organic anion transporter (OAT) family, of pharmaceutical interest. Recently, crystal structures of two bacterial representatives of the MFS family--the glycerol-3-phosphate transporter (GlpT) and lac-permease (LacY)--have been solved and, because of assumptions regarding the high structural conservation of this family, there is hope that the results can be applied to mammalian transporters as well. Based on crystallography, it has been suggested that a major conformational "switching" mechanism accounts for ligand transport by MFS proteins. This conformational switch would then allow periodic changes in the overall transporter configuration, resulting in its cyclic opening to the periplasm or cytoplasm. Following this lead, we have modeled a possible "switch" mechanism in GlpT, using the concept of rotation of protein domains as in the DynDom program17 and membranephilic constraints predicted by the MAPAS program.(23) We found that the minima of energies of intersubunit interactions support two alternate positions consistent with their transport properties. Thus, for GlpT, a "tilt" of 9 degrees -10 degrees rotation had the most favorable energetics of electrostatic interaction between the two halves of the transporter; moreover, this confirmation was sufficient to suggest transport of the ligand across the membrane. We conducted steered molecular dynamics simulations of the GlpT-ligand system to explore how glycerol-3-phosphate would be handled by the "tilted" structure, and obtained results generally consistent with experimental mutagenesis data. While biochemical data remain most consistent with a single-site alternating access model, our results raise the possibility that, while the

  6. Dengue Virus NS1 Protein Modulates Cellular Energy Metabolism by Increasing Glyceraldehyde-3-Phosphate Dehydrogenase Activity

    PubMed Central

    Allonso, Diego; Andrade, Iamara S.; Conde, Jonas N.; Coelho, Diego R.; Rocha, Daniele C. P.; da Silva, Manuela L.; Ventura, Gustavo T.

    2015-01-01

    ABSTRACT Dengue is one of the main public health concerns worldwide. Recent estimates indicate that over 390 million people are infected annually with the dengue virus (DENV), resulting in thousands of deaths. Among the DENV nonstructural proteins, the NS1 protein is the only one whose function during replication is still unknown. NS1 is a 46- to 55-kDa glycoprotein commonly found as both a membrane-associated homodimer and a soluble hexameric barrel-shaped lipoprotein. Despite its role in the pathogenic process, NS1 is essential for proper RNA accumulation and virus production. In the present study, we identified that glyceraldehyde-3-phosphate dehydrogenase (GAPDH) interacts with intracellular NS1. Molecular docking revealed that this interaction occurs through the hydrophobic protrusion of NS1 and the hydrophobic residues located at the opposite side of the catalytic site. Moreover, addition of purified recombinant NS1 enhanced the glycolytic activity of GAPDH in vitro. Interestingly, we observed that DENV infection promoted the relocalization of GAPDH to the perinuclear region, where NS1 is commonly found. Both DENV infection and expression of NS1 itself resulted in increased GAPDH activity. Our findings indicate that the NS1 protein acts to increase glycolytic flux and, consequently, energy production, which is consistent with the recent finding that DENV induces and requires glycolysis for proper replication. This is the first report to propose that NS1 is an important modulator of cellular energy metabolism. The data presented here provide new insights that may be useful for further drug design and the development of alternative antiviral therapies against DENV. IMPORTANCE Dengue represents a serious public health problem worldwide and is caused by infection with dengue virus (DENV). Estimates indicate that half of the global population is at risk of infection, with almost 400 million cases occurring per year. The NS1 glycoprotein is found in both the

  7. Dengue Virus NS1 Protein Modulates Cellular Energy Metabolism by Increasing Glyceraldehyde-3-Phosphate Dehydrogenase Activity.

    PubMed

    Allonso, Diego; Andrade, Iamara S; Conde, Jonas N; Coelho, Diego R; Rocha, Daniele C P; da Silva, Manuela L; Ventura, Gustavo T; Silva, Emiliana M; Mohana-Borges, Ronaldo

    2015-12-01

    Dengue is one of the main public health concerns worldwide. Recent estimates indicate that over 390 million people are infected annually with the dengue virus (DENV), resulting in thousands of deaths. Among the DENV nonstructural proteins, the NS1 protein is the only one whose function during replication is still unknown. NS1 is a 46- to 55-kDa glycoprotein commonly found as both a membrane-associated homodimer and a soluble hexameric barrel-shaped lipoprotein. Despite its role in the pathogenic process, NS1 is essential for proper RNA accumulation and virus production. In the present study, we identified that glyceraldehyde-3-phosphate dehydrogenase (GAPDH) interacts with intracellular NS1. Molecular docking revealed that this interaction occurs through the hydrophobic protrusion of NS1 and the hydrophobic residues located at the opposite side of the catalytic site. Moreover, addition of purified recombinant NS1 enhanced the glycolytic activity of GAPDH in vitro. Interestingly, we observed that DENV infection promoted the relocalization of GAPDH to the perinuclear region, where NS1 is commonly found. Both DENV infection and expression of NS1 itself resulted in increased GAPDH activity. Our findings indicate that the NS1 protein acts to increase glycolytic flux and, consequently, energy production, which is consistent with the recent finding that DENV induces and requires glycolysis for proper replication. This is the first report to propose that NS1 is an important modulator of cellular energy metabolism. The data presented here provide new insights that may be useful for further drug design and the development of alternative antiviral therapies against DENV. Dengue represents a serious public health problem worldwide and is caused by infection with dengue virus (DENV). Estimates indicate that half of the global population is at risk of infection, with almost 400 million cases occurring per year. The NS1 glycoprotein is found in both the intracellular and the

  8. N-(phosphonomethyl)glycine (glyphosate) tolerance in Euglena gracilis acquired by either overproduced or resistant 5-enolpyruvylshikimate-3-phosphate synthase.

    PubMed

    Reinbothe, S; Nelles, A; Parthier, B

    1991-06-01

    Photoautotrophic cells of Euglena gracilis can be adapted to N-(phosphonomethyl)glycine (glyphosate) by cultivation in media with progressively higher concentrations of the herbicide. Two different mechanisms of tolerance to the herbicide were observed. One is characterized by the overproduction and 40-fold accumulation of the target enzyme. 5-enolpyruvylshikimate-3-phosphate synthase, in cells adapted to 6 mM N-(phosphonomethyl)glycine. The other is connected with a herbicide-insensitive enzyme. No evidence was obtained for the involvement of the putative multifunctional arom protein previously reported to be involved in the biosynthesis of aromatic amino acids in Euglena. Cells adapted to N-(phosphonomethyl)glycine excreted shikimate and shikimate 3-phosphate into the medium: the amounts depended on the actual concentration of the herbicide. Two-dimensional gel electrophoresis and determination of 5-enolpyruvylshikimate-3-phosphate synthase activity in crude extracts, as well as after separation by non-denaturing gel electrophoresis, revealed that the overproduction of the enzyme in adapted cells correlates with the accumulation of a 59-kDa protein. Overproduction of this 59-kDa protein resulted from a selectively increased level of a mRNA coding for a 64.5-kDa polypeptide which appeared in adapted cells, as shown by cell-free translation in the wheat germ system. In contrast to this quantitative, adaptive type of tolerance, the second mechanism causing tolerance to N-(phosphonomethyl)glycine in the Euglena cell line NR 6/50 was probably related to a qualitatively altered 5-enolpyruvylshikimate-3-phosphate synthase, which could not be inhibited by even 2 mM N-(phosphonomethyl)glycine in vitro. In agreement with this observation, the putatively mutated cell line excreted neither shikimate nor shikimate 3-phosphate into the growth medium containing N-(phosphonomethyl)glycine, even if cultivated in the presence of 20 mM or 50 mM N-(phosphonomethyl)glycine.

  9. RNAi mediated down regulation of myo-inositol-3-phosphate synthase to generate low phytate rice.

    PubMed

    Ali, Nusrat; Paul, Soumitra; Gayen, Dipak; Sarkar, Sailendra Nath; Datta, Swapan K; Datta, Karabi

    2013-05-15

    Phytic acid (InsP6) is considered as the major source of phosphorus and inositol phosphates in cereal grains. Reduction of phytic acid level in cereal grains is desirable in view of its antinutrient properties to maximize mineral bioavailability and minimize the load of phosphorus waste management. We report here RNAi mediated seed-specific silencing of myo-inositol-3-phosphate synthase (MIPS) gene catalyzing the first step of phytic acid biosynthesis in rice. Moreover, we also studied the possible implications of MIPS silencing on myo-inositol and related metabolism, since, first step of phytic acid biosynthesis is also the rate limiting step of myo-inositol synthesis, catalyzed by MIPS. The resulting transgenic rice plants (T3) showed a 4.59 fold down regulation in MIPS gene expression, which corresponds to a significant decrease in phytate levels and a simultaneous increment in the amount of inorganic phosphate in the seeds. A diminution in the myo-inositol content of transgenic plants was also observed due to disruption of the first step of phytic acid biosynthetic pathway, which further reduced the level of ascorbate and altered abscisic acid (ABA) sensitivity of the transgenic plants. In addition, our results shows that in the transgenic plants, the lower phytate levels has led to an increment of divalent cations, of which a 1.6 fold increase in the iron concentration in milled rice seeds was noteworthy. This increase could be attributed to reduced chelation of divalent metal (iron) cations, which may correlate to higher iron bioavailability in the endosperm of rice grains. The present study evidently suggests that seed-specific silencing of MIPS in transgenic rice plants can yield substantial reduction in levels of phytic acid along with an increase in inorganic phosphate content. However, it was also demonstrated that the low phytate seeds had an undesirable diminution in levels of myo-inositol and ascorbate, which probably led to sensitiveness of seeds to

  10. Glyceraldehyde-3-phosphate dehydrogenase: a universal internal control for Western blots in prokaryotic and eukaryotic cells.

    PubMed

    Wu, Yonghong; Wu, Min; He, Guowei; Zhang, Xiao; Li, Weiguang; Gao, Yan; Li, Zhihui; Wang, Zhaoyan; Zhang, Chenggang

    2012-04-01

    In the current study, we examined the expression level of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) protein in a number of organisms and the stability of GAPDH under various conditions. Our results revealed that GAPDH is present in multiple Escherichia coli strains, the yeast strain GS115, Caenorhabditis elegans, rat PC12 cells, and both mouse and rat brain. Furthermore, GAPDH was stably expressed under different concentrations of inducer and at different times of induction in E. coli (BL21) cells and yeast GS115 cells. Stable expression of GAPDH protein was also observed in C.elegans and PC12 cells that were treated with different concentrations of paraquat or sodium sulfite, respectively. In addition, we were able to detect and identify the endogenous gapA protein in E.coli via immunoprecipitation and MALDI-TOF-MS analysis. Endogenous gapA protein and exogenously expressed (subcloned) GAPDH proteins were detected in E. coli BL21 but not for gapC. With the exception of gapC in E. coli, the various isoforms of GAPDH possessed enzymatic activity. Finally, sequence analysis revealed that the GAPDH proteins were 76% identical, with the exception of E. coli gapC. Taken together, our results indicate that GAPDH could be universally used as an internal control for the Western blot analysis of prokaryotic and eukaryotic samples. Crown Copyright © 2012. Published by Elsevier Inc. All rights reserved.

  11. Glyceraldehyde 3-phosphate dehydrogenase-telomere association correlates with redox status in Trypanosoma cruzi.

    PubMed

    Pariona-Llanos, Ricardo; Pavani, Raphael Souza; Reis, Marcelo; Noël, Vincent; Silber, Ariel Mariano; Armelin, Hugo Aguirre; Cano, Maria Isabel Nogueira; Elias, Maria Carolina

    2015-01-01

    Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is a classical metabolic enzyme involved in energy production and plays a role in additional nuclear functions, including transcriptional control, recognition of misincorporated nucleotides in DNA and maintenance of telomere structure. Here, we show that the recombinant protein T. cruzi GAPDH (rTcGAPDH) binds single-stranded telomeric DNA. We demonstrate that the binding of GAPDH to telomeric DNA correlates with the balance between oxidized and reduced forms of nicotinamide adenine dinucleotides (NAD+/NADH). We observed that GAPDH-telomere association and NAD+/NADH balance changed throughout the T. cruzi life cycle. For example, in replicative epimastigote forms of T. cruzi, which show similar intracellular concentrations of NAD+ and NADH, GAPDH binds to telomeric DNA in vivo and this binding activity is inhibited by exogenous NAD+. In contrast, in the T. cruzi non-proliferative trypomastigote forms, which show higher NAD+ concentration, GAPDH was absent from telomeres. In addition, NAD+ abolishes physical interaction between recombinant GAPDH and synthetic telomere oligonucleotide in a cell free system, mimicking exogenous NAD+ that reduces GAPDH-telomere interaction in vivo. We propose that the balance in the NAD+/NADH ratio during T. cruzi life cycle homeostatically regulates GAPDH telomere association, suggesting that in trypanosomes redox status locally modulates GAPDH association with telomeric DNA.

  12. Glyceraldehyde 3-Phosphate Dehydrogenase-Telomere Association Correlates with Redox Status in Trypanosoma cruzi

    PubMed Central

    Pariona-Llanos, Ricardo; Pavani, Raphael Souza; Reis, Marcelo; Noël, Vincent; Silber, Ariel Mariano; Armelin, Hugo Aguirre; Cano, Maria Isabel Nogueira; Elias, Maria Carolina

    2015-01-01

    Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is a classical metabolic enzyme involved in energy production and plays a role in additional nuclear functions, including transcriptional control, recognition of misincorporated nucleotides in DNA and maintenance of telomere structure. Here, we show that the recombinant protein T. cruzi GAPDH (rTcGAPDH) binds single-stranded telomeric DNA. We demonstrate that the binding of GAPDH to telomeric DNA correlates with the balance between oxidized and reduced forms of nicotinamide adenine dinucleotides (NAD+/NADH). We observed that GAPDH-telomere association and NAD+/NADH balance changed throughout the T. cruzi life cycle. For example, in replicative epimastigote forms of T. cruzi, which show similar intracellular concentrations of NAD+ and NADH, GAPDH binds to telomeric DNA in vivo and this binding activity is inhibited by exogenous NAD+. In contrast, in the T. cruzi non-proliferative trypomastigote forms, which show higher NAD+ concentration, GAPDH was absent from telomeres. In addition, NAD+ abolishes physical interaction between recombinant GAPDH and synthetic telomere oligonucleotide in a cell free system, mimicking exogenous NAD+ that reduces GAPDH-telomere interaction in vivo. We propose that the balance in the NAD+/NADH ratio during T. cruzi life cycle homeostatically regulates GAPDH telomere association, suggesting that in trypanosomes redox status locally modulates GAPDH association with telomeric DNA. PMID:25775131

  13. Plastid-expressed 5-enolpyruvylshikimate-3-phosphate synthase genes provide high level glyphosate tolerance in tobacco.

    PubMed

    Ye, G N; Hajdukiewicz, P T; Broyles, D; Rodriguez, D; Xu, C W; Nehra, N; Staub, J M

    2001-02-01

    Plastid transformation (transplastomic) technology has several potential advantages for biotechnological applications including the use of unmodified prokaryotic genes for engineering, potential high-level gene expression and gene containment due to maternal inheritance in most crop plants. However, the efficacy of a plastid-encoded trait may change depending on plastid number and tissue type. We report a feasibility study in tobacco plastids to achieve high-level herbicide resistance in both vegetative tissues and reproductive organs. We chose to test glyphosate resistance via over-expression in plastids of tolerant forms of 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS). Immunological, enzymatic and whole-plant assays were used to prove the efficacy of three different prokaryotic (Achromobacter, Agrobacterium and Bacillus) EPSPS genes. Using the Agrobacterium strain CP4 EPSPS as a model we identified translational control sequences that direct a 10,000-fold range of protein accumulation (to >10% total soluble protein in leaves). Plastid-expressed EPSPS could provide very high levels of glyphosate resistance, although levels of resistance in vegetative and reproductive tissues differed depending on EPSPS accumulation levels, and correlated to the plastid abundance in these tissues. Paradoxically, higher levels of plastid-expressed EPSPS protein accumulation were apparently required for efficacy than from a similar nuclear-encoded gene. Nevertheless, the demonstration of high-level glyphosate tolerance in vegetative and reproductive organs using transplastomic technology provides a necessary step for transfer of this technology to other crop species.

  14. Identification of Glyceraldehyde 3-Phosphate Dehydrogenase Sequence and Expression Profiles in Tree Shrew (Tupaia belangeri)

    PubMed Central

    Zheng, Yu; Wang, Yingjun; Smith, Wanli W.; Leng, Jing

    2014-01-01

    The tree shrews (Tupaia belangeri) diverged from the primate order (Primates) and are classified as Scandentia, a separate taxonomic group of mammals. The tree shrew has been suggested to use an animal model to study human disease but the genomic sequences of tree shrew is largely unidentified. Here we identified the full-length cDNA sequence of a housekeeping gene, Glyceraldehyde 3-phosphate Dehydrogenase (GAPDH), in tree shrew. We further constructed a phylogenetic family tree base on GAPDH molecules of various organisms and compared GAPDH sequences with human and other small experimental animals. These study revealed that tree shrew was closer to human than mouse, rat, rabbit and guinea pig. The Quantitative Reverse Transcription PCR and western blot analysis further demonstrated that GAPDH expressed in various tissues in tree shrew as a general conservative housekeeping proteins as in human. Our findings provide the novel genetic knowledge of the tree shrew and strong evidences that tree shrew can be an experimental model system to study human disorders. PMID:24887411

  15. Occurrence of a multimeric high-molecular-weight glyceraldehyde-3-phosphate dehydrogenase in human serum.

    PubMed

    Kunjithapatham, Rani; Geschwind, Jean-Francois; Devine, Lauren; Boronina, Tatiana N; O'Meally, Robert N; Cole, Robert N; Torbenson, Michael S; Ganapathy-Kanniappan, Shanmugasundaram

    2015-04-03

    Cellular glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a phylogenetically conserved, ubiquitous enzyme that plays an indispensable role in energy metabolism. Although a wealth of information is available on cellular GAPDH, there is a clear paucity of data on its extracellular counterpart (i.e., the secreted or extracellular GAPDH). Here, we show that the extracellular GAPDH in human serum is a multimeric, high-molecular-weight, yet glycolytically active enzyme. The high-molecular-weight multimers of serum GAPDH were identified by immunodetection on one- and two-dimensional gel electrophoresis using multiple antibodies specific for various epitopes of GAPDH. Partial purification of serum GAPDH by DEAE Affigel affinity/ion exchange chromatography further established the multimeric composition of serum GAPDH. In vitro data demonstrated that human cell lines secrete a multimeric, high-molecular-weight enzyme similar to that of serum GAPDH. Furthermore, LC-MS/MS analysis of extracellular GAPDH from human cell lines confirmed the presence of unique peptides of GAPDH in the high-molecular-weight subunits. Furthermore, data from pulse-chase experiments established the presence of high-molecular-weight subunits in the secreted, extracellular GAPDH. Taken together, our findings demonstrate the presence of a high-molecular-weight, enzymatically active secretory GAPDH in human serum that may have a hitherto unknown function in humans.

  16. Glyceraldehyde-3-Phosphate Dehydrogenase-Encoding Gene as a Useful Taxonomic Tool for Staphylococcus spp.

    PubMed Central

    Yugueros, Javier; Temprano, Alejandro; Berzal, Beatriz; Sánchez, María; Hernanz, Carmen; Luengo, José María; Naharro, Germán

    2000-01-01

    The gap gene of Staphylococcus aureus, encoding glyceraldehyde-3-phosphate dehydrogenase, was used as a target to amplify a 933-bp DNA fragment by PCR with a pair of primers 26 and 25 nucleotides in length. PCR products, detected by agarose gel electrophoresis, were also amplified from 12 Staphylococcus spp. analyzed previously. Hybridization with an internal 279-bp DNA fragment probe was positive in all PCR-positive samples. No PCR products were amplified when other gram-positive and gram-negative bacterial genera were analyzed using the same pair of primers. AluI digestion of PCR-generated products gave 12 different restriction fragment length polymorphism (RFLP) patterns, one for each species analyzed. However, we could detect two intraspecies RFLP patterns in Staphylococcus epidermidis, Staphylococcus hominis, and Staphylococcus simulans which were different from the other species. An identical RFLP pattern was observed for 112 S. aureus isolates from humans, cows, and sheep. The sensitivity of the PCR assays was very high, with a detection limit for S. aureus cells of 20 CFU when cells were suspended in saline. PCR amplification of the gap gene has the potential for rapid identification of at least 12 species belonging to the genus Staphylococcus, as it is highly specific. PMID:11101563

  17. Comparative molecular analysis of evolutionarily distant glyceraldehyde-3-phosphate dehydrogenase from Sardina pilchardus and Octopus vulgaris.

    PubMed

    Baibai, Tarik; Oukhattar, Laila; Mountassif, Driss; Assobhei, Omar; Serrano, Aurelio; Soukri, Abdelaziz

    2010-12-01

    The NAD(+)-dependent cytosolic glyceraldehyde-3-phosphate dehydrogenase (GAPDH, EC 1.2.1.12), which is recognized as a key to central carbon metabolism in glycolysis and gluconeogenesis and as an important allozymic polymorphic biomarker, was purified from muscles of two marine species: the skeletal muscle of Sardina pilchardus Walbaum (Teleost, Clupeida) and the incompressible arm muscle of Octopus vulgaris (Mollusca, Cephalopoda). Comparative biochemical studies have revealed that they differ in their subunit molecular masses and in pI values. Partial cDNA sequences corresponding to an internal region of the GapC genes from Sardina and Octopus were obtained by polymerase chain reaction using degenerate primers designed from highly conserved protein motifs. Alignments of the deduced amino acid sequences were used to establish the 3D structures of the active site of two enzymes as well as the phylogenetic relationships of the sardine and octopus enzymes. These two enzymes are the first two GAPDHs characterized so far from teleost fish and cephalopod, respectively. Interestingly, phylogenetic analyses indicated that the sardina GAPDH is in a cluster with the archetypical enzymes from other vertebrates, while the octopus GAPDH comes together with other molluscan sequences in a distant basal assembly closer to bacterial and fungal orthologs, thus suggesting their different evolutionary scenarios.

  18. Phosphatidic Acid Binds to Cytosolic Glyceraldehyde-3-phosphate Dehydrogenase and Promotes Its Cleavage in Arabidopsis *

    PubMed Central

    Kim, Sang-Chul; Guo, Liang; Wang, Xuemin

    2013-01-01

    Phosphatidic acid (PA) is a class of lipid messengers involved in a variety of physiological processes. To understand how PA mediates cell functions in plants, we used a PA affinity membrane assay to isolate PA-binding proteins from Camelina sativa followed by mass spectrometric sequencing. A cytosolic glyceraldehyde-3-phosphate dehydrogenase (GAPC) was identified to bind to PA, and detailed analysis was carried out subsequently using GAPC1 and GAPC1 from Arabidopsis. The PA and GAPC binding was abolished by the cation zinc whereas oxidation of GAPCs promoted the PA binding. PA had little impact on the GAPC catalytic activity in vitro, but the PA treatment of Arabidopsis seedlings induced proteolytic cleavage of GAPC2 and inhibited Arabidopsis seedling growth. The extent of PA inhibition was greater in GAPC-overexpressing than wild-type seedlings, but the greater PA inhibition was abolished by application of zinc to the seedling. The PA treatment also reduced the expression of genes involved in PA synthesis and utilization, and the PA-reduced gene expression was partially recovered by zinc treatment. These data suggest that PA binds to oxidized GAPDH and promotes its cleavage and that the PA and GAPC interaction may provide a signaling link coordinating carbohydrate and lipid metabolism. PMID:23504314

  19. Phosphoinositol 3-phosphate acts as a timer for reactive oxygen species production in the phagosome.

    PubMed

    Song, Zhi Min; Bouchab, Leïla; Hudik, Elodie; Le Bars, Romain; Nüsse, Oliver; Dupré-Crochet, Sophie

    2017-01-17

    Production of reactive oxygen species (ROS) in the phagosome by the NADPH oxidase is critical for mammalian immune defense against microbial infections and phosphoinositides are important regulators in this process. Phosphoinositol 3-phosphate (PI(3)P) regulates ROS production at the phagosome via p40(phox) by an unknown mechanism. This study tested the hypothesis that PI(3)P controls ROS production by regulating the presence of p40(phox) and p67(phox) at the phagosomal membrane. Pharmacologic inhibition of PI(3)P synthesis at the phagosome decreased the ROS production both in differentiated PLB-985 cells and human neutrophils. It also releases p67(phox), the key cytosolic subunit of the oxidase, and p40(phox) from the phagosome. The knockdown of the PI(3)P phosphatase MTM1 or Rubicon or both increases the level of PI(3)P at the phagosome. That increase enhances ROS production inside the phagosome and triggers an extended accumulation of p67(phox) at the phagosome. Furthermore, the overexpression of MTM1 at the phagosomal membrane induces the disappearance of PI(3)P from the phagosome and prevents sustained ROS production. In conclusion, PI(3)P, indeed, regulates ROS production by maintaining p40(phox) and p67(phox) at the phagosomal membrane.

  20. Expression of glyceraldehyde 3-phosphate dehydrogenase is enhanced in Leishmania spp naturally resistant to nitric oxide.

    PubMed

    Rios, M C; Silva, W R T; Azevedo, A F; Santos, P L; Teixeira, S A; Muscará, M N; Thomazzi, S M; Almeida, R P; Fernandes, R P M; Scher, R

    2015-06-29

    Leishmania spp are the causative agents of a spectrum of diseases termed leishmaniasis that affect mammals, including humans and dogs. Although reactive nitrogen species are employed in the control of parasitism by the immune system, it is known that Leishmania can withstand this oxidative stress. As the mechanism by which these species are resistant to nitric oxide (NO) is poorly understood, the main objective of this study was to evaluate the expression of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) in Leishmania amazonensis and Leishmania chagasi promastigotes showing natural resistance to NO. GAPDH transcript levels were quantified by real-time polymerase chain reaction amplification, and GAPDH activity (assessed by levels of NADH oxidation) was measured by spectrophotometry. The level of nitration in total protein was assessed by immunoblotting. The results demonstrated an increase in GAPDH expression in resistant isolates of both species compared to susceptible isolates. The increase in GAPDH expression led to an increase in the activity of GAPDH in L. amazonensis human isolates resistant to NO. The pattern of protein nitration did not differ between sensitive and resistant isolates. Our results suggest that changes in expression of GAPDH may be responsible, at least in part, to natural resistance to NO found in human and canine Leishmania spp.

  1. Glyceraldehyde-3-phosphate dehydrogenase-encoding gene as a useful taxonomic tool for Staphylococcus spp.

    PubMed

    Yugueros, J; Temprano, A; Berzal, B; Sánchez, M; Hernanz, C; Luengo, J M; Naharro, G

    2000-12-01

    The gap gene of Staphylococcus aureus, encoding glyceraldehyde-3-phosphate dehydrogenase, was used as a target to amplify a 933-bp DNA fragment by PCR with a pair of primers 26 and 25 nucleotides in length. PCR products, detected by agarose gel electrophoresis, were also amplified from 12 Staphylococcus spp. analyzed previously. Hybridization with an internal 279-bp DNA fragment probe was positive in all PCR-positive samples. No PCR products were amplified when other gram-positive and gram-negative bacterial genera were analyzed using the same pair of primers. AluI digestion of PCR-generated products gave 12 different restriction fragment length polymorphism (RFLP) patterns, one for each species analyzed. However, we could detect two intraspecies RFLP patterns in Staphylococcus epidermidis, Staphylococcus hominis, and Staphylococcus simulans which were different from the other species. An identical RFLP pattern was observed for 112 S. aureus isolates from humans, cows, and sheep. The sensitivity of the PCR assays was very high, with a detection limit for S. aureus cells of 20 CFU when cells were suspended in saline. PCR amplification of the gap gene has the potential for rapid identification of at least 12 species belonging to the genus Staphylococcus, as it is highly specific.

  2. Bioreaction Engineering Leading to Efficient Synthesis of L-Glyceraldehyd-3-Phosphate.

    PubMed

    Molla, Getachew S; Kinfu, Birhanu M; Chow, Jennifer; Streit, Wolfgang; Wohlgemuth, Roland; Liese, Andreas

    2017-03-01

    Enantiopure L-glyceraldehyde-3-phosphate (L-GAP) is a useful building block in natural biological and synthetic processes. A biocatalytic process using glycerol kinase from Cellulomonas sp. (EC 2.7.1.30) catalyzed phosphorylation of L-glyceraldehyde (L-GA) by ATP is used for the synthesis of L-GAP. L-GAP has a half-life of 6.86 h under reaction conditions. The activity of this enzyme depends on the Mg(2+) to ATP molar ratio showing maximum activity at the optimum molar ratio of 0.7. A kinetic model is developed and validated showing a 2D correlation of 99.9% between experimental and numerical data matrices. The enzyme exhibits inhibition by ADP, AMP, methylglyoxal and Ca(2+) , but not by L-GAP and inorganic orthophosphate. Moreover, equal amount of Ca(2+) exerts a different degree of inhibition relative to the activity without the addition of Ca(2+) depending on the Mg(2+) to ATP molar ratio. If the Mg(2+) to ATP molar ratio is set to be at the optimum value or less, inorganic hexametaphosphate (PPi6) suppresses the enzyme activity; otherwise PPi6 enhances the enzyme activity. Based on reaction engineering parameters such as conversion, selectivity and specific productivity, evaluation of different reactor types reveals that batchwise operation via stirred-tank reactor is the most efficient process for the synthesis of L-GAP.

  3. Lactobacillus reuteri glyceraldehyde-3-phosphate dehydrogenase functions in adhesion to intestinal epithelial cells.

    PubMed

    Zhang, Wen-Ming; Wang, Hai-Feng; Gao, Kan; Wang, Cong; Liu, Li; Liu, Jian-Xin

    2015-05-01

    This study was aimed to identify key surface proteins mediating the adhesion of lactobacilli to intestinal epithelial cells. By using Caco-2 and IPEC-J2 cells labeled with sulfo-NHS-biotin in the western blotting, a protein band of an approximately 37 kDa was detected on the surface layer of Lactobacillus reuteri strains ZJ616, ZJ617, ZJ621, and ZJ623 and Lactobacillus rhamnosus GG. Mass spectrometry analysis using the adhesion-related protein from L. reuteri ZJ617 showed that it was 100% homologous to the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) of L. reuteri JCM 1112 (GenBank: YP_001841377). The ability of L. reuteri ZJ617 to adhere to epithelial cells decreased significantly by treatment with LiCl or by blocking with an anti-GAPDH antibody, in comparison with the untreated strain (p < 0.05). Immunoelectron microscopic and immunofluorescence analyses confirmed that GAPDH is located on the surface layer of L. reuteri ZJ617. The results indicated that the GAPDH protein of L. reuteri ZJ617 acts as an adhesion component that plays an important role in binding to the intestinal epithelial cells.

  4. The influence of cytosolic phosphorylating glyceraldehyde 3-phosphate dehydrogenase (GAPC) on potato tuber metabolism.

    PubMed

    Hajirezaei, Mohammad-Reza; Biemelt, Sophia; Peisker, Martin; Lytovchenko, Anna; Fernie, Alisdair R; Sonnewald, Uwe

    2006-01-01

    The aim of this work was to investigate the importance of cytosolic phosphorylating glyceraldehyde 3-phosphate dehydrogenase (GAPC) in potato carbohydrate metabolism. For this purpose, the cytosolic isoform of phosphorylating GAPC was cloned and used for an antisense approach to generate transgenic potato plants that exhibited constitutively decreased GAPDH activity. Potato lines with decreased activities of phosphorylating GAPC exhibited no major changes in either whole-plant or tuber morphology. However, the levels of 3-phosphoglycerate were decreased in leaves of the transformants. A broad metabolic phenotyping of tubers from the transformants revealed an increase in sucrose and UDPglucose content, a decrease in the glycolytic intermediates 3-phosphoglycerate and phosphoenolpyruvate but little change in the levels of other metabolites. Moreover, the transformants displayed no differences in cold sweetening with respect to the wild type. Taken together these data suggest that phosphorylating GAPC plays only a minor role in the regulation of potato metabolism. The results presented here are discussed in relation to current models regarding primary metabolism in the potato tuber parenchyma.

  5. The Inositol-3-Phosphate Synthase Biosynthetic Enzyme Has Distinct Catalytic and Metabolic Roles

    PubMed Central

    Frej, Anna D.; Clark, Jonathan; Le Roy, Caroline I.; Lilla, Sergio; Thomason, Peter A.; Otto, Grant P.; Churchill, Grant; Insall, Robert H.; Claus, Sandrine P.; Hawkins, Phillip; Stephens, Len

    2016-01-01

    Inositol levels, maintained by the biosynthetic enzyme inositol-3-phosphate synthase (Ino1), are altered in a range of disorders, including bipolar disorder and Alzheimer's disease. To date, most inositol studies have focused on the molecular and cellular effects of inositol depletion without considering Ino1 levels. Here we employ a simple eukaryote, Dictyostelium discoideum, to demonstrate distinct effects of loss of Ino1 and inositol depletion. We show that loss of Ino1 results in an inositol auxotrophy that can be rescued only partially by exogenous inositol. Removal of inositol supplementation from the ino1− mutant resulted in a rapid 56% reduction in inositol levels, triggering the induction of autophagy, reduced cytokinesis, and substrate adhesion. Inositol depletion also caused a dramatic generalized decrease in phosphoinositide levels that was rescued by inositol supplementation. However, loss of Ino1 triggered broad metabolic changes consistent with the induction of a catabolic state that was not rescued by inositol supplementation. These data suggest a metabolic role for Ino1 that is independent of inositol biosynthesis. To characterize this role, an Ino1 binding partner containing SEL1L1 domains (Q54IX5) and having homology to mammalian macromolecular complex adaptor proteins was identified. Our findings therefore identify a new role for Ino1, independent of inositol biosynthesis, with broad effects on cell metabolism. PMID:26951199

  6. Short-term hypothermia activates hepatic mitochondrial sn-glycerol-3-phosphate dehydrogenase and thermogenic systems.

    PubMed

    Bobyleva, V; Pazienza, L; Muscatello, U; Kneer, N; Lardy, H

    2000-08-15

    The contribution of the sn-glycerol-3-phosphate (G-3-P) shuttle in the control of energy metabolism is well established. It is also known that its activity may be modulated by hormones involved in thermogenesis, such as thyroid hormones or dehydroepiandrosterone and its metabolites, that act by inducing de novo synthesis of mitochondrial G-3-P dehydrogenase (mGPDH). However, little is known as to the factors that may influence the activity without enzyme induction. In the present study we investigated the possible role of the G-3-P shuttle in the thermogenic response to different hypothermic stresses. It was found that a decrease of body temperature causes the liver rapidly to enhance mGPDH activity and G-3-P-dependent respiration. The enhancement, which does not result from de novo synthesis of enzymes, has the potential of increasing heat production both by decreased ATP synthesis during the oxidation of G-3-P and by activation of the glycolytic pathway.

  7. Negative regulation of phosphatidylinositol 3-phosphate levels in early-to-late endosome conversion

    PubMed Central

    Liu, Kai; Jian, Youli; Sun, Xiaojuan; Yang, Chengkui; Gao, Zhiyang; Zhang, Zhili; Liu, Xuezhao; Li, Yang; Xu, Jing; Jing, Yudong; Mitani, Shohei; He, Sudan

    2016-01-01

    Phosphatidylinositol 3-phosphate (PtdIns3P) plays a central role in endosome fusion, recycling, sorting, and early-to-late endosome conversion, but the mechanisms that determine how the correct endosomal PtdIns3P level is achieved remain largely elusive. Here we identify two new factors, SORF-1 and SORF-2, as essential PtdIns3P regulators in Caenorhabditis elegans. Loss of sorf-1 or sorf-2 leads to greatly elevated endosomal PtdIns3P, which drives excessive fusion of early endosomes. sorf-1 and sorf-2 function coordinately with Rab switching genes to inhibit synthesis of PtdIns3P, allowing its turnover for endosome conversion. SORF-1 and SORF-2 act in a complex with BEC-1/Beclin1, and their loss causes elevated activity of the phosphatidylinositol 3-kinase (PI3K) complex. In mammalian cells, inactivation of WDR91 and WDR81, the homologs of SORF-1 and SORF-2, induces Beclin1-dependent enlargement of PtdIns3P-enriched endosomes and defective degradation of epidermal growth factor receptor. WDR91 and WDR81 interact with Beclin1 and inhibit PI3K complex activity. These findings reveal a conserved mechanism that controls appropriate PtdIns3P levels in early-to-late endosome conversion. PMID:26783301

  8. A glyceraldehyde-3-phosphate dehydrogenase homolog in Borrelia burgdorferi and Borrelia hermsii.

    PubMed Central

    Anda, P; Gebbia, J A; Backenson, P B; Coleman, J L; Benach, J L

    1996-01-01

    A polyreactive monoclonal antibody recognized a 38.5-kDa polypeptide with amino-terminal sequence identity to conserved regions of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in Borrelia burgdorferi, the Lyme disease agent, and Borrelia hermsii, an agent of American relapsing fever. This monoclonal antibody also recognized GAPDH from other pathogenic spirochetes and other prokaryotes and eukaryotes as well. GAPDH activity was detected in sonicates of both B. burgdorferi and B. hermsii but not in live, intact organisms, indicating the possibility of a subsurface localization for the Borrelia GAPDH activity. Degenerate primers constructed from highly conserved regions of gapdh of other prokaryotes successfully amplified this gene homolog in both B. burgdorferi and B. hermsii. Nuclei acid and deduced amino acid sequence analysis of the 838-bp probes for each borrelia indicated 93.9% identity between B. burgdorferi and B. hermsii at the amino acid level. Amino acid identities of B. burgdorferi and B. hermsii with Bacillus stearothermophilus were 59.2% and 58.8% respectively. Southern hybridization studies indicated that the gene encoding GAPDH is located on the chromosome of each borrella. In other bacterial species, GAPDH has other functions in addition to its traditional enzymatic role in the glycolytic pathway. GAPDH may play a similar role in borrelias. PMID:8557349

  9. Endosomal Phosphatidylinositol 3-Phosphate Promotes Gephyrin Clustering and GABAergic Neurotransmission at Inhibitory Postsynapses*♦

    PubMed Central

    Rhee, Hong Jun; Subramanian, Devaraj; Paraskevopoulou, Foteini; Mueller, Rainer; Schultz, Carsten; Brose, Nils; Rhee, Jeong-Seop; Betz, Heinrich

    2017-01-01

    The formation of neuronal synapses and the dynamic regulation of their efficacy depend on the proper assembly of the postsynaptic neurotransmitter receptor apparatus. Receptor recruitment to inhibitory GABAergic postsynapses requires the scaffold protein gephyrin and the guanine nucleotide exchange factor collybistin (Cb). In vitro, the pleckstrin homology domain of Cb binds phosphoinositides, specifically phosphatidylinositol 3-phosphate (PI3P). However, whether PI3P is required for inhibitory postsynapse formation is currently unknown. Here, we investigated the role of PI3P at developing GABAergic postsynapses by using a membrane-permeant PI3P derivative, time-lapse confocal imaging, electrophysiology, as well as knockdown and overexpression of PI3P-metabolizing enzymes. Our results provide the first in cellula evidence that PI3P located at early/sorting endosomes regulates the postsynaptic clustering of gephyrin and GABAA receptors and the strength of inhibitory, but not excitatory, postsynapses in cultured hippocampal neurons. In human embryonic kidney 293 cells, stimulation of gephyrin cluster formation by PI3P depends on Cb. We therefore conclude that the endosomal pool of PI3P, generated by the class III phosphatidylinositol 3-kinase, is important for the Cb-mediated recruitment of gephyrin and GABAA receptors to developing inhibitory postsynapses and thus the formation of postsynaptic membrane specializations. PMID:27941024

  10. Degradation of glyceraldehyde-3-phosphate dehydrogenase induced by acetylleucine chloromethyl ketone in U937 cells.

    PubMed

    Yamaguchi, Mitsune; Tsuchiya, Yukihiro; Chikuma, Toshiyuki; Hojo, Hiroshi

    2002-05-15

    We examined whether any changes were induced in cellular proteins by an inhibitor of acylpeptide hydrolase (ACPH) (EC 3.4.19.1), acetylleucine chloromethyl ketone (ALCK), which was shown in our previous report to induce apoptosis of human U937 cells. Extract prepared from U937 cells in 0.05% Triton X-100-PBS was incubated with ALCK at 37 degrees, and then analyzed using SDS-PAGE. A 36kDa protein in the cell extract was decreased markedly during the incubation period. This protein was purified and identified as glyceraldehyde-3-phosphate dehydrogenase (GAPDH, EC 1.2.1.12) by its specific enzyme activity, N-terminal amino acid sequence, and Western blotting. Incubation of purified GAPDH with ALCK resulted in a decrease of GAPDH activity, but not in a decrease in the amount of GAPDH. The ALCK-induced GAPDH decrease in the cell extract was abrogated by co-incubation with a serine protease inhibitor, diisopropyl fluorophosphate, suggesting that GAPDH was first inactivated by ALCK, and subsequently degraded by a serine protease(s). GAPDH degradation was also observed in U937 cell cultures in the presence of ALCK. The significance of GAPDH inhibition in the apoptotic process is discussed.

  11. D-glyceraldehyde-3-phosphate dehydrogenase. Properties of the enzyme modified at arginine residues.

    PubMed

    Nagradova, N K; Schmalhausen, E V; Levashov, P A; Asryants, R A; Muronetz, V I

    1996-01-01

    Examination of the properties of Escherichia coli and rabbit muscle D-glyceraldehyde-3-phosphate dehydrogenase (GPDHs) modified by 2,3-butanedione has shown that both tetrameric enzymes are stabilized, on selective modification of arginine residues (probably Arg 231), in an asymmetric state with only two active centers capable of performing the dehydrogenase reaction. The functionally incompetent active centers can be alkylated by iodoacetate or iodoacetamide in the case of E. coli enzyme, but are inaccessible for these reagents in the case of rabbit muscle D-GPDH. These results are consistent with the idea that the two homologous enzymes share common principles of the protein design, but differ somewhat in their active centers geometries. Modification of the arginine procedures marked changes in the shape of the charge transfer complex spectrum in the region of 300-370 nm, suggestive of the alterations in the microenvironment of the nicotinamide ring of NAD(+), although the coenzyme binding characteristics remain largely unaltered. On arginine modification, the enzyme becomes insensitive to the effect of AMP on the kinetic parameters of p-nitrophenyl acetate hydrolysis reaction.

  12. Isolation of a functional, insulin regulatable glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene

    SciTech Connect

    Alexander-Bridges, M.; Ramaika, C.; Lomanto, M.; Florence, B.; Ercolani, L.

    1987-05-01

    Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a glycolytic enzyme which is regulated by insulin in 3T3 adipocytes and liver. They have isolated a 10 Kb BAM fragment which contains the entire GAPDH coding sequence and 1 Kb of the 5' flanking region. This clone has been mapped and sequenced to show the presence of a TATAA box; ATG, TAA, ATAA sites; and introns. When transiently expressed in L cells, the gene encodes a full length mRNA and functional protein. Thus, they have isolated a functional gene and not a pseudogene. When the gene is expressed in insulin-sensitive cells, the human GAPDH mRNA level was increased 3-fold in H35 hepatoma cells similar to the fold effect seen for endogenous rat mRNA in the same experiment. A 600 bp fragment of the GAPDH gene 5' flanking sequence was subcloned into a vector containing the chloramphenicol acetyl transferase (CAT) gene and cotransfected with the neomycin-resistant gene in H35 hepatoma cells to select stable lines. RNA isolated from control- and insulin-treated transfected cells show an appropriately sized S1 nuclease-protected fragment indicating that both the control species and insulin-stimulated species are primed off the GAPDH protomer. These studies indicate that insulin regulates GAPDH gene expression through an interaction with specific DNA sequences in rat hepatoma cells.

  13. Chemical Synthesis and Molecular Recognition of Phosphatase-Resistant Analogues of Phosphatidylinositol-3-phosphate

    PubMed Central

    Xu, Yong; Lee, Stephanie A.; Kutateladze, Tatiana G.; Sbrissa, Diego; Shisheva, Assia; Prestwich, Glenn D.

    2008-01-01

    The remodeling of phosphatidylinositol polyphosphates in cellular membranes by phosphatases and kinases orchestrates the signaling by these lipids in space and time. In order to provide chemical tools to study of the changes in cell physiology mediated by these lipids, three new metabolically-stabilized (ms) analogues of phosphatidylinositol-3-phosphate (PtdIns(3)P were synthesized. We describe herein the total asymmetric synthesis of 3-methylphosphonate, 3-monofluoromethylphosphonate and 3-phosphorothioate analogues of PtdIns(3)P. From differentially protected D-myo-inositol key intermediates, a versatile phosphoramidite reagent was employed in the synthesis of PtdIns(3)P analogues with diacylglyceryl moieties containing dioleoyl, dipalmitoyl and dibutyryl chains. In addition, we introduce a new phosphorlyation reagent, monofluoromethylphosphonyl chloride, which has general applications for the preparation of “pKa-matched” monofluorophosphonates. These ms-PtdIns(3)P analogues exhibited reduced binding activities with 15N-labelled FYVE and PX domains, as significant 1H and 15N chemical shift changes in the FYVE domain were induced by titrating ms-PtdIns(3)Ps into membrane-mimetic dodecylphosphocholine (DPC) micelles. In addition, the PtdIns(3)P analogues with dioleyl and dipalmitoyl chains were substrates for the 5-kinase enzyme PIKfyve; the corresponding phosphorylated ms-PI(3,5)P2 products were detected by radio-TLC analysis. PMID:16417379

  14. Evidence for thiol/disulfide exchange reactions between tubulin and glyceraldehyde-3-phosphate dehydrogenase.

    PubMed

    Landino, Lisa M; Hagedorn, Tara D; Kennett, Kelly L

    2014-12-01

    While thiol redox reactions are a common mechanism to regulate protein structure and function, protein disulfide bond formation is a marker of oxidative stress that has been linked to neurodegeneration. Both tubulin and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) contain multiple cysteines that have been identified as targets for oxidation to disulfides, S-nitrosation and S-glutathionylation. We show that GAPDH is one of three prominent brain microtubule-associated proteins (MAPs), in addition to MAP-2 and tau, with reactive cysteines. We detected a threefold to fourfold increase in tubulin cysteine oxidation by hydrogen peroxide in the presence of rabbit muscle GAPDH by 5-iodoacetamidofluorescein labeling and by Western blot detection of higher molecular weight inter-chain tubulin disulfides. In thiol/disulfide exchange experiments, tubulin restored ∼50% of oxidized GAPDH cysteines and the equilibrium favored reduced GAPDH. Further, we report that oxidized GAPDH is repaired by the thioredoxin reductase system (TRS). Restoration of GAPDH activity after reduction by both tubulin and the TRS was time-dependent suggesting conformational changes near the active site cysteine149. The addition of brain MAPs to oxidized tubulin reduced tubulin disulfides and labeling of MAP-2 and of GAPDH decreased. Because the extent of tubulin repair of oxidized GAPDH was dependent on buffer strength, we conclude that electrostatics influence thiol/disulfide exchange between the two proteins. The novel interactions presented herein may protect GAPDH from inhibition under oxidative stress conditions.

  15. Cytosolic phosphorylating glyceraldehyde-3-phosphate dehydrogenases affect Arabidopsis cellular metabolism and promote seed oil accumulation.

    PubMed

    Guo, Liang; Ma, Fangfang; Wei, Fang; Fanella, Brian; Allen, Doug K; Wang, Xuemin

    2014-07-01

    The cytosolic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPC) catalyzes a key reaction in glycolysis, but its contribution to plant metabolism and growth are not well defined. Here, we show that two cytosolic GAPCs play important roles in cellular metabolism and seed oil accumulation. Knockout or overexpression of GAPCs caused significant changes in the level of intermediates in the glycolytic pathway and the ratios of ATP/ADP and NAD(P)H/NAD(P). Two double knockout seeds had ∼3% of dry weight decrease in oil content compared with that of the wild type. In transgenic seeds under the constitutive 35S promoter, oil content was increased up to 42% of dry weight compared with 36% in the wild type and the fatty acid composition was altered; however, these transgenic lines exhibited decreased fertility. Seed-specific overexpression lines had >3% increase in seed oil without compromised seed yield or fecundity. The results demonstrate that GAPC levels play important roles in the overall cellular production of reductants, energy, and carbohydrate metabolites and that GAPC levels are directly correlated with seed oil accumulation. Changes in cellular metabolites and cofactor levels highlight the complexity and tolerance of Arabidopsis thaliana cells to the metabolic perturbation. Further implications for metabolic engineering of seed oil production are discussed. © 2014 American Society of Plant Biologists. All rights reserved.

  16. Nucleotide variability in the 5-enolpyruvylshikimate-3-phosphate synthase gene from Eleusine indica (L.) Gaertn.

    PubMed

    Chong, J L; Wickneswari, R; Ismail, B S; Salmijah, S

    2008-02-01

    This study reports the results of the partial DNA sequence analysis of the 5-enolpyruvyl-shikimate-3-phosphate synthase (EPSPS) gene in glyphosate-resistant (R) and glyphosate-susceptible (S) biotypes of Eleusine indica (L.) Gaertn from Peninsular Malaysia. Sequencing results revealed point mutation at nucleotide position 875 in the R biotypes of Bidor, Chaah and Temerloh. In the Chaah R population, substitution of cytosine (C) to adenine (A) resulted in the change of threonine (Thr106) to proline (Pro106) and from C to thymidine (T) in the Bidor R population, leading to serine (Ser106) from Pro106. As for the Temerloh R, C was substituted by T resulting in the change of Pro106 to Ser106. A new mutation previously undetected in the Temerloh R was revealed with C being substituted with A, resulting in the change of Pro106 to Thr106 indicating multiple founding events rather than to the spread of a single resistant allele. There was no point mutation recorded at nucleotide position 875 previously demonstrated to play a pivotal role in conferring glyphosate resistance to E. indica for the Lenggeng, Kuala Selangor, Melaka R populations. Thus, there may be another resistance mechanism yet undiscovered in the resistant Lenggeng, Kuala Selangor and Melaka populations.

  17. Glyphosate inhibition of 5-enolpyruvylshikimate 3-phosphate synthease from suspension-cultured cells of Nicotiana silvestris

    SciTech Connect

    Rubin, J.L.; Gaines, C.G.; Jensen, R.A.

    1984-07-01

    Treatment of isogenic suspension-cultured cells of Nicotiana silvestris Speg, et Comes with glyphosate (N-(phosphonomethyl)glycine) led to elevated levels of intracellular shikimate (364-fold increase by 1.0 millimolar glyphosate). In the presence of glyphosate, it is likely that most molecules of shikimate originate from the action of 3-deoxy-d-arabino-heptulosonate 7-phosphate (DAHP) synthase-Mn since this isozyme, in contrast to the DAHP synthase-Co isozyme, is insensitive to inhibition by glyphosate. 5-Enolpyruvylshikimate 3-phosphate (EPSP) synthase (EC 2.5.1.19) from N. silvestris was sensitive to micromolar concentrations of glyphosate and possessed a single inhibitor binding site. Rigorous kinetic studies of EPSP synthase required resolution from the multiple phosphatase activities present in crude extracts, a result achieved by ion-exchange column chromatography. Although EPSP synthase exhibited a broad pH profile (50% of maximal activity between pH 6.2 and 8.5), sensitivity to glyphosate increased dramatically with increasing pH within this range. In accordance with these data and the pK/sub a/ values of glyphosate, it is likely that the ionic form of glyphosate inhibiting EPSP synthase is COO/sup -/CH/sub 2/NH/sub 2//sup +/CH/sub 2/PO/sub 3//sup 2 -/, and that a completely ionized phosphono group is essential for inhibition. At pH 7.0, inhibition was competitive with respect to phosphoenolpyruvate (K/sub i/ = 1.25 micromolar) and uncompetitive with respect to shikimate-3-P (K/sub i/ = 18.3 micromolar). All data were consistent with a mechanism of inhibition in which glyphosate competes with PEP for binding to an (enzyme:shikimate-3-P) complex and ultimately forms the dead-end complex of (enzyme:shikimate-3-P:glyphosate). 36 references, 8 figures, 1 table.

  18. Halophilic class I aldolase and glyceraldehyde-3-phosphate dehydrogenase: some salt-dependent structural features.

    PubMed

    Krishnan, G; Altekar, W

    1993-01-26

    Aldolase and glyceraldehyde-3-phosphate dehydrogenase from the extremely halophilic archaebacterium Haloarcula vallismortis are stable only in high concentrations of KCl present within the physiological environment. Data concerning the structural changes in the two enzymes as a result of lowering of salt concentration and changes in pH were obtained by monitoring the intrinsic protein fluorescence in the presence of quenchers. When the KCl concentrations were lowered below 2 M or in the presence of 6 M guanidine hydrochloride, the emission maximum shifted to a longer wavelength, indicating enhanced exposure of tryptophyl residues to the solvent. The spectral characteristics of the two proteins in guanidine hydrochloride and 0.4 M KCl were identical. However, these denatured states appear to be different than those observed after acid denaturation. Further perturbation of fluorescence was observed due to I-, and application of the Stern-Volmer law showed that the total fluorescence was available to the quenchers only in 0.4 M KCl solutions. The unfolding of proteins in 0.4 M KCl was a gradual process which was accompanied by a time-dependent loss in enzyme activity. The activity loss was complete within 30 min for aldolase whereas in the case of GAPDH nearly 3 h was required for the destruction of activity. For both enzymes, inactivation and protein denaturation were strongly correlated. The data on activity and thermostability measurements of the two enzymes in varying concentrations of KCl and potassium phosphate revealed that though both proteins are halophilic, the forces in the maintenance of their stability could be different.(ABSTRACT TRUNCATED AT 250 WORDS)

  19. Resistance of Francisella Novicida to Fosmidomycin Associated with Mutations in the Glycerol-3-Phosphate Transporter

    PubMed Central

    Mackie, Ryan S.; McKenney, Elizabeth S.; van Hoek, Monique L.

    2012-01-01

    The methylerythritol phosphate (MEP) pathway is essential in most prokaryotes and some lower eukaryotes but absent from human cells, and is a validated target for antimicrobial drug development. The formation of MEP is catalyzed by 1-deoxy-d-xylulose 5-phosphate reductoisomerase (DXR). MEP pathway genes have been identified in many category A and B biothreat agents, including Francisella tularensis, which causes the zoonosis tularemia. Fosmidomycin (Fos) inhibits purified Francisella DXR. This compound also inhibits the growth of F. tularensis NIH B38, F. novicida and F. tularensis subsp. holarctica LVS bacteria. Related compounds such as FR900098 and the lipophilic prodrug of FR900098 (compound 1) have been developed to improve the bioavailability of these DXR inhibitors. In performing disk-inhibition assays with these compounds, we observed breakthrough colonies of F. novicida in the presence of Fos, suggesting spontaneous development of Fos resistance (FosR). FosR bacteria had decreased sensitivity to both Fos and FR900098. The two most likely targets for the development of mutants would be the DXR enzyme itself or the glycerol-3-phosphate transporter (GlpT) that allows entry of Fos into the bacteria. Sensitivity of FosR F. novicida bacteria to compound 1 was not abated suggesting that spontaneous resistance is not due to mutation of DXR. We thus predicted that the glpT transporter may be mutated leading to this resistant phenotype. Supporting this, transposon insertion mutants at the glpT locus were also found to be resistant to Fos. DNA sequencing of four different spontaneous FosR colonies demonstrated a variety of deletions in the glpT coding region. The overall frequency of FosR mutations in F. novicida was determined to be 6.3 × 10−8. Thus we conclude that one mechanism of resistance of F. novicida to Fos is caused by mutations in GlpT. This is the first description of spontaneous mutations in Francisella leading to FosR. PMID:22905031

  20. Glycerol-3-phosphate metabolism plays a role in stress response in the red alga Pyropia haitanensis.

    PubMed

    Lai, Xiao-Juan; Yang, Rui; Luo, Qi-Jun; Chen, Juan-Juan; Chen, Hai-Min; Yan, Xiao-Jun

    2015-04-01

    Glycerol-3-phosphate (G3P) has been suggested as a novel regulator of plant defense signaling, however, its role in algal resistance remains largely unknown. The glycerol kinase (also designated as NHO1) and NAD-dependent G3P dehydrogenase (GPDH) are two key enzymes involved in the G3P biosynthesis. In our study, we cloned the full-length cDNA of NHO1 (NHO1Ph ) and GPDH (GPDHP h ) from the red alga Pyropia haitanensis (denoted as NHO1Ph and GPDHP h ) and examined their expression level under flagellin peptide 22 (flg22) stimulation or heat stress. We also measured the level of G3P and floridoside (a downstream product of G3P in P. haitanensis) under flg22 stimulation or heat stress. Both NHO1Ph and GPDHP h shared high sequence identity and structural conservation with their orthologs from different species, especially from red algae. Phylogenetic analysis showed that NHO1s and GPDHs from red algae were closely related to those from animals. Under flg22 stimulation or heat stress, the expression levels of NHO1Ph and GPDHP h were up-regulated, G3P levels increased, and the contents of floridoside decreased. But the floridoside level increased in the recovery period after heat stress. Taken together, we found that G3P metabolism was associated with the flg22-induced defense response and heat stress response in P. haitanensis, indicating the general conservation of defense response in angiosperms and algae. Furthermore, floridoside might also participate in the stress resistance of P. haitanensis. © 2015 Phycological Society of America.

  1. Cellular recovery of glyceraldehyde-3-phosphate dehydrogenase activity and thiol status after exposure to hydroperoxides

    SciTech Connect

    Brodie, A.E.; Reed, D.J. )

    1990-01-01

    The activity of the thiol-dependent enzyme glyceraldehyde-3-phosphate dehydrogenase (GPD), in vertebrate cells, was modulated by a change in the intracellular thiol:disulfide redox status. Human lung carcinoma cells (A549) were incubated with 1-120 mM H2O2, 1-120 mM t-butyl hydroperoxide, 1-6 mM ethacrynic acid, or 0.1-10 mM N-ethylmaleimide for 5 min. Loss of reduced protein thiols, as measured by binding of the thiol reagent iodoacetic acid to GPD, and loss of GPD enzymatic activity occurred in a dose-dependent manner. Incubation of the cells, following oxidative treatment, in saline for 30 min or with 20 mM dithiothreitol (DTT) partially reversed both changes in GPD. The enzymatic recovery of GPD activity was observed either without addition of thiols to the medium or by incubation of a sonicated cell mixture with 2 mM cysteine, cystine, cysteamine, or glutathione (GSH); GSSG had no effect. Treatment of cells with buthionine sulfoximine (BSO) to decrease cellular GSH by varying amounts caused a dose-related increase in sensitivity of GPD activity to inactivation by H2O2 and decreased cellular ability for subsequent recovery. GPD responded in a similar fashion with oxidative treatment of another lung carcinoma cell line (A427) as well as normal lung tissue from human and rat. These findings indicate that the cellular thiol redox status can be important in determining GPD enzymatic activity.

  2. Oxidation of an Exposed Methionine Instigates the Aggregation of Glyceraldehyde-3-phosphate Dehydrogenase*

    PubMed Central

    Samson, Andre L.; Knaupp, Anja S.; Kass, Itamar; Kleifeld, Oded; Marijanovic, Emilia M.; Hughes, Victoria A.; Lupton, Chris J.; Buckle, Ashley M.; Bottomley, Stephen P.; Medcalf, Robert L.

    2014-01-01

    Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a ubiquitous and abundant protein that participates in cellular energy production. GAPDH normally exists in a soluble form; however, following necrosis, GAPDH and numerous other intracellular proteins convert into an insoluble disulfide-cross-linked state via the process of “nucleocytoplasmic coagulation.” Here, free radical-induced aggregation of GAPDH was studied as an in vitro model of nucleocytoplasmic coagulation. Despite the fact that disulfide cross-linking is a prominent feature of GAPDH aggregation, our data show that it is not a primary rate-determining step. To identify the true instigating event of GAPDH misfolding, we mapped the post-translational modifications that arise during its aggregation. Solvent accessibility and energy calculations of the mapped modifications within the context of the high resolution native GAPDH structure suggested that oxidation of methionine 46 may instigate aggregation. We confirmed this by mutating methionine 46 to leucine, which rendered GAPDH highly resistant to free radical-induced aggregation. Molecular dynamics simulations suggest that oxidation of methionine 46 triggers a local increase in the conformational plasticity of GAPDH that likely promotes further oxidation and eventual aggregation. Hence, methionine 46 represents a “linchpin” whereby its oxidation is a primary event permissive for the subsequent misfolding, aggregation, and disulfide cross-linking of GAPDH. A critical role for linchpin residues in nucleocytoplasmic coagulation and other forms of free radical-induced protein misfolding should now be investigated. Furthermore, because disulfide-cross-linked aggregates of GAPDH arise in many disorders and because methionine 46 is irrelevant to native GAPDH function, mutation of methionine 46 in models of disease should allow the unequivocal assessment of whether GAPDH aggregation influences disease progression. PMID:25086035

  3. Amperometric triglyceride bionanosensor based on nanoparticles of lipase, glycerol kinase, glycerol-3-phosphate oxidase.

    PubMed

    Pundir, C S; Aggarwal, V

    2017-01-15

    The nanoparticles (NPs) aggregates of lipase from porcine pancreas, glycerol kinase (GK) from Cellulomonas sp. and glycerol-3-phosphate oxidase (GPO) from Aerococcus viridanss were prepared by desolvation and glutaraldehyde crosslinking and functionalized by cysteamine. These enzyme nanoparticles (ENPs) were characterized by transmission electron microscopy (TEM) and Fourier transform infra red (FTIR) spectroscopy. The functionalzed ENPs aggregates were co-immobilized covalently onto polycrystalline Au electrode through thiolated bond. An improved amperometric triglyceride (TG) bionanosensor was constructed using this ENPs modified Au electrode as working electrode. Biosensor showed optimum current at 1.2 V within 5s, at pH 6.5 and 35 °C.A linear relationship was obtained between current (mA) and triolein concentration in lower concentration range,10-100 mg/dL and higher concentration range, 100-500 mg/dL. Limit of detection (LOD) of bionanosensor was 1.0 μg/ml. Percent analytical recovery of added trolein (50 and 100 mg/dL) in serum was 95.2 ± 0.5 and 96.0 ± 0.17. Within and between batch coefficients of variation (CV) were 2.33% and 2.15% respectively. A good correlation (R(2) = 0.99) was obtained between TG values in sera measured by present biosensor and standard enzymic colorimetric method with the regression equation: y= (0.993x + 0.967). ENPs/Au electrode was used 180 times over a period of 3 months with 50% loss in its initial activity, when stored dry at 4 °C.

  4. Inhibition of glyceraldehyde-3-phosphate dehydrogenase in tissues of the rat by acrylamide and related compounds.

    PubMed

    Vyas, I; Lowndes, H E; Howland, R D

    1985-01-01

    In previous investigations acrylamide was found to inhibit several enzymes of glycolysis both in vitro and in vivo. The present study examines the characteristics of the in vitro inhibition of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and compares the in vivo effects of acrylamide on GAPDH activity to other analogs. Inhibition of GAPDH produced by acrylamide was characteristic of an irreversible or slowly reversible mechanism. In vivo, GAPDH activity was determined in sciatic nerve, brain, skeletal muscle and liver after cumulative doses of 250, 350 or 500 mg/kg of acrylamide. Specific activities were significantly lower in extensor muscle and liver after the 250 mg/kg dose. Activities in brain and sciatic nerve tended to be decreased but the differences were not statistically significant. Specific activity of GAPDH was decreased in medulla pons, cerebellum and the rest of the brain after a 350 mg/kg cumulative dose of acrylamide, although protein concentrations were not different from those in controls. The maximum decrease was about 20%. Treatment with acrylamide, methylene-bis-acrylamide (non-neurotoxic), or N-isopropylacrylamide (neurotoxic) significantly decreased the weight of the cortex and associated brain areas as well as general body weights. No signs of developing neuropathy were observed during treatment with methylene-bis-acrylamide to a cumulative dose (8.1 mmoles/kg) equivalent to that of acrylamide causing frank paralysis. Although the compound exhibited some ability to inhibit GAPDH in vitro, no decrease in GAPDH activity was found in rat brain. Treatment with N-isopropylacrylamide resulted in progressive neurologic impairment. After treatment to a cumulative dose of the compound causing a severe hind-limb paralysis (9.2 mmoles/kg), a small but significant decrease in GAPDH was found in the three areas of brain examined.

  5. Vaccine efficacy of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from Edwardsiella ictaluri against E. tarda in tilapia.

    PubMed

    Trung Cao, Thanh; Tsai, Ming-An; Yang, Chung-Da; Wang, Pei-Chyi; Kuo, Tsun-Yung; Gabriel Chen, Hsu-Chung; Chen, Shih-Chu

    2014-01-01

    Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), derived from the outer-membrane protein (OMP) fraction, has been used as a potential candidate for vaccine development. The gene-encoding 37 kDa GAPDH outer membrane protein (OMP) from Edwardsiella ictaluri was amplified using polymerase chain reaction (PCR) and was cloned and expressed in Escherichia coli BL21 (DE3). Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), Western blotting, and nucleotide and amino acid sequencing were used to analyze the expressed antigenic protein and gene encoding this protein. Comparative DNA and protein sequence analysis of GAPDH from E. ictaluri GAPDHs from several Gram-negative bacterial species within the Enterobacteriaceae family revealed that the GAPDHs within this group are highly conserved and share a sequence similarity of 75-100% with E. ictaluri GDPDH. Rabbit antiserum raised against the E. ictaluri recombinant GAPDH (rGAPDH) protein recognized purified GADPH, indicating that it has a strong immunogenicity. Tilapia fish were intraperitoneally immunized with formalin-killed E. ictaluri whole cells, and rGAPDH (30 μg fish(-1)) from E. ictaluri, both of which were emulsified in ISA 763A adjuvant. At 3 months after immunization, fish were challenged with the E. tarda strain to assess vaccine efficacy; the relative percent survival (RPS) values were found to exceed 71.4%. The specific mean antibody titer log2 level of groups vaccinated with rGAPDH at 3 months was significantly higher than that of non-vaccinated fish (control group). Therefore, this recombinant protein can be considered a multi-purpose candidate vaccine against several pathogenic bacteria.

  6. Glyceraldehyde-3-phosphate Dehydrogenase (GAPDH) Aggregation Causes Mitochondrial Dysfunction during Oxidative Stress-induced Cell Death*

    PubMed Central

    Itakura, Masanori; Kubo, Takeya; Kaneshige, Akihiro; Harada, Naoki; Izawa, Takeshi; Azuma, Yasu-Taka; Kuwamura, Mitsuru; Yamaji, Ryouichi; Takeuchi, Tadayoshi

    2017-01-01

    Glycolytic glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a multifunctional protein that also mediates cell death under oxidative stress. We reported previously that the active-site cysteine (Cys-152) of GAPDH plays an essential role in oxidative stress-induced aggregation of GAPDH associated with cell death, and a C152A-GAPDH mutant rescues nitric oxide (NO)-induced cell death by interfering with the aggregation of wild type (WT)-GAPDH. However, the detailed mechanism underlying GAPDH aggregate-induced cell death remains elusive. Here we report that NO-induced GAPDH aggregation specifically causes mitochondrial dysfunction. First, we observed a correlation between NO-induced GAPDH aggregation and mitochondrial dysfunction, when GAPDH aggregation occurred at mitochondria in SH-SY5Y cells. In isolated mitochondria, aggregates of WT-GAPDH directly induced mitochondrial swelling and depolarization, whereas mixtures containing aggregates of C152A-GAPDH reduced mitochondrial dysfunction. Additionally, treatment with cyclosporin A improved WT-GAPDH aggregate-induced swelling and depolarization. In doxycycline-inducible SH-SY5Y cells, overexpression of WT-GAPDH augmented NO-induced mitochondrial dysfunction and increased mitochondrial GAPDH aggregation, whereas induced overexpression of C152A-GAPDH significantly suppressed mitochondrial impairment. Further, NO-induced cytochrome c release into the cytosol and nuclear translocation of apoptosis-inducing factor from mitochondria were both augmented in cells overexpressing WT-GAPDH but ameliorated in C152A-GAPDH-overexpressing cells. Interestingly, GAPDH aggregates induced necrotic cell death via a permeability transition pore (PTP) opening. The expression of either WT- or C152A-GAPDH did not affect other cell death pathways associated with protein aggregation, such as proteasome inhibition, gene expression induced by endoplasmic reticulum stress, or autophagy. Collectively, these results suggest that NO-induced GAPDH

  7. Glyceraldehyde-3-phosphate Dehydrogenase Aggregates Accelerate Amyloid-β Amyloidogenesis in Alzheimer Disease.

    PubMed

    Itakura, Masanori; Nakajima, Hidemitsu; Kubo, Takeya; Semi, Yuko; Kume, Satoshi; Higashida, Shusaku; Kaneshige, Akihiro; Kuwamura, Mitsuru; Harada, Naoki; Kita, Akinori; Azuma, Yasu-Taka; Yamaji, Ryoichi; Inui, Takashi; Takeuchi, Tadayoshi

    2015-10-23

    Alzheimer disease (AD) is a progressive neurodegenerative disorder characterized by loss of neurons and formation of pathological extracellular deposits induced by amyloid-β peptide (Aβ). Numerous studies have established Aβ amyloidogenesis as a hallmark of AD pathogenesis, particularly with respect to mitochondrial dysfunction. We have previously shown that glycolytic glyceraldehyde-3-phosphate dehydrogenase (GAPDH) forms amyloid-like aggregates upon exposure to oxidative stress and that these aggregates contribute to neuronal cell death. Here, we report that GAPDH aggregates accelerate Aβ amyloidogenesis and subsequent neuronal cell death both in vitro and in vivo. Co-incubation of Aβ40 with small amounts of GAPDH aggregates significantly enhanced Aβ40 amyloidogenesis, as assessed by in vitro thioflavin-T assays. Similarly, structural analyses using Congo red staining, circular dichroism, and atomic force microscopy revealed that GAPDH aggregates induced Aβ40 amyloidogenesis. In PC12 cells, GAPDH aggregates augmented Aβ40-induced cell death, concomitant with disruption of mitochondrial membrane potential. Furthermore, mice injected intracerebroventricularly with Aβ40 co-incubated with GAPDH aggregates exhibited Aβ40-induced pyramidal cell death and gliosis in the hippocampal CA3 region. These observations were accompanied by nuclear translocation of apoptosis-inducing factor and cytosolic release of cytochrome c from mitochondria. Finally, in the 3×Tg-AD mouse model of AD, GAPDH/Aβ co-aggregation and mitochondrial dysfunction were consistently detected in an age-dependent manner, and Aβ aggregate formation was attenuated by GAPDH siRNA treatment. Thus, this study suggests that GAPDH aggregates accelerate Aβ amyloidogenesis, subsequently leading to mitochondrial dysfunction and neuronal cell death in the pathogenesis of AD. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  8. Real-time amplification of glyceraldehyde-3-phosphate dehydrogenase gene for quality control of leukopoor platelets.

    PubMed

    Chen, Ding-Ping; Sun, Chien-Feng; Ning, Hsiao-Chen; Peng, Chien-Ting; Wang, Wei-Ting; Tseng, Ching-Ping

    2014-02-01

    Leukoreduction of blood products is crucial to prevent white blood cell (WBC)-associated complications during transfusion. Of the widely accepted methods for quantifying WBCs in blood components, Nageotte hemocytometry is time-consuming and laborious whereas a specialized instrument is required for flow cytometry. A reliable and affordable method to assess WBC count in blood products is of particular interest. Real-time polymerase chain reaction (PCR) of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was developed for quantifying WBCs in leukopoor platelets (LPPs). After normalization by the cell-free prefiltrated and postfiltrated plasma DNA, the relative copy number of GAPDH gene in the platelet (PLT) concentrate and its corresponding LPPs was calculated according to the equation of 2(-ΔΔCt) of which Ct is defined as the threshold cycle. The percentage and the number of WBCs that remained in LPPs were consequently determined. This method was compared to Nageotte hemocytometry and was validated by using serially diluted PLT concentrate and 10 pairs of PLT concentrate-LPP samples. Consistent with the removal of WBCs after filtration, the Ct values for the LPP samples were increased when compared to their corresponding PLT concentrate. As revealed by real-time PCR of GAPDH gene, there is a correlation between the calculated and theoretical WBC count in the serially diluted PLT concentrate (correlation coefficient, 0.9532). The WBC counts for the 10 LPP samples were comparable between Nageotte and real-time PCR method and were all below 3.3 × 10(6) WBCs/L. The real-time PCR method we report in this study is applicable for routine quality assurance during leukoreduction process. © 2013 American Association of Blood Banks.

  9. Thioredoxin-dependent regulation of photosynthetic glyceraldehyde-3-phosphate dehydrogenase: autonomous vs. CP12-dependent mechanisms.

    PubMed

    Trost, P; Fermani, S; Marri, L; Zaffagnini, M; Falini, G; Scagliarini, S; Pupillo, P; Sparla, F

    2006-09-01

    Regulation of the Calvin-Benson cycle under varying light/dark conditions is a common property of oxygenic photosynthetic organisms and photosynthetic glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is one of the targets of this complex regulatory system. In cyanobacteria and most algae, photosynthetic GAPDH is a homotetramer of GapA subunits which do not contain regulatory domains. In these organisms, dark-inhibition of the Calvin-Benson cycle involves the formation of a kinetically inhibited supramolecular complex between GAPDH, the regulatory peptide CP12 and phosphoribulokinase. Conditions prevailing in the dark, i.e. oxidation of thioredoxins and low NADP(H)/NAD(H) ratio promote aggregation. Although this regulatory system has been inherited in higher plants, these phototrophs contain in addition a second type of GAPDH subunits (GapB) resulting from the fusion of GapA with the C-terminal half of CP12. Heterotetrameric A(2)B(2)-GAPDH constitutes the major photosynthetic GAPDH isoform of higher plants chloroplasts and coexists with CP12 and A(4)-GAPDH. GapB subunits of A(2)B(2)-GAPDH have inherited from CP12 a regulatory domain (CTE for C-terminal extension) which makes the enzyme sensitive to thioredoxins and pyridine nucleotides, resembling the GAPDH/CP12/PRK system. The two systems are similar in other respects: oxidizing conditions and low NADP(H)/NAD(H) ratios promote aggregation of A(2)B(2)-GAPDH into strongly inactivated A(8)B(8)-GAPDH hexadecamers, and both CP12 and CTE specifically affect the NADPH-dependent activity of GAPDH. The alternative, lower activity with NADH is always unaffected. Based on the crystal structure of spinach A(4)-GAPDH and the analysis of site-specific mutants, a model of the autonomous (CP12-independent) regulatory mechanism of A(2)B(2)-GAPDH is proposed. Both CP12 and CTE seem to regulate different photosynthetic GAPDH isoforms according to a common and ancient molecular mechanism.

  10. Glyceraldehyde-3-phosphate Dehydrogenase Aggregates Accelerate Amyloid-β Amyloidogenesis in Alzheimer Disease*

    PubMed Central

    Itakura, Masanori; Nakajima, Hidemitsu; Kubo, Takeya; Semi, Yuko; Kume, Satoshi; Higashida, Shusaku; Kaneshige, Akihiro; Kuwamura, Mitsuru; Harada, Naoki; Kita, Akinori; Azuma, Yasu-Taka; Yamaji, Ryoichi; Inui, Takashi; Takeuchi, Tadayoshi

    2015-01-01

    Alzheimer disease (AD) is a progressive neurodegenerative disorder characterized by loss of neurons and formation of pathological extracellular deposits induced by amyloid-β peptide (Aβ). Numerous studies have established Aβ amyloidogenesis as a hallmark of AD pathogenesis, particularly with respect to mitochondrial dysfunction. We have previously shown that glycolytic glyceraldehyde-3-phosphate dehydrogenase (GAPDH) forms amyloid-like aggregates upon exposure to oxidative stress and that these aggregates contribute to neuronal cell death. Here, we report that GAPDH aggregates accelerate Aβ amyloidogenesis and subsequent neuronal cell death both in vitro and in vivo. Co-incubation of Aβ40 with small amounts of GAPDH aggregates significantly enhanced Aβ40 amyloidogenesis, as assessed by in vitro thioflavin-T assays. Similarly, structural analyses using Congo red staining, circular dichroism, and atomic force microscopy revealed that GAPDH aggregates induced Aβ40 amyloidogenesis. In PC12 cells, GAPDH aggregates augmented Aβ40-induced cell death, concomitant with disruption of mitochondrial membrane potential. Furthermore, mice injected intracerebroventricularly with Aβ40 co-incubated with GAPDH aggregates exhibited Aβ40-induced pyramidal cell death and gliosis in the hippocampal CA3 region. These observations were accompanied by nuclear translocation of apoptosis-inducing factor and cytosolic release of cytochrome c from mitochondria. Finally, in the 3×Tg-AD mouse model of AD, GAPDH/Aβ co-aggregation and mitochondrial dysfunction were consistently detected in an age-dependent manner, and Aβ aggregate formation was attenuated by GAPDH siRNA treatment. Thus, this study suggests that GAPDH aggregates accelerate Aβ amyloidogenesis, subsequently leading to mitochondrial dysfunction and neuronal cell death in the pathogenesis of AD. PMID:26359500

  11. The Plastidial Glyceraldehyde-3-Phosphate Dehydrogenase Is Critical for Viable Pollen Development in Arabidopsis1[W

    PubMed Central

    Muñoz-Bertomeu, Jesús; Cascales-Miñana, Borja; Irles-Segura, Asunción; Mateu, Isabel; Nunes-Nesi, Adriano; Fernie, Alisdair R.; Segura, Juan; Ros, Roc

    2010-01-01

    Plant metabolism is highly coordinated with development. However, an understanding of the whole picture of metabolism and its interactions with plant development is scarce. In this work, we show that the deficiency in the plastidial glycolytic glyceraldehyde-3-phosphate dehydrogenase (GAPCp) leads to male sterility in Arabidopsis (Arabidopsis thaliana). Pollen from homozygous gapcp double mutant plants (gapcp1gapcp2) displayed shrunken and collapsed forms and were unable to germinate when cultured in vitro. The pollen alterations observed in gapcp1gapcp2 were attributed to a disorganized tapetum layer. Accordingly, the expression of several of the genes involved in tapetum development was down-regulated in gapcp1gapcp2. The fertility of gapcp1gapcp2 was rescued by transforming this mutant with a construct carrying the GAPCp1 cDNA under the control of its native promoter (pGAPCp1::GAPCp1c). However, the GAPCp1 or GAPCp2 cDNA under the control of the 35S promoter (p35S::GAPCp), which is poorly expressed in the tapetum, did not complement the mutant fertility. Mutant GAPCp isoforms deficient in the catalytic activity of the enzyme were unable to complement the sterile phenotype of gapcp1gapcp2, thus confirming that both the expression and catalytic activity of GAPCp in anthers are necessary for mature pollen development. A metabolomic study in flower buds indicated that the most important difference between the sterile (gapcp1gapcp2, gapcp1gapcp2-p35S::GAPCp) and the fertile (wild-type plants, gapcp1gapcp2-pGAPCp1::GAPCp1c) lines was the increase in the signaling molecule trehalose. This work corroborates the importance of plastidial glycolysis in plant metabolism and provides evidence for the crucial role of GAPCps in pollen development. It additionally brings new insights into the complex interactions between metabolism and development. PMID:20107025

  12. PCR-mediated recombination of the amplification products of the Hibiscus tiliaceus cytosolic glyceraldehyde-3-phosphate dehydrogenase gene.

    PubMed

    Wu, Linghui; Tang, Tian; Zhou, Renchao; Shi, Suhua

    2007-03-31

    PCR-mediated recombination describes the process of in vitro chimera formation from related template sequences present in a single PCR amplification. The high levels of genetic redundancy in eukaryotic genomes should make recombination artifacts occur readily. However, few evolutionary biologists adequately consider this phenomenon when studying gene lineages. The cytosolic glyceraldehyde-3-phosphate dehydrogenase gene (GapC), which encodes a NADP-dependent nonphosphorylating glyceraldehyde-3-phosphate dehydrogenase in the cytosol, is a classical low-copy nuclear gene marker and is commonly used in molecular evolutionary studies. Here, we report on the occurrence of PCR-mediated recombination in the GapC gene family of Hibiscus tiliaceus. The study suggests that recombinant areas appear to be correlated with DNA template secondary structures. Our observations highlight that recombination artifacts should be considered when studying specific and allelic phylogenies. The authors suggest that nested PCR be used to suppress PCR-mediated recombination.

  13. Arsenite release on enzymic transformation of arsonomethyl substrate analogues: a potentially lethal synthesis by glycerol-3-phosphate dehydrogenase.

    PubMed

    Mutenda, E K; Sparkes, M J; Dixon, H B

    1995-09-15

    The isosteric arsenical analogue of glycerol 3-phosphate, 3,4-dihydroxybutylarsonic acid, is a good substrate for rabbit muscle glycerol-3-phosphate dehydrogenase. Its oxidation is accompanied by release of arsenite. This release seems to be due to a spontaneous elimination of arsenite by 3-oxoalkylarsonic acids, as it is also observed in (1) the oxidation of 3-hydroxypropylarsonic acid by yeast alcohol dehydrogenase, (2) treatment of 3,4-dihydroxybutylarsonic acid with periodate and (3) nonenzymic transamination of the glutamate analogue 2-amino-4-arsonobutyric acid. Enzymic formation of 3-oxoalkylarsonic acids in cells can therefore be lethal, as arsenite is poisonous to most organisms because of its high affinity for dithiols such as dihydrolipoyl groups.

  14. 4-Hydroxy-2-Nonenal-Modified Glyceraldehyde-3-Phosphate Dehydrogenase Is Degraded by Cathepsin G in Rat Neutrophils

    PubMed Central

    Tsuchiya, Yukihiro; Okada, Go; Kobayashi, Shigeki; Chikuma, Toshiyuki; Hojo, Hiroshi

    2011-01-01

    Degradation of oxidized or oxidatively modified proteins is an essential part of the antioxidant defenses of cells. 4-Hydroxy-2-nonenal, a major reactive aldehyde formed by lipid peroxidation, causes many types of cellular damage. It has been reported that 4-hydroxy-2-nonenal-modified proteins are degraded by the ubiquitin-proteasome pathway or, in some cases, by the lysosomal pathway. However, our previous studies using U937 cells showed that 4-hydroxy-2-nonenal-modified glyceraldehyde-3-phosphate dehydrogenase is degraded by cathepsin G. In the present study, we isolated the 4-hydroxy-2-nonenal-modified glyceraldehyde-3-phosphate dehydrogenase-degrading enzyme from rat neutrophils to an active protein fraction of 28 kDa. Using the specific antibody, the 28 kDa protein was identified as cathepsin G. Moreover, the degradation activity was inhibited by cathepsin G inhibitors. These results suggest that cathepsin G plays a crucial role in the degradation of 4-hydroxy-2-nonenal-modified glyceraldehyde-3-phosphate dehydrogenase. PMID:21904640

  15. The Effect of 3% Phosphate Ascorbyl Gel on Bond Strength of Composite Resin to Enamel treated with 35% Hydrogen Peroxide.

    PubMed

    de Castro, Milena de Fátima Schalcher; Silva, Alice Carvalho; Franco, Marcela Mayana Pereira; Silva, Ana Paula Brito; Bramante, Fausto da Silva; da Silva, Monica Barros; Lima, Darlon Martins; Pereira, Adriana de Fátima Vasconcelos

    2015-05-01

    To evaluate the effect of 3% phosphate ascorbyl gel (PA) in different times onto the microshear bond strength of composite resin (CR) to bovine enamel treated with 35% hydrogen peroxide (HP). Thirty enamel blocks of bovine incisors were made and divided into 5 groups (n = 6) with three specimens per group (n = 18), according to treatment: G1= No bleaching + CR; G2 = HP + CR after 15d; G3 = HP + CR after 24 hours; G4 = HP + PA (15 min) + CR after 24 hours; G5 = HP + PA (2 hours) + CR after 24 hours. The resin cylinders were made by Tygon matrices. Microshear bond strength test was performed using universal testing machine with a 50N load at a speed of 0.5 mm/min. Fracture modes were assessed by a stereomicroscope 40 ×. Microshear bond strength values were submitted to the analysis of variance (ANOVA) one-way and Tukey test (p < 0.05). G1 had significant results when compared to G3 and G5 (p < 0.01). However, G2, G3, G4 and G5 have showed no significant differences among groups (p > 0.05). Failure modes were categorized into adhesive (90%) and mixed (10%). The use of 3% phosphate ascorbyl gel for 15 minutes was able to improve bond strength of composite resin to bleached bovine enamel, but when 3% phosphate ascorbyl gel was applied during 40 minutes it negatively interfered in the adhesion of the resin to bleached bovine enamel.

  16. The Glycerol-3-Phosphate Acyltransferase TbGAT is Dispensable for Viability and the Synthesis of Glycerolipids in Trypanosoma brucei.

    PubMed

    Patel, Nipul; Pirani, Karim A; Zhu, Tongtong; Cheung-See-Kit, Melanie; Lee, Sungsu; Chen, Daniel G; Zufferey, Rachel

    2016-09-01

    Glycerolipids are the main constituents of biological membranes in Trypanosoma brucei, which causes sleeping sickness in humans. Importantly, they occur as a structural component of the glycosylphosphatidylinositol lipid anchor of the abundant cell surface glycoproteins procyclin in procyclic forms and variant surface glycoprotein in bloodstream form, that play crucial roles for the development of the parasite in the insect vector and the mammalian host, respectively. The present work reports the characterization of the glycerol-3-phosphate acyltransferase TbGAT that initiates the biosynthesis of ester glycerolipids. TbGAT restored glycerol-3-phosphate acyltransferase activity when expressed in a Leishmania major deletion strain lacking this activity and exhibited preference for medium length, unsaturated fatty acyl-CoAs. TbGAT localized to the endoplasmic reticulum membrane with its N-terminal domain facing the cytosol. Despite that a TbGAT null mutant in T. brucei procyclic forms lacked glycerol-3-phosphate acyltransferase activity, it remained viable and exhibited similar growth rate as the wild type. TbGAT was dispensable for the biosynthesis of phosphatidylcholine, phosphatidylinositol, phosphatidylserine, and GPI-anchored protein procyclin. However, the null mutant exhibited a slight decrease in phosphatidylethanolamine biosynthesis that was compensated with a modest increase in production of ether phosphatidylcholine. Our data suggest that an alternative initial acyltransferase takes over TbGAT's function in its absence. © 2016 The Author(s) Journal of Eukaryotic Microbiology © 2016 International Society of Protistologists.

  17. The Glycerol-3-Phosphate Acyltransferase TbGAT Is Dispensable for Viability and the Synthesis of Glycerolipids in Trypanosoma brucei

    PubMed Central

    Patel, Nipul; Pirani, Karim A.; Zhu, Tongtong; Cheung-See-Kit, Melanie; Lee, Sungsu; Chen, Daniel G.; Zufferey, Rachel

    2016-01-01

    Glycerolipids are the main constituents of biological membranes in Trypanosoma brucei, which causes sleeping sickness in humans. Importantly, they occur as a structural component of the glycosylphosphatidylinositol lipid anchor of the abundant cell surface glycoproteins procyclin in procyclic forms and variant surface glycoprotein in bloodstream form, that play crucial roles for the development of the parasite in the insect vector and the mammalian host, respectively. The present work reports the characterization of the glycerol-3-phosphate acyltransferase TbGAT that initiates the biosynthesis of ester glycerolipids. TbGAT restored glycerol-3-phosphate acyltransferase activity when expressed in a Leishmania major deletion strain lacking this activity and exhibited preference for medium length, unsaturated fatty acyl-CoAs. TbGAT localized to the endoplasmic reticulum membrane with its N-terminal domain facing the cytosol. Despite that a TbGAT null mutant in T. brucei procyclic forms lacked glycerol-3-phosphate acyltransferase activity, it remained viable and exhibited similar growth rate as the wild type. TbGAT was dispensable for the biosynthesis of phosphatidylcholine, phosphatidylinositol, phosphatidylserine and GPI-anchored protein procyclin. However, the null mutant exhibited a slight decrease in phosphatidylethanolamine biosynthesis that was compensated with a modest increase in production of ether phosphatidylcholine. Our data suggest that an alternative initial acyltransferase takes over TbGAT’s function in its absence. PMID:26909872

  18. Sulfur mustard induced nuclear translocation of glyceraldehyde-3-phosphate-dehydrogenase (GAPDH).

    PubMed

    Steinritz, Dirk; Weber, Jana; Balszuweit, Frank; Thiermann, Horst; Schmidt, Annette

    2013-12-05

    Sulfur Mustard (SM) is a vesicant chemical warfare agent, which is acutely toxic to a variety of organ systems including skin, eyes, respiratory system and bone marrow. The underlying molecular pathomechanism was mainly attributed to the alkylating properties of SM. However, recent studies have revealed that cellular responses to SM exposure are of more complex nature and include increased protein expression and protein modifications that can be used as biomarkers. In order to confirm already known biomarkers, to detect potential new ones and to further elucidate the pathomechanism of SM, we conducted large-scale proteomic experiments based on a human keratinocyte cell line (HaCaT) exposed to SM. Surprisingly, our analysis identified glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) as one of the up-regulated proteins after exposure of HaCaT cells to SM. In this paper we demonstrate the sulfur mustard induced nuclear translocation of GAPDH in HaCaT cells by 2D gel-electrophoresis (2D GE), immunocytochemistry (ICC), Western Blot (WB) and a combination thereof. 2D GE in combination with MALDI-TOF MS/MS analysis identified GAPDH as an up-regulated protein after SM exposure. Immunocytochemistry revealed a distinct nuclear translocation of GAPDH after exposure to 300μM SM. This finding was confirmed by fractionated WB analysis. 2D GE and subsequent immunoblot staining of GAPDH demonstrated two different spot locations of GAPH (pI 7.0 and pI 8.5) that are related to cytosolic or nuclear GAPDH respectively. After exposure to 300μM SM a significant increase of nuclear GAPDH at pI 8.5 occurred. Nuclear GAPDH has been associated with apoptosis, detection of structural DNA alterations, DNA repair and regulation of genomic integrity and telomere structure. The results of our study add new aspects to the pathophysiology of sulfur mustard toxicity, yet further studies will be necessary to reveal the specific function of nuclear GAPDH in the pathomechanism of sulfur mustard.

  19. Glycerol-3-phosphate phosphatase/PGP: Role in intermediate metabolism and target for cardiometabolic diseases.

    PubMed

    Possik, Elite; Madiraju, S R Murthy; Prentki, Marc

    2017-08-04

    Metabolic diseases, including obesity, type 2 diabetes, and metabolic syndrome arise because of disturbances in glucose and fat metabolism, which impact associated physiological events such as insulin secretion and action, fat storage and oxidation. Even though, decades of research has contributed to our current understanding of the components involved in glucose and fat metabolism and their regulation, that led to the development of many therapeutics, there are still many unanswered questions. Glycerol-3-phosphate (Gro3P), which is formed during glycolysis, is at the intersection of glucose and fat metabolism, and the availability of this metabolite can regulate energy and intermediary metabolism in mammalian cells. During the course of evolution, mammalian cells are assumed to have lost the capacity to directly hydrolyze Gro3P to glycerol, until the recent discovery from our laboratory showing that a previously known mammalian enzyme, phosphoglycolate phosphatase (PGP), can function as Gro3P phosphatase (G3PP) and regulate this metabolite levels. Emerging evidence indicates that G3PP/PGP is an evolutionarily conserved "multi-tasking" enzyme that belongs to the superfamily of haloacid dehalogenase-like phosphatase enzymes, and is capable of hydrolyzing Gro3P, an abundant physiologically relevant substrate, as well as other metabolites including 2-phosphoglycolate, 4-phospherythronate and 2-phospholactate, which are present in much smaller amounts in cells, under normal conditions. G3PP, by regulating Gro3P levels, plays a critical role in intermediary metabolism, including glycolysis, glucose oxidation, cellular redox and ATP production, gluconeogenesis, esterification of fatty acids towards glycerolipid synthesis and fatty acid oxidation. Because of G3PP's ability to regulate energy and intermediary metabolism as well as physiological functions such as insulin secretion, hepatic glucose production, and fat synthesis, storage and oxidation, the pathophysiological

  20. Effects of lactate dehydrogenase suppression and glycerol-3-phosphate dehydrogenase overexpression on cellular metabolism.

    PubMed

    Jeong, Dae-won; Cho, Il Taeg; Kim, Tae Soo; Bae, Gun Won; Kim, Ik-Hwan; Kim, Ick Young

    2006-03-01

    In order to conduct a physiological functional study of lactate dehydrogenase (LDH) and glycerol-3-phosphate dehydrogenase (GPDH), we engineered a CHO dhfr(-) cell, by overexpressing either the anti-sense LDH-A RNA (anti-LDH cells) or GPDH (GP3 cells), or both (GP3/anti-LDH cells). LDH activity in the cell cytosol, and lactate content and pHe change in the growth media were found to decrease according to the order: cell lines GP3/anti-LDH > anti-LDH > GP3 > CHO. Intracellular ATP contents, representing the extent of respiration rate, also decreased, according to a rank order as follows: GP3 > CHO > GP3/anti-LDH > anti-LDH. We also attempted to identify and characterize any physiological changes occurring in the cells which harbored diverse metabolic pathways. First, anti-LDH cells with heightened respiration rates were found to display a higher degree of sensitivity to the prooxidant tert-butyl hydroperoxide (tBOOH), and the mitochondrial complex III inhibitor, antimycin A, than the GPDH-expressing cells (GP3 and GP3/anti-LDH), which have a lower respiration rate. Second, the anti-sense LDH-A RNA-expressing cells (anti-LDH and GP3/anti-LDH) evidenced a higher degree of resistance to apoptosis by cell-cell contact inhibition, and a faster doubling time ( approximately 19 h compared with approximately 26 h) than the CHO and GP3 cells. Additionally, cell growth in an extended culture under HCO(3) (-)-free conditions to induce a steep acidification could be maintained with the anti-sense LDH-A RNA-expressing cells, but could not be maintained with the CHO and GP3 cells. Third, we observed that the most appropriate cell line for the optical production of a certain therapeutic protein (Tissue-Plasminogen Activator) was the GP3/anti-LDH cells. Collectively, our data indicate a variety of physiological roles for LDH and GPDH, including cellular acidosis, oxidoresistance, apoptosis by both acidosis and cell-cell contact inhibition, cell growth, and the generation of

  1. Glyceraldehyde 3-phosphate dehydrogenase negatively regulates human immunodeficiency virus type 1 infection

    PubMed Central

    2012-01-01

    Background Host proteins are incorporated inside human immunodeficiency virus type 1 (HIV-1) virions during assembly and can either positively or negatively regulate HIV-1 infection. Although the identification efficiency of host proteins is improved by mass spectrometry, how those host proteins affect HIV-1 replication has not yet been fully clarified. Results In this study, we show that virion-associated glyceraldehyde 3-phosphate dehydrogenase (GAPDH) does not allosterically inactivate HIV-1 reverse transcriptase (RT) but decreases the efficiency of reverse transcription reactions by decreasing the packaging efficiency of lysyl-tRNA synthetase (LysRS) and tRNALys3 into HIV-1 virions. Two-dimensional (2D) gel electrophoresis demonstrated that some isozymes of GAPDH with different isoelectric points were expressed in HIV-1-producing CEM/LAV-1 cells, and a proportion of GAPDH was selectively incorporated into the virions. Suppression of GAPDH expression by RNA interference in CEM/LAV-1 cells resulted in decreased GAPDH packaging inside the virions, and the GAPDH-packaging-defective virus maintained at least control levels of viral production but increased the infectivity. Quantitative analysis of reverse transcription products indicated that the levels of early cDNA products of the GAPDH-packaging-defective virus were higher than those of the control virus owing to the higher packaging efficiencies of LysRS and tRNALys3 into the virions rather than the GAPDH-dependent negative allosteric modulation for RT. Furthermore, immunoprecipitation assay using an anti-GAPDH antibody showed that GAPDH directly interacted with Pr55gag and p160gag-pol and the overexpression of LysRS in HIV-1-producing cells resulted in a decrease in the efficiency of GAPDH packaging in HIV particles. In contrast, the viruses produced from cells expressing a high level of GAPDH showed decreased infectivity in TZM-bl cells and reverse transcription efficiency in TZM-bl cells and peripheral blood

  2. Thermal unfolding of phosphorylating D-glyceraldehyde-3-phosphate dehydrogenase studied by differential scanning calorimetry.

    PubMed

    Levashov, P; Orlov, V; Boschi-Muller, S; Talfournier, F; Asryants, R; Bulatnikov, I; Muronetz, V; Branlant, G; Nagradova, N

    1999-08-17

    Thermal unfolding parameters were determined for a two-domain tetrameric enzyme, phosphorylating D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and for its isolated NAD(+)-binding domain. At pH 8.0, the transition temperatures (t(max)) for the apoforms of the native Bacillus stearothermophilus GAPDH and the isolated domain were 78.3 degrees C and 61.9 degrees C, with calorimetric enthalpies (DeltaH(cal)) of 4415 and 437 kJ/mol (or 30.7 and 22.1 J/g), respectively. In the presence of nearly saturating NAD(+) concentrations, the t(max) and the DeltaH(cal) increased by 13.6 degrees C and by 2365 kJ/mol, respectively, for the native apoenzyme, and by 2.8 degrees C and 109 kJ/mol for the isolated domain. These results indicate that interdomain interactions are essential for NAD(+) to produce its stabilizing effect on the structure of the native enzyme. The thermal stability of the isolated NAD(+)-binding domain increased considerably upon transition from pH 6.0 to 8.0. By contrast, native GAPDH exhibited greater stability at pH 6.0; similar pH-dependencies of thermal stability were displayed by GAPDHs isolated from rabbit muscle and Escherichia coli. The binding of NAD(+) to rabbit muscle apoenzyme increased t(max) and DeltaH(cal) and diminished the widths of the DSC curves; the effect was found to grow progressively with increasing coenzyme concentrations. Alkylation of the essential Cys149 with iodoacetamide destabilized the apoenzyme and altered the effect of NAD(+). Replacement of Cys149 by Ser or by Ala in the B. stearothermophilus GAPDH produced some stabilization, the effect of added NAD(+) being basically similar to that observed with the wild-type enzyme. These data indicate that neither the ion pairing between Cys149 and His176 nor the charge transfer interaction between Cys149 and NAD(+) make any significant contribution to the stabilization of the enzyme's native tertiary structure and the accomplishment of NAD(+)-induced conformational changes. The H

  3. Crystal Structures of Group B Streptococcus Glyceraldehyde-3-Phosphate Dehydrogenase: Apo-Form, Binary and Ternary Complexes

    PubMed Central

    Schormann, Norbert; Ayres, Chapelle A.; Fry, Alexandra; Green, Todd J.; Banerjee, Surajit; Ulett, Glen C.

    2016-01-01

    Glyceraldehyde 3-phosphate dehydrogenase or GAPDH is an evolutionarily conserved glycolytic enzyme. It catalyzes the two step oxidative phosphorylation of D-glyceraldehyde 3-phosphate into 1,3-bisphosphoglycerate using inorganic phosphate and NAD+ as cofactor. GAPDH of Group B Streptococcus is a major virulence factor and a potential vaccine candidate. Moreover, since GAPDH activity is essential for bacterial growth it may serve as a possible drug target. Crystal structures of Group B Streptococcus GAPDH in the apo-form, two different binary complexes and the ternary complex are described here. The two binary complexes contained NAD+ bound to 2 (mixed-holo) or 4 (holo) subunits of the tetrameric protein. The structure of the mixed-holo complex reveals the effects of NAD+ binding on the conformation of the protein. In the ternary complex, the phosphate group of the substrate was bound to the new Pi site in all four subunits. Comparison with the structure of human GAPDH showed several differences near the adenosyl binding pocket in Group B Streptococcus GAPDH. The structures also reveal at least three surface-exposed areas that differ in amino acid sequence compared to the corresponding areas of human GAPDH. PMID:27875551

  4. Crystal Structures of Group B Streptococcus Glyceraldehyde-3-Phosphate Dehydrogenase: Apo-Form, Binary and Ternary Complexes.

    PubMed

    Schormann, Norbert; Ayres, Chapelle A; Fry, Alexandra; Green, Todd J; Banerjee, Surajit; Ulett, Glen C; Chattopadhyay, Debasish

    2016-01-01

    Glyceraldehyde 3-phosphate dehydrogenase or GAPDH is an evolutionarily conserved glycolytic enzyme. It catalyzes the two step oxidative phosphorylation of D-glyceraldehyde 3-phosphate into 1,3-bisphosphoglycerate using inorganic phosphate and NAD+ as cofactor. GAPDH of Group B Streptococcus is a major virulence factor and a potential vaccine candidate. Moreover, since GAPDH activity is essential for bacterial growth it may serve as a possible drug target. Crystal structures of Group B Streptococcus GAPDH in the apo-form, two different binary complexes and the ternary complex are described here. The two binary complexes contained NAD+ bound to 2 (mixed-holo) or 4 (holo) subunits of the tetrameric protein. The structure of the mixed-holo complex reveals the effects of NAD+ binding on the conformation of the protein. In the ternary complex, the phosphate group of the substrate was bound to the new Pi site in all four subunits. Comparison with the structure of human GAPDH showed several differences near the adenosyl binding pocket in Group B Streptococcus GAPDH. The structures also reveal at least three surface-exposed areas that differ in amino acid sequence compared to the corresponding areas of human GAPDH.

  5. Cloning and characterization of CmGPD1, the Candida magnoliae homologue of glycerol-3-phosphate dehydrogenase.

    PubMed

    Lee, Dae-Hee; Kim, Myoung-Dong; Ryu, Yeon-Woo; Seo, Jin-Ho

    2008-12-01

    Glycerol-3-phosphate dehydrogenase (GPDH) plays a central role in glycerol metabolism. A genomic CmGPD1 gene encoding NADH-dependent GPDH was isolated from Candida magnoliae producing a significant amount of glycerol. The gene encodes a polypeptide of 360 amino acids, which shows high homology with known NADH-dependent GPDHs of other species. The CmGPD1 gene was expressed in recombinant Escherichia coli with the maltose-binding protein (MBP) fusion system and purified to homogeneity using simple affinity chromatography. The purified CmGpd1p without the MBP fusion displayed an apparent molecular mass of 40 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. The CmGpd1p enzyme exhibited a K(cat)/K(m) value of 195 min(-1) mM(-1) for dihydroxyacetone phosphate whereas K(cat)/K(m) for glycerol-3-phosphate is 0.385 min(-1) mM(-1). In a complementation study, CmGpd1p rescued the ability of glycerol synthesis and salt tolerance in a Saccharomyces cerevisiae GPD1DeltaGPD2Delta mutant strain. The overall results indicated that CmGPD1 encodes a functional homologue of S. cerevisiae GPDH.

  6. Identification of a second two-component signal transduction system that controls fosfomycin tolerance and glycerol-3-phosphate uptake.

    PubMed

    Kurabayashi, Kumiko; Hirakawa, Yuko; Tanimoto, Koichi; Tomita, Haruyoshi; Hirakawa, Hidetada

    2015-03-01

    Particular interest in fosfomycin has resurfaced because it is a highly beneficial antibiotic for the treatment of refractory infectious diseases caused by pathogens that are resistant to other commonly used antibiotics. The biological cost to cells of resistance to fosfomycin because of chromosomal mutation is high. We previously found that a bacterial two-component system, CpxAR, induces fosfomycin tolerance in enterohemorrhagic Escherichia coli (EHEC) O157:H7. This mechanism does not rely on irreversible genetic modification and allows EHEC to relieve the fitness burden that results from fosfomycin resistance in the absence of fosfomycin. Here we show that another two-component system, TorSRT, which was originally characterized as a regulatory system for anaerobic respiration utilizing trimethylamine-N-oxide (TMAO), also induces fosfomycin tolerance. Activation of the Tor regulatory pathway by overexpression of torR, which encodes the response regulator, or addition of TMAO increased fosfomycin tolerance in EHEC. We also show that phosphorylated TorR directly represses the expression of glpT, a gene that encodes a symporter of fosfomycin and glycerol-3-phosphate, and activation of the TorR protein results in the reduced uptake of fosfomycin by cells. However, cells in which the Tor pathway was activated had an impaired growth phenotype when cultured with glycerol-3-phosphate as a carbon substrate. These observations suggest that the TorSRT pathway is the second two-component system to reversibly control fosfomycin tolerance and glycerol-3-phosphate uptake in EHEC, and this may be beneficial for bacteria by alleviating the biological cost. We expect that this mechanism could be a potential target to enhance the utility of fosfomycin as chemotherapy against multidrug-resistant pathogens. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  7. Disparate evolution of yeasts and filamentous fungi indicated by phylogenetic analysis of glyceraldehyde-3-phosphate dehydrogenase genes.

    PubMed Central

    Smith, T L

    1989-01-01

    Genes encoding glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12) from several evolutionarily disparate organisms were used to construct a phylogenetic tree by evolutionary parsimony. The GAPDH tree indicates that, in contrast to the presently accepted taxonomy of fungi, the yeasts Saccharomyces cerevisiae and Zygosaccharomyces rouxii evolved separately from the filamentous ascomycetes (such as Aspergillus nidulans) with which these yeasts are classified. According to this tree, the Saccharomyces-like yeasts evolved very early in the course of eukaryotic evolution, whereas both ascomycete and basidiomycete filamentous fungi diverged much later through a common ancestor. PMID:2674943

  8. Anaerobic energy-yielding reaction associated with transhydrogenation from glycerol 3-phosphate to fumarate by an Escherichia coli system.

    PubMed Central

    Miki, K; Lin, E C

    1975-01-01

    A particulate subcellular fraction from Escherichia coli K-12 induced in anaerobic sn-glycerol 3-phosphate (G3P) dehydrogenase and fumarate reductase can catalyze under anaerobic conditions the transfer of hydrogens from G3P to fumarate, with attendant generation of high-energy phosphate. The phsophorylation process is more sensitive than the transhydrogenation process to inhibition by the detergent Triton X-100. The same is true with respect to sensitivity to sodium azide, carbonyl cyanide m-chlorophenylhydrazone and N,N'-dicyclohexylcarbodiimide. Such a preparation derived from cells with beta-galactoside permease can accumulate thiomethyl beta-D-galactoside anaerobically, and the accumulation can be stimulated twofold by adding G3P and fumarate. Mutants lacking the membrane-associated Mg2+-dependent adenosine triphosphatase cannot grow anaerobically on glycerol with fumarate as the hydrogen acceptor, although they can grow aerobically on glycerol alone. PMID:127785

  9. Anaerobic energy-yielding reaction associated with transhydrogenation from glycerol 3-phosphate to fumarate by an Escherichia coli system.

    PubMed

    Miki, K; Lin, E C

    1975-12-01

    A particulate subcellular fraction from Escherichia coli K-12 induced in anaerobic sn-glycerol 3-phosphate (G3P) dehydrogenase and fumarate reductase can catalyze under anaerobic conditions the transfer of hydrogens from G3P to fumarate, with attendant generation of high-energy phosphate. The phsophorylation process is more sensitive than the transhydrogenation process to inhibition by the detergent Triton X-100. The same is true with respect to sensitivity to sodium azide, carbonyl cyanide m-chlorophenylhydrazone and N,N'-dicyclohexylcarbodiimide. Such a preparation derived from cells with beta-galactoside permease can accumulate thiomethyl beta-D-galactoside anaerobically, and the accumulation can be stimulated twofold by adding G3P and fumarate. Mutants lacking the membrane-associated Mg2+-dependent adenosine triphosphatase cannot grow anaerobically on glycerol with fumarate as the hydrogen acceptor, although they can grow aerobically on glycerol alone.

  10. Cloning, heterologous expression and biochemical characterization of plastidial sn-glycerol-3-phosphate acyltransferase from Helianthus annuus.

    PubMed

    Payá-Milans, Miriam; Venegas-Calerón, Mónica; Salas, Joaquín J; Garcés, Rafael; Martínez-Force, Enrique

    2015-03-01

    The acyl-[acyl carrier protein]:sn-1-glycerol-3-phosphate acyltransferase (GPAT; E.C. 2.3.1.15) catalyzes the first step of glycerolipid assembly within the stroma of the chloroplast. In the present study, the sunflower (Helianthus annuus, L.) stromal GPAT was cloned, sequenced and characterized. We identified a single ORF of 1344base pairs that encoded a GPAT sharing strong sequence homology with the plastidial GPAT from Arabidopsis thaliana (ATS1, At1g32200). Gene expression studies showed that the highest transcript levels occurred in green tissues in which chloroplasts are abundant. The corresponding mature protein was heterologously overexpressed in Escherichia coli for purification and biochemical characterization. In vitro assays using radiolabelled acyl-ACPs and glycerol-3-phosphate as substrates revealed a strong preference for oleic versus palmitic acid, and weak activity towards stearic acid. The positional fatty acid composition of relevant chloroplast phospholipids from sunflower leaves did not reflect the in vitro GPAT specificity, suggesting a more complex scenario with mixed substrates at different concentrations, competition with other acyl-ACP consuming enzymatic reactions, etc. In summary, this study has confirmed the affinity of this enzyme which would partly explain the resistance to cold temperatures observed in sunflower plants.

  11. Characterization and partial purification of acyl-CoA:glycerol 3-phosphate acyltransferase from sunflower (Helianthus annuus L.) developing seeds.

    PubMed

    Ruiz-López, Noemí; Garcés, Rafael; Harwood, John L; Martínez-Force, Enrique

    2010-01-01

    The glycerol 3-phosphate acyltransferase (GPAT, EC 2.3.1.15) from sunflower (Helianthus annuus L.) microsomes has been characterised and partially purified. The in vitro determination of activity was optimized, and the maximum value for GPAT activity identified between 15 and 20 days after flowering. The apparent Michaelis-Menten K(m) for the glycerol 3-phosphate was 354 muM. The preferred substrates were palmitoyl-CoA = linoleoyl-CoA > oleoyl-CoA with the lowest activity using stearoyl-CoA. High solubilisation was achieved using 0.75% Tween80 and the solubilised GPAT was partially purified by ion-exchange chromatography using a Hi-Trap DEAE FF column, followed by gel filtration chromatography using a Superose 12 HR column. The fraction containing the GPAT activity was analysed by SDS-PAGE and contained a major band of 60.1 kDa. Finally, evidence is provided which shows the role of GPAT in the asymmetrical distribution, between positions sn-1 and sn-3, of saturated fatty acids in highly saturated sunflower triacylglycerols. This work provides background information on the sunflower endoplasmic reticulum GPAT which may prove valuable for future modification of oil deposition in this important crop.

  12. A critical role of plastidial glycolytic glyceraldehyde-3-phosphate dehydrogenase in the control of plant metabolism and development

    PubMed Central

    Muñoz-Bertomeu, Jesús; Cascales-Miñana, Borja; Alaiz, Manuel; Segura, Juan

    2010-01-01

    Glycolysis is a central metabolic pathway that provides energy and generates precursors for the synthesis of primary metabolites such as amino acids and fatty acids.1–3 In plants, glycolysis occurs in the cytosol and plastids, which complicates the understanding of this essential process.1 As a result, the contribution of each glycolytic pathway to the specific primary metabolite production and the degree of integration of both pathways is still unresolved. The glycolytic glyceraldehyde-3-phosphate dehydrogenase (GAPDH) catalyzes the conversion of glyceraldehyde-3-phosphate to 1,3-bisphosphoglycerate. Both cytosolic (GAPCs) and plastidial (GAPCps) GAPDH activities have been described biochemically. But, up to now, little attention had been paid to GAPCps, probably because they have been considered as “minor isoforms” that catalyze a reversible reaction in plastids where it has been assumed that key glycolytic intermediates are in equilibrium with the cytosol. In the associated study,4 we have elucidated the crucial role of Arabidopsis GAPCps in the control of primary metabolism in plants. GAPCps deficiency affects amino acid and sugar metabolism and impairs plant development. Specifically, GAPCp deficiency affects the serine supply to roots, provoking a drastic phenotype of arrested root development. Also, we show that the phosphorylated serine biosynthesis pathway is critical to supply serine to non-photosynthetic organs such as roots. These studies provide new insights of the contribution of plastidial glycolysis to plant metabolism and evidence the complex interactions existing between metabolism and development. PMID:20592814

  13. Metabolic engineering of enhanced glycerol-3-phosphate synthesis to increase lipid production in Synechocystis sp. PCC 6803.

    PubMed

    Wang, Xi; Xiong, Xiaochao; Sa, Na; Roje, Sanja; Chen, Shulin

    2016-07-01

    With the growing attention to global warming and energy sustainability, biosynthesis of lipids by photosynthetic microorganisms has attracted more interest for the production of renewable transportation fuels. Recently, the cyanobacterium Synechocystis sp. PCC 6803 has been widely used for biofuel production through metabolic engineering because of its efficient photosynthesis and well-developed genetic tools. In lipid biosynthesis, glycerol-3-phosphate (G3P) is a key node for both CO2 fixation and lipid metabolism in cyanobacteria. However, few studies have explored the use of G3P synthesis to improve photosynthetic lipid production. In this study, metabolic engineering combined with flux balance analysis (FBA) was conducted to reveal the effect of G3P synthesis on lipid production. Heterologous genes that encoded glycerol-3-phosphate dehydrogenase (GPD) and diacylglycerol acyltransferase (DGAT) were engineered into Synechocystis sp. PCC 6803 to enhance G3P supply and lipid production. The resultant recombinant Synechocystis produced higher levels of lipids without a significant reduction in cell growth. Compared with the wild-type strain, lipid content and productivity of the engineered cyanobacteria increased by up to 36 and 31 %, respectively, under autotrophic conditions. Lipid production under mixotrophic conditions of the engineered cyanobacteria was also investigated. This work demonstrated that enhanced G3P synthesis was an important factor in photosynthetic lipid production and that introducing heterologous GPD and DGAT genes was an effective strategy to increase lipid production in Synechocystis sp. PCC 6803.

  14. Synergistic interaction of glyceraldehydes-3-phosphate dehydrogenase and ArsJ, a novel organoarsenical efflux permease, confers arsenate resistance.

    PubMed

    Chen, Jian; Yoshinaga, Masafumi; Garbinski, Luis D; Rosen, Barry P

    2016-06-01

    Microbial biotransformations are major contributors to the arsenic biogeocycle. In parallel with transformations of inorganic arsenic, organoarsenicals pathways have recently been recognized as important components of global cycling of arsenic. The well-characterized pathway of resistance to arsenate is reduction coupled to arsenite efflux. Here, we describe a new pathway of arsenate resistance involving biosynthesis and extrusion of an unusual pentavalent organoarsenical. A number of arsenic resistance (ars) operons have two genes of unknown function that are linked in these operons. One, gapdh, encodes the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase. The other, arsJ, encodes a major facilitator superfamily (MFS) protein. The two genes were cloned from the chromosome of Pseudomonas aeruginosa. When expressed together, but not alone, in Escherichia coli, gapdh and arsJ specifically conferred resistance to arsenate and decreased accumulation of As(V). Everted membrane vesicles from cells expressing arsJ accumulated As(V) in the presence of purified GAPDH, D-glceraldehylde 3-phosphate (G3P) and NAD(+) . GAPDH forms the unstable organoarsenical 1-arseno-3-phosphoglycerate (1As3PGA). We propose that ArsJ is an efflux permease that extrudes 1As3PGA from cells, where it rapidly dissociates into As(V) and 3-phosphoglycerate (3PGA), creating a novel pathway of arsenate resistance. © 2016 John Wiley & Sons Ltd.

  15. Structural basis for regulation of stability and activity in glyceraldehyde-3-phosphate dehydrogenases. Differential scanning calorimetry and molecular dynamics.

    PubMed

    Makshakova, Olga N; Semenyuk, Pavel I; Kuravsky, Mikhail L; Ermakova, Elena A; Zuev, Yuriy F; Muronetz, Vladimir I

    2015-05-01

    Tissue specific isoforms of human glyceraldehyde-3-phosphate dehydrogenase, somatic (GAPD) and sperm-specific (GAPDS), have been reported to display different levels of both stability and catalytic activity. Here we apply MD simulations to investigate molecular basis of this phenomenon. The protein is a tetramer where each subunit consists of two domains - catalytic and NAD-binding one. We demonstrated key residues responsible for intersubunit and interdomain interactions. Effect of several residues was studied by point mutations. Overall we considered three mutations (Glu96Gln, Glu244Gln and Asp311Asn) disrupting GAPDS-specific salt bridges. Comparison of calculated interaction energies with calorimetric enthalpies confirmed that intersubunit interactions were responsible for enhanced thermostability of GAPDS whereas interdomain interactions had indirect influence on intersubunit contacts. Mutation Asp311Asn was around 10Å far from the active center and corresponded to the closest natural substitution in the isoenzymes. MD simulations revealed that this residue had slight interaction with catalytic residues but influenced the hydrogen bond net and dynamics in active site. These effects can be responsible for a strong influence of this residue on catalytic activity. Overall, our results provide new insight into glyceraldehyde-3-phosphate dehydrogenase structure-function relationships and can be used for the engineering of mutant proteins with modified properties and for development of new inhibitors with indirect influence on the catalytic site. Copyright © 2015 Elsevier Inc. All rights reserved.

  16. Cloning and characterisation of the glyceraldehyde 3-phosphate dehydrogenase gene of Candida bombicola and use of its promoter.

    PubMed

    Van Bogaert, Inge N A; De Maeseneire, Sofie L; Develter, Dirk; Soetaert, Wim; Vandamme, Erick J

    2008-10-01

    The glyceraldehyde-3-phosphate dehydrogenase gene (GPD) of the sophorolipid producing yeast Candida bombicola was isolated using degenerated PCR and genome walking. The obtained 3,740 bp contain the 1,008 bases of the coding sequence and 1,613 and 783 bp of the upstream and downstream regions, respectively. The corresponding protein shows high homology to the other known GPD genes and is 74% identical to the gyceraldehyde-3-phosphate dehydrogenase of Yarrowia lipolytica. The particular interest in the C. bombicola GPD gene sequence originates from the potential use of its promoter for high and constitutive expression of homologous and heterologous genes. Southern blot analysis did not give any indication for the presence of multiple GPD genes and it can therefore be expected that the promoter can be used for efficient and high expression. This hypothesis was further confirmed by the biased codon usage in the GPD gene. GDP promoter fragments of different lengths were used to construct hygromycin resistance cassettes. The constructs were used for the transformation of C. bombicola and all of them, even the ones with only 190 bp of the GPD promoter, were able to render the cells resistant to hygromycin. The efficacy of a short GPD promoter can be a convenient characteristic for the construction of compact expression cassettes or vectors for C. bombicola. The GenBank accession number of the sequence described in this article is EU315245.

  17. Effects of substrates and phosphate on INT (2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyl tetrazolium chloride) and CTC (5-cyano-2,3-ditolyl tetrazolium chloride) reduction in Escherichia coli

    NASA Technical Reports Server (NTRS)

    Smith, J. J.; McFeters, G. A.

    1996-01-01

    The effects of substrates of primary aerobic dehydrogenases, and inorganic phosphate on aerobic INT and CTC reduction in Escherichia coli were examined. In general, INT produced less formazan than CTC, but INT (+) cell counts remained near values of CTC (+) cells. INT and CTC (+) cell numbers were higher than plate counts on R2A medium using succinate, formate, lactate, casamino acids, glucose, glycerol (INT only) and no substrate. Formate resulted in the greatest amount of INT and CTC formazan. Reduction of both INT and CTC was inhibited above 10 mmol l-1 phosphate, and this appeared to be related to decreased rates of O2 consumption. Formation of fluorescent CTC (+), but not INT (+) cells was also inhibited in a concentration dependent manner by phosphate above 10 mmol l-1. From light microscopic observations it appeared CTC formed increasing amounts of poorly or non-fluorescent formazan with increasing phosphate. Therefore, use of phosphate buffer in excess of 10 mmol l-1 may not be appropriate in CTC and INT reduction assays.

  18. Effects of substrates and phosphate on INT (2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyl tetrazolium chloride) and CTC (5-cyano-2,3-ditolyl tetrazolium chloride) reduction in Escherichia coli

    NASA Technical Reports Server (NTRS)

    Smith, J. J.; McFeters, G. A.

    1996-01-01

    The effects of substrates of primary aerobic dehydrogenases, and inorganic phosphate on aerobic INT and CTC reduction in Escherichia coli were examined. In general, INT produced less formazan than CTC, but INT (+) cell counts remained near values of CTC (+) cells. INT and CTC (+) cell numbers were higher than plate counts on R2A medium using succinate, formate, lactate, casamino acids, glucose, glycerol (INT only) and no substrate. Formate resulted in the greatest amount of INT and CTC formazan. Reduction of both INT and CTC was inhibited above 10 mmol l-1 phosphate, and this appeared to be related to decreased rates of O2 consumption. Formation of fluorescent CTC (+), but not INT (+) cells was also inhibited in a concentration dependent manner by phosphate above 10 mmol l-1. From light microscopic observations it appeared CTC formed increasing amounts of poorly or non-fluorescent formazan with increasing phosphate. Therefore, use of phosphate buffer in excess of 10 mmol l-1 may not be appropriate in CTC and INT reduction assays.

  19. Chemical mechanism of glycerol 3-phosphate phosphatase: pH-dependent changes in the rate-limiting step.

    PubMed

    Larrouy-Maumus, Gérald; Kelly, Geoff; de Carvalho, Luiz Pedro Sório

    2014-01-14

    The halo-acid dehalogenase (HAD) superfamily comprises a large number of enzymes that share a conserved core domain responsible for a diverse array of chemical transformations (e.g., phosphonatase, dehalogenase, phosphohexomutase, and phosphatase) and a cap domain that controls substrate specificity. Phosphate hydrolysis is thought to proceed via an aspartyl-phosphate intermediate, and X-ray crystallography has shown that protein active site conformational changes are required for catalytic competency. Using a combination of steady-state and pre-steady-state kinetics, pL-rate studies, solvent kinetic isotope effects, (18)O molecular isotope exchange, and partition experiments, we provide a detailed description of the chemical mechanism of a glycerol 3-phosphate phosphatase. This phosphatase has been recently recognized as a rate-limiting factor in lipid polar head recycling in Mycobacterium tuberculosis [Larrouy-Maumus, G., et al. (2013) Proc. Natl. Acad. Sci. 110 (28), 11320-11325]. Our results clearly establish the existence of an aspartyl-phosphate intermediate in this newly discovered member of the HAD superfamily. No ionizable groups are rate-limiting from pH 5.5 to 9.5, consistent with the pK values of the catalytic aspartate residues. The formation and decay of this intermediate are partially rate-limiting below pH 7.0, and a conformational change preceding catalysis is rate-limiting above pH 7.0.

  20. Citrin/mitochondrial glycerol-3-phosphate dehydrogenase double knock-out mice recapitulate features of human citrin deficiency.

    PubMed

    Saheki, Takeyori; Iijima, Mikio; Li, Meng Xian; Kobayashi, Keiko; Horiuchi, Masahisa; Ushikai, Miharu; Okumura, Fumihiko; Meng, Xiao Jian; Inoue, Ituro; Tajima, Atsushi; Moriyama, Mitsuaki; Eto, Kazuhiro; Kadowaki, Takashi; Sinasac, David S; Tsui, Lap-Chee; Tsuji, Mihoko; Okano, Akira; Kobayashi, Tsuyoshi

    2007-08-24

    Citrin is the liver-type mitochondrial aspartate-glutamate carrier that participates in urea, protein, and nucleotide biosynthetic pathways by supplying aspartate from mitochondria to the cytosol. Citrin also plays a role in transporting cytosolic NADH reducing equivalents into mitochondria as a component of the malate-aspartate shuttle. In humans, loss-of-function mutations in the SLC25A13 gene encoding citrin cause both adult-onset type II citrullinemia and neonatal intrahepatic cholestasis, collectively referred to as human citrin deficiency. Citrin knock-out mice fail to display features of human citrin deficiency. Based on the hypothesis that an enhanced glycerol phosphate shuttle activity may be compensating for the loss of citrin function in the mouse, we have generated mice with a combined disruption of the genes for citrin and mitochondrial glycerol 3-phosphate dehydrogenase. The resulting double knock-out mice demonstrated citrullinemia, hyperammonemia that was further elevated by oral sucrose administration, hypoglycemia, and a fatty liver, all features of human citrin deficiency. An increased hepatic lactate/pyruvate ratio in the double knock-out mice compared with controls was also further elevated by the oral sucrose administration, suggesting that an altered cytosolic NADH/NAD(+) ratio is closely associated with the hyperammonemia observed. Microarray analyses identified over 100 genes that were differentially expressed in the double knock-out mice compared with wild-type controls, revealing genes potentially involved in compensatory or downstream effects of the combined mutations. Together, our data indicate that the more severe phenotype present in the citrin/mitochondrial glycerol-3-phosphate dehydrogenase double knock-out mice represents a more accurate model of human citrin deficiency than citrin knock-out mice.

  1. Glyceraldehyde-3-phosphate ferredoxin oxidoreductase (GAPOR) and nonphosphorylating glyceraldehyde-3-phosphate dehydrogenase (GAPN), key enzymes of the respective modified Embden-Meyerhof pathways in the hyperthermophilic crenarchaeota Pyrobaculum aerophilum and Aeropyrum pernix.

    PubMed

    Reher, Matthias; Gebhard, Susanne; Schönheit, Peter

    2007-08-01

    The growth of Pyrobaculum aerophilum on yeast extract and nitrate was stimulated by the addition of maltose. Extracts of maltose/yeast extract/nitrate-grown cells contained all enzyme activities of a modified Embden-Meyerhof (EM) pathway, including ATP-dependent glucokinase, phosphoglucose isomerase, ATP-dependent 6-phosphofructokinase, fructose-1,6-phosphate aldolase, triose-phosphate isomerase, GAPOR, phosphoglycerate mutase, enolase and pyruvate kinase. The activity of GAPOR was stimulated about fourfold by maltose, indicating a role in sugar degradation. GAPOR was purified 200-fold to homogeneity and characterized as a 67 kDa monomeric, extremely thermostable protein. The enzyme showed high specificity for glyceraldehyde-3-phosphate and did not use glyceraldehyde, acetaldehyde or formaldehyde as substrates. By matrix-assisted laser desorption/ionization-time of flight analysis of the purified enzyme, ORF PA1029 was identified as a coding gene, gapor, in the sequenced genome of Pyrobaculum aerophilum. The data indicate that the (micro)aerophilic Pyrobaculum aerophilum contains a functional GAPOR as part of a modified EM pathway. Cells of the strictly aerobic crenarchaeon Aeropyrum pernix also contain enzyme activities of a modified EM pathway similar to that of Pyrobaculum aerophilum, except that a GAPN activity replaces GAPOR activity.

  2. Mechanism for increased hepatic glycerol synthesis in the citrin/mitochondrial glycerol-3-phosphate dehydrogenase double-knockout mouse: Urine glycerol and glycerol 3-phosphate as potential diagnostic markers of human citrin deficiency.

    PubMed

    Moriyama, Mitsuaki; Fujimoto, Yuki; Rikimaru, Shizuka; Ushikai, Miharu; Kuroda, Eishi; Kawabe, Kenji; Takano, Katsura; Asakawa, Akihiro; Inui, Akio; Eto, Kazuhiro; Kadowaki, Takashi; Sinasac, David S; Okano, Yoshiyuki; Yazaki, Masahide; Ikeda, Shu-Ichi; Zhang, Chunhua; Song, Yuan-Zong; Sakamoto, Osamu; Kure, Shigeo; Mitsubuchi, Hiroshi; Endo, Fumio; Horiuchi, Masahisa; Nakamura, Yoichi; Yamamura, Ken-Ichi; Saheki, Takeyori

    2015-09-01

    The mitochondrial aspartate-glutamate carrier isoform 2 (citrin) and mitochondrial glycerol-3-phosphate dehydrogenase (mGPD) double-knockout mouse has been a useful model of human citrin deficiency. One of the most prominent findings has been markedly increased hepatic glycerol 3-phosphate (G3P) following oral administration of a sucrose solution. We aimed to investigate whether this change is detectable outside of the liver, and to explore the mechanism underlying the increased hepatic G3P in these mice. We measured G3P and its metabolite glycerol in plasma and urine of the mice under various conditions. Glycerol synthesis from fructose was also studied using the liver perfusion system. The citrin/mGPD double-knockout mice showed increased urine G3P and glycerol under normal, fed conditions. We also found increased plasma glycerol under fasted conditions, while oral administration of different carbohydrates or ethanol led to substantially increased plasma glycerol. Fructose infusion to the perfused liver of the double-knockout mice augmented hepatic glycerol synthesis, and was accompanied by a concomitant increase in the lactate/pyruvate (L/P) ratio. Co-infusion of either pyruvate or phenazine methosulfate, a cytosolic oxidant, with fructose corrected the high L/P ratio, leading to reduced glycerol synthesis. Overall, these findings suggest that hepatic glycerol synthesis is cytosolic NADH/NAD(+) ratio-dependent and reveal a likely regulatory mechanism for hepatic glycerol synthesis following a high carbohydrate load in citrin-deficient patients. Therefore, urine G3P and glycerol may represent potential diagnostic markers for human citrin deficiency. Copyright © 2015 Elsevier B.V. All rights reserved.

  3. Characterization of two proteins of Staphylococcus aureus isolated from bovine clinical mastitis with homology to glyceraldehyde-3-phosphate dehydrogenase.

    PubMed

    Goji, Noriko; Potter, Andrew A; Perez-Casal, Jose

    2004-04-19

    Staphylococcus aureus is the most common causative agent of bovine mastitis and vaccines developed to control this disease showed limited protection due in part to the lack of common antigens among the mastitis isolates. We isolated and identified two genes encoding proteins with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activity from a S. aureus strain isolated from bovine clinical mastitis. The GapB and GapC proteins share considerable homology to the GapB and GapC products of human strains of S. aureus. These two proteins could be distinguished by their different GAPDH activities and binding to bovine transferrin properties. Both gapB and gapC genes were conserved in 11 strains tested, and the GapC protein was present on the surface of all S. aureus strains.

  4. Autonomous folding of the excised coenzyme-binding domain of D-glyceraldehyde 3-phosphate dehydrogenase from Thermotoga maritima.

    PubMed Central

    Jecht, M.; Tomschy, A.; Kirschner, K.; Jaenicke, R.

    1994-01-01

    An important question in protein folding is whether compact substructures or domains are autonomous units of folding and assembly. The protomer of the tetrameric D-glyceraldehyde-3-phosphate dehydrogenase from the hyperthermophilic bacterium Thermotoga maritima has a complex coenzyme-binding domain, in which residues 1-146 form a compact substructure with the last 31 residues (313-333). Here it is shown that the gene of a single-chain protein can be expressed in Escherichia coli after deleting the 163 codons corresponding to the interspersed catalytic domain (150-312). The purified gene product is a soluble, monomeric protein that binds both NAD+ and NADH strongly and possesses the same unfolding transition induced by guanidinium chloride as the native tetramer. The autonomous folding of the coenzyme-binding domain has interesting implications for the folding, assembly, function, and evolution of the native enzyme. PMID:8019412

  5. The sweet side of RNA regulation: glyceraldehyde-3-phosphate dehydrogenase as a noncanonical RNA-binding protein

    PubMed Central

    White, Michael R.; Garcin, Elsa D.

    2016-01-01

    The glycolytic protein, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), has a vast array of extraglycolytic cellular functions, including interactions with nucleic acids. GAPDH has been implicated in the translocation of transfer RNA (tRNA), the regulation of cellular messenger RNA (mRNA) stability and translation, as well as the regulation of replication and gene expression of many single-stranded RNA viruses. A growing body of evidence supports GAPDH–RNA interactions serving as part of a larger coordination between intermediary metabolism and RNA biogenesis. Despite the established role of GAPDH in nucleic acid regulation, it is still unclear how and where GAPDH binds to its RNA targets, highlighted by the absence of any conserved RNA-binding sequences. This review will summarize our current understanding of GAPDH-mediated regulation of RNA function. PMID:26564736

  6. Analysis of l-glycerol-3-phosphate dehydrogenase mutants in Drosophila melanogaster: complementation for intracellular degradation of the mutant polypeptide.

    PubMed

    Bewley, G C; DeZurik, J M; Pagelson, G

    1980-01-01

    Null and low activity alleles at the genetic locus coding for L-Glycerol-3-phosphate dehydrogenase (alpha-GPDH, NAD+ oxidoreductase, E.C. 1.1.1.8) in Drosophila melanogaster have been analyzed by a combination of rocket immunoelectrophoresis, interallelic complementation, and two-dimensional gel electrophoresis. In addition to proving information on the molecular weight, charged state, and steady state level of CRM in each of these mutants, it is suggested that each mutation has resulted in a genetic lesion within the structural element, Gpdh+. CRM levels appear to be the result of differential sensitivity to the normal intracellular degradative process and the CRM- mutants represent "hypersensitive" alleles, such that the mutant polypeptide does not accumulate in the intracellular environment.

  7. Cytoplastic Glyceraldehyde-3-Phosphate Dehydrogenases Interact with ATG3 to Negatively Regulate Autophagy and Immunity in Nicotiana benthamiana

    PubMed Central

    Han, Shaojie; Wang, Yan; Zheng, Xiyin; Jia, Qi; Zhao, Jinping; Bai, Fan; Hong, Yiguo; Liu, Yule

    2015-01-01

    Autophagy as a conserved catabolic pathway can respond to reactive oxygen species (ROS) and plays an important role in degrading oxidized proteins in plants under various stress conditions. However, how ROS regulates autophagy in response to oxidative stresses is largely unknown. Here, we show that autophagy-related protein 3 (ATG3) interacts with the cytosolic glyceraldehyde-3-phosphate dehydrogenases (GAPCs) to regulate autophagy in Nicotiana benthamiana plants. We found that oxidative stress inhibits the interaction of ATG3 with GAPCs. Silencing of GAPCs significantly activates ATG3-dependent autophagy, while overexpression of GAPCs suppresses autophagy in N. benthamiana plants. Moreover, silencing of GAPCs enhances N gene-mediated cell death and plant resistance against both incompatible pathogens Tobacco mosaic virus and Pseudomonas syringae pv tomato DC3000, as well as compatible pathogen P. syringae pv tabaci. These results indicate that GAPCs have multiple functions in the regulation of autophagy, hypersensitive response, and plant innate immunity. PMID:25829441

  8. Non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase is post-translationally phosphorylated in heterotrophic cells of wheat (Triticum aestivum).

    PubMed

    Bustos, Diego M; Iglesias, Alberto A

    2002-10-23

    In wheat, non-phosphorylating, NADP-dependent glyceraldehyde-3-phosphate dehydrogenase (GAPN) was found to be encoded by one gene giving rise to a single protein. However, Western blots revealed two different subunits of about 58 and 60 kDa in endosperm and shoots. The latter was attributed to in vivo phosphorylation of shoot GAPN. No modification occurred in leaves, where the enzyme is composed by a single 58 kDa polypeptide. GAPN partially purified from shoots and endosperm was dephosphorylated in vitro with alkaline phosphatase. Phosphorylated GAPN exhibited similar affinity for substrates but a lower V(max) compared to the non-phosphorylated enzyme. Results suggest that reversible phosphorylation of GAPN could regulate NADPH production in the cytosol of heterotrophic plant cells.

  9. Homocysteine induces glyceraldehyde-3-phosphate dehydrogenase acetylation and apoptosis in the neuroblastoma cell line Neuro2a

    PubMed Central

    Fang, M.; Jin, A.; Zhao, Y.; Liu, X.

    2016-01-01

    High plasma levels of homocysteine (Hcy) promote the progression of neurodegenerative diseases. However, the mechanism by which Hcy mediates neurotoxicity has not been elucidated. We observed that upon incubation with Hcy, the viability of a neuroblastoma cell line Neuro2a declined in a dose-dependent manner, and apoptosis was induced within 48 h. The median effective concentration (EC50) of Hcy was approximately 5 mM. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) nuclear translocation and acylation has been implicated in the regulation of apoptosis. We found that nuclear translocation and acetylation of GAPDH increased in the presence of 5 mM Hcy and that higher levels of acetyltransferase p300/CBP were detected in Neuro2a cells. These findings implicate the involvement of GAPDH in the mechanism whereby Hcy induces apoptosis in neurons. This study highlights a potentially important pathway in neurodegenerative disorders, and a novel target pathway for neuroprotective therapy. PMID:26785692

  10. Genome-wide identification and characterization of Glyceraldehyde-3-phosphate dehydrogenase genes family in wheat (Triticum aestivum).

    PubMed

    Zeng, Lingfeng; Deng, Rong; Guo, Ziping; Yang, Shushen; Deng, Xiping

    2016-03-16

    Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a central enzyme in glycolysi, we performed genome-wide identification of GAPDH genes in wheat and analyzed their structural characteristics and expression patterns under abiotic stress in wheat. A total of 22 GAPDH genes were identified in wheat cv. Chinese spring; the phylogenetic and structure analysis showed that these GAPDH genes could be divided into four distinct subfamilies. The expression profiles of GAPDH genes showed tissue specificity all over plant development stages. The qRT-PCR results revealed that wheat GAPDHs were involved in several abiotic stress response. Wheat carried 22 GAPDH genes, representing four types of plant GAPDHs (gapA/B, gapC, gapCp and gapN). Whole genome duplication and segmental duplication might account for the expansion of wheat GAPDHs. Expression analysis implied that GAPDHs play roles in plants abiotic stress tolerance.

  11. Over-expression of PsGPD, a mushroom glyceraldehyde-3-phosphate dehydrogenase gene, enhances salt tolerance in rice plants.

    PubMed

    Cho, Jung-Il; Lim, Hye-Min; Siddiqui, Zamin Shaheed; Park, Sung-Han; Kim, A-Ram; Kwon, Taek-Ryoun; Lee, Seong-Kon; Park, Soo-Chul; Jeong, Mi-Jeong; Lee, Gang-Seob

    2014-08-01

    Transgenic potatoes expressing glyceraldehyde-3-phosphate dehydrogenase (GPD), isolated from the oyster mushroom, Pleurotus sajor-caju, had increased tolerance to salt stress (Jeong et al. Biochem Biophys Res Commun 278:192-196, 2000). To examine the physiological mechanisms enhancing salt tolerance in GPD-transgenic rice plants, the salt tolerance of five GPD transgenic rice lines (T1-T5) derived from Dongjin rice cultivar were evaluated in a fixed 150 mM saline environment in comparison to two known wild-type rice cultivars, Dongjin (salt sensitive) and Pokali (salt tolerant). Transgenic lines, T2, T3, and T5, had a substantial increase in biomass and relative water content compared to Dongjin. Stomatal conductance and osmotic potential were higher in the GPD transgenic lines and were similar to those in Pokali. The results are discussed based on the comparative physiological response of GPD transgenic lines with those of the salt-sensitive and salt-tolerant rice cultivars.

  12. Glyceraldehyde 3-phosphate dehydrogenase augments the intercellular transmission and toxicity of polyglutamine aggregates in a cell model of Huntington disease.

    PubMed

    Mikhaylova, Elena R; Lazarev, Vladimir F; Nikotina, Alina D; Margulis, Boris A; Guzhova, Irina V

    2016-03-01

    The common feature of Huntington disease is the accumulation of oligomers or aggregates of mutant huntingtin protein (mHTT), which causes the death of a subset of striatal neuronal populations. The cytotoxic species can leave neurons and migrate to other groups of cells penetrating and damaging them in a prion-like manner. We hypothesized that the glycolytic enzyme glyceraldehyde 3-phosphate dehydrogenase (GAPDH), previously shown to elevate the aggregation of mHTT, is associated with an increased efficiency of intercellular propagation of mHTT. GAPDH, on its own or together with polyglutamine species, was shown to be released into the extracellular milieu mainly from dying cells as assessed by a novel enzyme immunoassay, western blotting, and ultrafiltration. The conditioned medium of cells with growing GAPDH-polyQ aggregates was toxic to naïve cells, whereas depletion of the aggregates from the medium lowered this cytotoxicity. The GAPDH component of the aggregates was found to increase their toxicity by two-fold in comparison with polyQ alone. Furthermore, GAPDH-polyQ complexes were shown to penetrate acceptor cells and to increase the capacity of polyQ to prionize its intracellular homolog containing a repeat of 25 glutamine residues. Finally, inhibitors of intracellular transport showed that polyQ-GAPDH complexes, as well as GAPDH itself, penetrated cells using clathrin-mediated endocytosis. This suggested a pivotal role of the enzyme in the intercellular transmission of Huntington disease pathogenicity. In conclusion, GAPDH occurring in complexes with polyglutamine strengthens the prion-like activity and toxicity of the migrating aggregates. Aggregating polygluatmine tracts were shown to release from the cells over-expressing mutant huntingtin in a complex with glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The enzyme enhances the intracellular transport of aggregates to healthy cells, prionization of normal cellular proteins and finally cell death, thus

  13. Catalytic residues and an electrostatic sandwich that promote enolpyruvyl shikimate 3-phosphate synthase (AroA) catalysis.

    PubMed

    Berti, Paul J; Chindemi, Paul

    2009-05-05

    Enolpyruvylshikimate 3-phosphate synthase (EPSP synthase, AroA) catalyzes the sixth step in aromatic amino acid biosynthesis. It forms EPSP from shikimate 3-phosphate (S3P) and phosphoenolpyruvate (PEP) in an addition/elimination reaction that proceeds through a tetrahedral intermediate. In spite of numerous mechanistic studies, the catalytic roles of specific amino acid residues remain an open question. Recent experimental evidence for cationic intermediates or cationic transition states, and a consideration of the catalytic imperative, have guided this study on the catalytic roles of Lys22 (K22), Asp313 (D313), and Glu341 (E341). Steady-state and pre-steady-state kinetics and protein stability studies showed that mutations of D313 and E341 caused k(cat) to decrease up to 30,000-fold and 76,000-fold, respectively, while the effects on K(M) were modest, never more than 40-fold. Thus, both are identified as catalytic residues. In an active site that is overwhelmingly positively charged, the D313 and E341 side chains are positioned to form an "electrostatic sandwich" around the positive charge at C2 in cationic intermediates/transition states, stabilizing them and thereby promoting catalysis. Mutation of K22 showed large effects on K(M,S3P) (100-fold), K(M,PEP) (>760-fold), and up to 120-fold on k(cat). Thus, K22 had roles in both substrate-binding and transition-state stabilization. These results support the identification of E341 and K22 as general acid/base catalytic residues.

  14. Hydrogen Sulfide Regulates the Cytosolic/Nuclear Partitioning of Glyceraldehyde-3-Phosphate Dehydrogenase by Enhancing its Nuclear Localization.

    PubMed

    Aroca, Angeles; Schneider, Markus; Scheibe, Renate; Gotor, Cecilia; Romero, Luis C

    2017-06-01

    Hydrogen sulfide is an important signaling molecule comparable with nitric oxide and hydrogen peroxide in plants. The underlying mechanism of its action is unknown, although it has been proposed to be S-sulfhydration. This post-translational modification converts the thiol groups of cysteines within proteins to persulfides, resulting in functional changes of the proteins. In Arabidopsis thaliana, S-sulfhydrated proteins have been identified, including the cytosolic isoforms of glyceraldehyde-3-phosphate dehydrogenase GapC1 and GapC2. In this work, we studied the regulation of sulfide on the subcellular localization of these proteins using two different approaches. We generated GapC1-green fluorescent protein (GFP) and GapC2-GFP transgenic plants in both the wild type and the des1 mutant defective in the l-cysteine desulfhydrase DES1, responsible for the generation of sulfide in the cytosol. The GFP signal was detected in the cytoplasm and the nucleus of epidermal cells, although with reduced nuclear localization in des1 compared with the wild type, and exogenous sulfide treatment resulted in similar signals in nuclei in both backgrounds. The second approach consisted of the immunoblot analysis of the GapC endogenous proteins in enriched nuclear and cytosolic protein extracts, and similar results were obtained. A significant reduction in the total amount of GapC in des1 in comparison with the wild type was determined and exogenous sulfide significantly increased the protein levels in the nuclei in both plants, with a stronger response in the wild type. Moreover, the presence of an S-sulfhydrated cysteine residue on GapC1 was demonstrated by mass spectrometry. We conclude that sulfide enhances the nuclear localization of glyceraldehyde-3-phosphate dehydrogenase. © The Author 2017. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

  15. cDNA, genomic sequence cloning and overexpression of glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH) from the Giant Panda.

    PubMed

    Hou, Wan-Ru; Hou, Yi-Ling; Du, Yu-Jie; Zhang, Tian; Hao, Yan-Zhe

    2010-01-01

    GAPDH (glyceraldehyde-3-phosphate dehydrogenase) is a key enzyme of the glycolytic pathway and it is related to the occurrence of some diseases. The cDNA and the genomic sequence of GAPDH were cloned successfully from the Giant Panda (Ailuropoda melanoleuca) using the RT-PCR technology and Touchdown-PCR, respectively. Both sequences were analyzed preliminarily. The cDNA of GAPDH cloned from the Giant Panda is 1191 bp in size, contains an open reading frame of 1002 bp encoding 333 amino acids. The genomic sequence is 3941 bp in length and was found to possess 10 exons and 9 introns. Alignment analysis indicates that the nucleotide sequence and the deduced amino acid sequence are highly conserved in some mammalian species, including Homo sapiens, Mu musculus, Rattus norvegicus, Canis lupus familiaris and Bos taurus. The homologies for the nucleotide sequences of the Giant Panda GAPDH to that of these species are 90.67, 90.92, 90.62, 95.01 and 92.32% respectively, while the homologies for the amino acid sequences are 94.93, 95.5, 95.8, 98.8 and 97.0%. Primary structure analysis revealed that the molecular weight of the putative GAPDH protein is 35.7899 kDa with a theoretical pI of 8.21. Topology prediction showed that there is one Glyceraldehyde 3-phosphate dehydrogenase active site, two N-glycosylation sites, four Casein kinase II phosphorylation sites, seven Protein kinase C phosphorylation sites and eight N-myristoylation sites in the GAPDH protein of the Giant Panda. The GAPDH gene was overexpressed in E. coli BL21. The results indicated that the fusion of GAPDH with the N-terminally His-tagged form gave rise to the accumulation of an expected 43 kDa polypeptide. The SDS-PAGE analysis also showed that the recombinant GAPDH was soluble and thus could be used for further functional studies.

  16. The role of glycerol-3-phosphate dehydrogenase 1 in the progression of fatty liver after acute ethanol administration in mice.

    PubMed

    Sato, Tomoki; Morita, Akihito; Mori, Nobuko; Miura, Shinji

    2014-02-21

    Acute ethanol consumption leads to the accumulation of triglycerides (TGs) in hepatocytes. The increase in lipogenesis and reduction of fatty acid oxidation are implicated as the mechanisms underlying ethanol-induced hepatic TG accumulation. Although glycerol-3-phosphate (Gro3P), formed by glycerol kinase (GYK) or glycerol-3-phosphate dehydrogenase 1 (GPD1), is also required for TG synthesis, the roles of GYK and GPD1 have been the subject of some debate. In this study, we examine (1) the expression of genes involved in Gro3P production in the liver of C57BL/6J mice in the context of hepatic TG accumulation after acute ethanol intake, and (2) the role of GPD1 in the progression of ethanol-induced fatty liver using GPD1 null mice. As a result, in C57BL/6J mice, ethanol-induced hepatic TG accumulation began within 2h and was 1.7-fold greater than that observed in the control group after 6h. The up-regulation of GPD1 began 2h after administering ethanol, and significantly increased 6h later with the concomitant escalation in the glycolytic gene expression. The incorporation of (14)C-labelled glucose into TG glycerol moieties increased during the same period. On the other hand, in GPD1 null mice carrying normal GYK activity, no significant increase in hepatic TG level was observed after acute ethanol intake. In conclusion, GPD1 and glycolytic gene expression is up-regulated by ethanol, and GPD1-mediated incorporation of glucose into TG glycerol moieties together with increased lipogenesis, is suggested to play an important role in ethanol-induced hepatic TG accumulation.

  17. Purification and properties of glycerol-3-phosphate dehydrogenase from the liver of the hibernating ground squirrel, Urocitellus richardsonii.

    PubMed

    Ruberto, Anthony A; Childers, Christine L; Storey, Kenneth B

    2016-12-01

    Cytosolic glycerol-3-phosphate dehydrogenase (G3PDH, EC 1.1.1.8) is an important branch point enzyme connecting lipid metabolism and carbohydrate metabolism. We investigated the dynamic nature of G3PDH by purifying the enzyme from the liver of Richardson's ground squirrel (Urocitellus richardsonii), a hibernating species, and analyzing its structural and functional changes during hibernation. Kinetic parameters of purified G3PDH from ground squirrel liver were characterized at 37, 22 and 5°C and compared between euthermic and hibernating states. Relative to euthermic liver G3PDH, hibernator liver G3PDH had a decreased affinity for its substrate, glycerol-3-phosphate (G3P), at 37°C and 22°C. However, at 5°C, there was a significant increase in the affinity for G3P in the hibernating form of the enzyme, relative to the euthermic form. Furthermore, the structure of G3PDH in the species' hibernating state showed greater thermal stability compared to its structure in the euthermic state. Western blot analysis revealed greater tyrosine phosphorylation in hibernator G3PDH as compared to euthermic G3PDH. In addition, using the protein sequence of the hibernating thirteen-lined ground squirrel (Ictidomys tridecemlineatus) and bioinformatics tools, a three-dimensional model of G3PDH was built to identify the potential phosphorylation site ((83)Tyr) responsible for the differential phosphorylation between euthermic and hibernator G3PDH. The structural and functional changes in G3PDH support the enzyme's function at a low core body temperature experienced during the species hibernating season. Copyright © 2016 Elsevier Inc. All rights reserved.

  18. Expression, purification, crystallization and preliminary X-ray analysis of an NADP-dependent glyceraldehyde-3-phosphate dehydrogenase from Helicobacter pylori

    SciTech Connect

    Elliott, Paul R.; Evans, Daniel; Greenwood, Jacqueline A.; Moody, Peter C. E.

    2008-08-01

    Glyceraldehyde-3-phosphate dehydrogenase A has been cloned, expressed and purified. Apoprotein crystals have been grown which diffracted to 1.75 Å resolution and belonged to space group P2{sub 1}; holo crystals were grown in the presence of NADP, diffracted to 2.6 Å resolution and belonged to space group P3{sub 2}. The classical glycolytic pathway contains an NAD-dependent glyceraldehyde-3-phosphate dehydrogenase, with NADP-dependent forms reserved for photosynthetic organisms and archaea. Here, the cloning, expression, purification, crystallization and preliminary X-ray analysis of an NADP-dependent glyceraldehyde-3-phosphate dehydrogenase from Helicobacter pylori is reported; crystals of the protein were grown both in the presence and the absence of NADP.

  19. Photolabeling identifies an interaction between phosphatidylcholine and glycerol-3-phosphate dehydrogenase (Gut2p) in yeast mitochondria.

    PubMed

    Janssen, Marjolein J F W; van Voorst, Frank; Ploeger, Ginette E J; Larsen, Peter Mose; Larsen, Martin R; de Kroon, Anton I P M; de Kruijff, Ben

    2002-05-07

    In search of mitochondrial proteins interacting with phosphatidylcholine (PC), a photolabeling approach was applied, in which photoactivatable probes were incorporated into isolated yeast mitochondria. Only a limited number of proteins were labeled upon photoactivation, using either the PC analogue [125I]TID-PC or the small hydrophobic probe [125I]TID-BE. The most prominent difference was the very specific labeling of a 70 kDa protein by [125I]TID-PC. Mass spectrometric analysis of a tryptic digest of the corresponding 2D-gel spot identified the protein as the GUT2 gene product, the FAD-dependent mitochondrial glycerol-3-phosphate dehydrogenase. This was confirmed by the lack of specific labeling in mitochondria from a gut2 deletion strain. Only under conditions where the inner membrane was accessible to the probe, Gut2p was labeled by [125I]TID-PC, in parallel with increased labeling of the phosphate carrier (P(i)C) in the inner membrane. A hemagglutinin-tagged version of Gut2p was shown to be membrane-bound. Carbonate extraction released the protein from the membrane, whereas a high concentration of NaCl did not, demonstrating that Gut2p is a peripheral membrane protein bound to the inner membrane via hydrophobic interactions. The significance of the observed interactions between Gut2p and PC is discussed.

  20. Glycerol-3-Phosphate Acyltransferase Contributes to Triacylglycerol Biosynthesis, Lipid Droplet Formation, and Host Invasion in Metarhizium robertsii

    PubMed Central

    Gao, Qiang; Shang, Yanfang; Huang, Wei

    2013-01-01

    Enzymes involved in the triacylglycerol (TAG) biosynthesis have been well studied in the model organisms of yeasts and animals. Among these, the isoforms of glycerol-3-phosphate acyltransferase (GPAT) redundantly catalyze the first and rate-limiting step in glycerolipid synthesis. Here, we report the functions of mrGAT, a GPAT ortholog, in an insect-pathogenic fungus, Metarhizium robertsii. Unlike in yeasts and animals, a single copy of the mrGAT gene is present in the fungal genome and the gene deletion mutant is viable. Compared to the wild type and the gene-rescued mutant, the ΔmrGAT mutant demonstrated reduced abilities to produce conidia and synthesize TAG, glycerol, and total lipids. More importantly, we found that mrGAT is localized to the endoplasmic reticulum and directly linked to the formation of lipid droplets (LDs) in fungal cells. Insect bioassay results showed that mrGAT is required for full fungal virulence by aiding fungal penetration of host cuticles. Data from this study not only advance our understanding of GPAT functions in fungi but also suggest that filamentous fungi such as M. robertsii can serve as a good model to elucidate the role of the glycerol phosphate pathway in fungal physiology, particularly to determine the mechanistic connection of GPAT to LD formation. PMID:24077712

  1. Isolation and properties of cytoplasmic alpha-glycerol 3-phosphate dehydrogenase from the pectoral muscle of the fruit bat, Eidolon helvum.

    PubMed

    Agboola, Femi Kayode; Thomson, Alan; Afolayan, Adeyinka

    2003-03-31

    Cytoplasmic alpha-glycerol-3-phosphate dehydrogenase from fruit-bat-breast muscle was purified by ion-exchange and affinity chromatography. The specific activity of the purified enzyme was approximately 120 units/mg of protein. The apparent molecular weight of the native enzyme, as determined by gel filtration on Sephadex G-100 was 59,500 +/- 650 daltons; its subunit size was estimated to be 35,700 +/- 140 by SDS-polyacrylamide gel electrophoresis. The true Michaelis-Menten constants for all substrates at pH 7.5 were 3.9 +/- 0.7 mM, 0.65 +/- 0.05 mM, 0.26 +/- 0.06 mM, and 0.005 +/- 0.0004 mM for L-glycerol-3-phosphate, NAD(+), DHAP, and NADH, respectively. The true Michaelis-Menten constants at pH 10.0 were 2.30 +/- 0.21 mM and 0.20 +/- 0.01 mM for L-glycerol-3-phosphate and NAD(+), respectively. The turnover number, k(cat), of the forward reaction was 1.9 +/- 0.2 x 10(4)s(-1). The treatment of the enzyme with 5,5'-dithiobis-2-nitrobenzoic acid (DTNB) under denaturing conditions indicated that there were a total of eight cysteine residues, while only two of these residues were reactive towards DTNB in the native enzyme. The overall results of the in vitro experiments suggest that alpha-glycerol-3-phosphate dehydrogenase of the fruit bat preferentially catalyses the reduction of dihydroxyacetone phosphate to glycerol-3-phosphate.

  2. Modeling of Glycerol-3-Phosphate Transporter Suggests a Potential ‘Tilt’ Mechanism involved in its Function

    PubMed Central

    Tsigelny, Igor F.; Greenberg, Jerry; Kouznetsova, Valentina; Nigam, Sanjay K.

    2009-01-01

    Many major facilitator superfamily (MFS) transporters have similar 12-transmembrane α-helical topologies with two six-helix halves connected by a long loop. In humans, these transporters participate in key physiological processes and are also, as in the case of members of the organic anion transporter (OAT) family, of pharmaceutical interest. Recently, crystal structures of two bacterial representatives of the MFS family — the glycerol-3-phosphate transporter (GlpT) and lac-permease (LacY) — have been solved and, because of assumptions regarding the high structural conservation of this family, there is hope that the results can be applied to mammalian transporters as well. Based on crystallography, it has been suggested that a major conformational “switching” mechanism accounts for ligand transport by MFS proteins. This conformational switch would then allow periodic changes in the overall transporter configuration, resulting in its cyclic opening to the periplasm or cytoplasm. Following this lead, we have modeled a possible “switch” mechanism in GlpT, using the concept of rotation of protein domains as in the DynDom program17 and membranephilic constraints predicted by the MAPAS program.23 We found that the minima of energies of intersubunit interactions support two alternate positions consistent with their transport properties. Thus, for GlpT, a “tilt” of 9°–10° rotation had the most favorable energetics of electrostatic interaction between the two halves of the transporter; moreover, this confirmation was sufficient to suggest transport of the ligand across the membrane. We conducted steered molecular dynamics simulations of the GlpT-ligand system to explore how glycerol-3-phosphate would be handled by the “tilted” structure, and obtained results generally consistent with experimental mutagenesis data. While biochemical data remain most consistent with a single-site alternating access model, our results raise the possibility that, while

  3. The role of glycerol-3-phosphate dehydrogenase 1 in the progression of fatty liver after acute ethanol administration in mice

    SciTech Connect

    Sato, Tomoki; Morita, Akihito; Mori, Nobuko; Miura, Shinji

    2014-02-21

    Highlights: • Ethanol administration increased GPD1 mRNA expression. • Ethanol administration increased glucose incorporation into TG glycerol moieties. • No increase in hepatic TG levels was observed in ethanol-injected GPD1 null mice. • We propose that GPD1 is required for ethanol-induced TG accumulation in the liver. - Abstract: Acute ethanol consumption leads to the accumulation of triglycerides (TGs) in hepatocytes. The increase in lipogenesis and reduction of fatty acid oxidation are implicated as the mechanisms underlying ethanol-induced hepatic TG accumulation. Although glycerol-3-phosphate (Gro3P), formed by glycerol kinase (GYK) or glycerol-3-phosphate dehydrogenase 1 (GPD1), is also required for TG synthesis, the roles of GYK and GPD1 have been the subject of some debate. In this study, we examine (1) the expression of genes involved in Gro3P production in the liver of C57BL/6J mice in the context of hepatic TG accumulation after acute ethanol intake, and (2) the role of GPD1 in the progression of ethanol-induced fatty liver using GPD1 null mice. As a result, in C57BL/6J mice, ethanol-induced hepatic TG accumulation began within 2 h and was 1.7-fold greater than that observed in the control group after 6 h. The up-regulation of GPD1 began 2 h after administering ethanol, and significantly increased 6 h later with the concomitant escalation in the glycolytic gene expression. The incorporation of {sup 14}C-labelled glucose into TG glycerol moieties increased during the same period. On the other hand, in GPD1 null mice carrying normal GYK activity, no significant increase in hepatic TG level was observed after acute ethanol intake. In conclusion, GPD1 and glycolytic gene expression is up-regulated by ethanol, and GPD1-mediated incorporation of glucose into TG glycerol moieties together with increased lipogenesis, is suggested to play an important role in ethanol-induced hepatic TG accumulation.

  4. Evidence for a reactive gamma-carboxyl group (Glu-418) at the herbicide glyphosate binding site of 5-enolpyruvylshikimate-3-phosphate synthase from Escherichia coli.

    PubMed

    Huynh, Q K

    1988-08-25

    Incubation of 5-enolpyruvylshikimate-3-phosphate synthase, a target for the nonselective herbicide glyphosate (N-(phosphonomethyl)glycine), with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide in the presence of glycine ethyl ester resulted in a time-dependent loss of enzyme activity. The inactivation followed pseudo-first order kinetics, with a second order rate constant of 2.2 M-1 min-1 at pH 5.5 and 25 degrees C. The inactivation is prevented by preincubation of the enzyme with a combination of the substrate shikimate 3-phosphate plus glyphosate, but not by shikimate 3-phosphate, phosphoenolpyruvate, or glyphosate alone. Increasing the concentration of glyphosate during preincubation resulted in decreasing the rate of inactivation of the enzyme. Complete inactivation of the enzyme required the modification of 4 carboxyl groups per molecule of the enzyme. However, statistical analysis of the residual activity and the extent of modification showed that among the 4 modifiable carboxyl groups, only 1 is critical for activity. Tryptic mapping of the enzyme modified in the absence of shikimate 3-phosphate and glyphosate by reverse phase chromatography resulted in the isolation of a [14C]glycine ethyl ester-containing peptide that was absent in the enzyme modified in the presence of shikimate 3-phosphate and glyphosate. By amino acid sequencing of this labeled peptide, the modified critical carboxyl group was identified as Glu-418. The above results suggest that Glu-418 is the most accessible reactive carboxyl group under these conditions and is located at or close to the glyphosate binding site.

  5. The sequential 2',3'-cyclic phosphodiesterase and 3'-phosphate/5'-OH ligation steps of the RtcB RNA splicing pathway are GTP-dependent.

    PubMed

    Chakravarty, Anupam K; Shuman, Stewart

    2012-09-01

    The RNA ligase RtcB splices broken RNAs with 5'-OH and either 2',3'-cyclic phosphate or 3'-phosphate ends. The 3'-phosphate ligase activity requires GTP and entails the formation of covalent RtcB-(histidinyl)-GMP and polynucleotide-(3')pp(5')G intermediates. There are currently two models for how RtcB executes the strand sealing step. Scheme 1 holds that the RNA 5'-OH end attacks the 3'-phosphorus of the N(3')pp(5')G end to form a 3',5'-phosphodiester and release GMP. Scheme 2 posits that the N(3')pp(5')G end is converted to a 2',3'-cyclic phosphodiester, which is then attacked directly by the 5'-OH RNA end to form a 3',5'-phosphodiester. Here we show that the sealing of a 2',3'-cyclic phosphate end by RtcB requires GTP, is contingent on formation of the RtcB-GMP adduct, and involves a kinetically valid RNA(3')pp(5')G intermediate. Moreover, we find that RtcB catalyzes the hydrolysis of a 2',3'-cyclic phosphate to a 3'-phosphate at a rate that is at least as fast as the rate of ligation. These results weigh in favor of scheme 1. The cyclic phosphodiesterase activity of RtcB depends on GTP and the formation of the RtcB-GMP adduct, signifying that RtcB guanylylation precedes the cyclic phosphodiesterase and 3'-phosphate ligase steps of the RNA splicing pathway.

  6. Glycerol-3-phosphate acyltransferase 4 is essential for the normal development of reproductive organs and the embryo in Brassica napus.

    PubMed

    Chen, Xue; Chen, Guanqun; Truksa, Martin; Snyder, Crystal L; Shah, Saleh; Weselake, Randall J

    2014-08-01

    The enzyme sn-glycerol-3-phosphate acyltransferase 4 (GPAT4) is involved in the biosynthesis of plant lipid poly-esters. The present study further characterizes the enzymatic activities of three endoplasmic reticulum-bound GPAT4 isoforms of Brassica napus and examines their roles in the development of reproductive organs and the embryo. All three BnGPAT4 isoforms exhibited sn-2 acyltransferase and phosphatase activities with dicarboxylic acid-CoA as acyl donor. When non-substituted acyl-CoA was used as acyl donor, the rate of acylation was considerably lower and phosphatase activity was not manifested. RNA interference (RNAi)-mediated down-regulation of all GPAT4 homologues in B. napus under the control of the napin promoter caused abnormal development of several reproductive organs and reduced seed set. Microscopic examination and reciprocal crosses revealed that both pollen grains and developing embryo sacs of the B. napus gpat4 lines were affected. The gpat4 mature embryos showed decreased cutin content and altered monomer composition. The defective embryo development further affected the oil body morphology, oil content, and fatty acid composition in gpat4 seeds. These results suggest that GPAT4 has a critical role in the development of reproductive organs and the seed of B. napus. © The Author 2014. Published by Oxford University Press on behalf of the Society for Experimental Biology.

  7. Export of malaria proteins requires co-translational processing of the PEXEL motif independent of phosphatidylinositol-3-phosphate binding

    PubMed Central

    Boddey, Justin A.; O'Neill, Matthew T.; Lopaticki, Sash; Carvalho, Teresa G.; Hodder, Anthony N.; Nebl, Thomas; Wawra, Stephan; van West, Pieter; Ebrahimzadeh, Zeinab; Richard, Dave; Flemming, Sven; Spielmann, Tobias; Przyborski, Jude; Babon, Jeff J.; Cowman, Alan F.

    2016-01-01

    Plasmodium falciparum exports proteins into erythrocytes using the Plasmodium export element (PEXEL) motif, which is cleaved in the endoplasmic reticulum (ER) by plasmepsin V (PMV). A recent study reported that phosphatidylinositol-3-phosphate (PI(3)P) concentrated in the ER binds to PEXEL motifs and is required for export independent of PMV, and that PEXEL motifs are functionally interchangeable with RxLR motifs of oomycete effectors. Here we show that the PEXEL does not bind PI(3)P, and that this lipid is not concentrated in the ER. We find that RxLR motifs cannot mediate export in P. falciparum. Parasites expressing a mutated version of KAHRP, with the PEXEL motif repositioned near the signal sequence, prevented PMV cleavage. This mutant possessed the putative PI(3)P-binding residues but is not exported. Reinstatement of PEXEL to its original location restores processing by PMV and export. These results challenge the PI(3)P hypothesis and provide evidence that PEXEL position is conserved for co-translational processing and export. PMID:26832821

  8. Constitutive Optimized Production of Streptokinase in Saccharomyces cerevisiae Utilizing Glyceraldehyde 3-Phosphate Dehydrogenase Promoter of Pichia pastoris

    PubMed Central

    Vellanki, Ravi N.; Doddapaneni, Kiran K.; Anubrolu, Naveen; Mangamoori, Lakshmi N.

    2013-01-01

    A novel expression vector constructed from genes of Pichia pastoris was applied for heterologous gene expression in Saccharomyces cerevisiae. Recombinant streptokinase (SK) was synthesized by cloning the region encoding mature SK under the control of glyceraldehyde 3-phosphate dehydrogenase (GAP) promoter of Pichia pastoris in Saccharomyces cerevisiae. SK was intracellularly expressed constitutively, as evidenced by lyticase-nitroanilide and caseinolytic assays. The functional activity was confirmed by plasminogen activation assay and in vitro clot lysis assay. Stability and absence of toxicity to the host with the recombinant expression vector as evidenced by southern analysis and growth profile indicate the application of this expression system for large-scale production of SK. Two-stage statistical approach, Plackett-Burman (PB) design and response surface methodology (RSM) was used for SK production medium optimization. In the first stage, carbon and organic nitrogen sources were qualitatively screened by PB design and in the second stage there was quantitative optimization of four process variables, yeast extract, dextrose, pH, and temperature, by RSM. PB design resulted in dextrose and peptone as best carbon and nitrogen sources for SK production. RSM method, proved as an efficient technique for optimizing process conditions which resulted in 110% increase in SK production, 2352 IU/mL, than for unoptimized conditions. PMID:24171161

  9. Early hypergravity exposure effects calbindin-D28k and inositol-3-phosphate expression in Purkinje cells.

    PubMed

    Bouët, Valentine; Dijk, Freark; Ijkema-Paassen, Jos; Wubbels, René J; van der Want, Johannes J; Gramsbergen, Albert

    In this study the effects of hypergravity were analyzed on cerebellar Purkinje cells during early development in rats. The cerebellum is a key structure in the control and the adaptation of posture and anti-gravity activities. This holds particularly when external conditions are modified. Three groups of rats were conceived, born and reared in hypergravity (2g). At postnatal day 5 (P5), P10 or P15, they were exposed to normal gravity and at P40, the cerebella were investigated on the expression of calbindin-D28k and inositol-3-phosphate (IP3) in Purkinje cells. Control animals were bred in the same conditions but at 1g. Immunoreactivity of Purkinje cells was studied in lobules III and IX of the vermis. Lobule IX of the vermis is one of the targets of primary otolithic vestibular projections, and lobule III served as a control, being much less related with vestibular inputs. The results show that hypergravity induces a decrease in calbindin and IP3 labeling in 20% of Purkinje cells of lobule IX without any change in lobule III. Animals transferred from 2g to 1g at P5 or P10 showed the most pronounced effects and much less at P15. This study demonstrates that early development of the cerebellum is highly sensitive to changes in gravity. Ages until P10 are critical for the development of vestibulo-cerebellar connections, and in particularly the calcium signaling in Purkinje cells.

  10. Glycerol-3-phosphate acyltransferase 4 is essential for the normal development of reproductive organs and the embryo in Brassica napus

    PubMed Central

    Chen, Xue; Chen, Guanqun; Truksa, Martin; Snyder, Crystal L.; Shah, Saleh; Weselake, Randall J.

    2014-01-01

    The enzyme sn-glycerol-3-phosphate acyltransferase 4 (GPAT4) is involved in the biosynthesis of plant lipid poly-esters. The present study further characterizes the enzymatic activities of three endoplasmic reticulum-bound GPAT4 isoforms of Brassica napus and examines their roles in the development of reproductive organs and the embryo. All three BnGPAT4 isoforms exhibited sn-2 acyltransferase and phosphatase activities with dicarboxylic acid-CoA as acyl donor. When non-substituted acyl-CoA was used as acyl donor, the rate of acylation was considerably lower and phosphatase activity was not manifested. RNA interference (RNAi)-mediated down-regulation of all GPAT4 homologues in B. napus under the control of the napin promoter caused abnormal development of several reproductive organs and reduced seed set. Microscopic examination and reciprocal crosses revealed that both pollen grains and developing embryo sacs of the B. napus gpat4 lines were affected. The gpat4 mature embryos showed decreased cutin content and altered monomer composition. The defective embryo development further affected the oil body morphology, oil content, and fatty acid composition in gpat4 seeds. These results suggest that GPAT4 has a critical role in the development of reproductive organs and the seed of B. napus. PMID:24821955

  11. Characterization of the highly active fragment of glyceraldehyde-3-phosphate dehydrogenase gene promoter for recombinant protein expression in Pleurotus ostreatus.

    PubMed

    Yin, Chaomin; Zheng, Liesheng; Zhu, Jihong; Chen, Liguo; Ma, Aimin

    2015-03-01

    Developing efficient native promoters is important for improving recombinant protein expression by fungal genetic engineering. The promoter region of glyceraldehyde-3-phosphate dehydrogenase gene in Pleurotus ostreatus (Pogpd) was isolated and optimized by upstream truncation. The activities of these promoters with different lengths were further confirmed by fluorescence, quantitative real-time PCR and Western blot analysis. A truncated Pogpd-P2 fragment (795 bp) drove enhanced green fluorescence protein (egfp) gene expression in P. ostreatus much more efficiently than full-length Pogpd-P1. Further truncating Pogpd-P2 to 603, 403 and 231 bp reduced the eGFP expression significantly. However, the 403-bp fragment between -356 bp and the start codon was the minimal but sufficient promoter element for eGFP expression. Compact native promoters for genetic engineering of P. ostreatus were successfully developed and validated in this study. This will broaden the preexisting repertoire of fungal promoters for biotechnology application. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  12. Phosphatidylinositol 3-phosphate 5-kinase (PIKfyve) is an AMPK target participating in contraction-stimulated glucose uptake in skeletal muscle.

    PubMed

    Liu, Yang; Lai, Yu-Chiang; Hill, Elaine V; Tyteca, Donatienne; Carpentier, Sarah; Ingvaldsen, Ada; Vertommen, Didier; Lantier, Louise; Foretz, Marc; Dequiedt, Franck; Courtoy, Pierre J; Erneux, Christophe; Viollet, Benoît; Shepherd, Peter R; Tavaré, Jeremy M; Jensen, Jørgen; Rider, Mark H

    2013-10-15

    PIKfyve (FYVE domain-containing phosphatidylinositol 3-phosphate 5-kinase), the lipid kinase that phosphorylates PtdIns3P to PtdIns(3,5)P2, has been implicated in insulin-stimulated glucose uptake. We investigated whether PIKfyve could also be involved in contraction/AMPK (AMP-activated protein kinase)-stimulated glucose uptake in skeletal muscle. Incubation of rat epitrochlearis muscles with YM201636, a selective PIKfyve inhibitor, reduced contraction- and AICAriboside (5-amino-4-imidazolecarboxamide riboside)-stimulated glucose uptake. Consistently, PIKfyve knockdown in C2C12 myotubes reduced AICAriboside-stimulated glucose transport. Furthermore, muscle contraction increased PtdIns(3,5)P2 levels and PIKfyve phosphorylation. AMPK phosphorylated PIKfyve at Ser307 both in vitro and in intact cells. Following subcellular fractionation, PIKfyve recovery in a crude intracellular membrane fraction was increased in contracting versus resting muscles. Also in opossum kidney cells, wild-type, but not S307A mutant, PIKfyve was recruited to endosomal vesicles in response to AMPK activation. We propose that PIKfyve activity is required for the stimulation of skeletal muscle glucose uptake by contraction/AMPK activation. PIKfyve is a new AMPK substrate whose phosphorylation at Ser307 could promote PIKfyve translocation to endosomes for PtdIns(3,5)P2 synthesis to facilitate GLUT4 (glucose transporter 4) translocation.

  13. Increased mitochondrial glycerol-3-phosphate acyltransferase protein and enzyme activity in rat epididymal fat upon cessation of wheel running.

    PubMed

    Kump, David S; Laye, Matthew J; Booth, Frank W

    2006-03-01

    Triacylglycerol synthesis in rat epididymal fat overshoots sedentary levels at 10, 29, and 53 h of physical inactivity after 21 days of wheel running. The purposes of the present study were to determine 1) whether this effect is also observed after an acute bout of physical activity and 2) what enzymatic changes might contribute to this effect. We show that more than one bout of physical activity, such as that which occurs with 21 days of wheel running, is necessary for palmitic acid incorporation into triacylglyceride (triglyceride synthesis) to overshoot sedentary values, which suggests that pretranslational mechanisms may be responsible for this overshoot effect. Ten hours after 21 days of wheel running, activity of the mitochondrial glycerol-3-phosphate acyltransferase-1 (mtGPAT1) isoform, a key regulator of triacylglycerol synthesis, overshot sedentary values by 48% and remained higher than sedentary values at 29 and 53 h of reduced physical activity. The overshoot in mtGPAT1 activity was accompanied by an increase in mtGPAT protein level. Cyclic AMP response element-binding protein-binding protein level was higher in sedentary 29 h after 21 days of wheel running. AMP kinase-alpha Thr(172) phosphorylation was increased immediately after treadmill running, but decreased to sedentary values by 5 h after activity. Casein kinase-2alpha protein level and activity were unchanged. We conclude that an increase in mtGPAT protein might contribute to the overshoot in triacylglycerol synthesis.

  14. Glycerol-3-phosphate O-acyltransferase is required for PBAN-induced sex pheromone biosynthesis in Bombyx mori.

    PubMed

    Du, Mengfang; Liu, Xiaoguang; Liu, Xiaoming; Yin, Xinming; Han, Shuangyin; Song, Qisheng; An, Shiheng

    2015-01-29

    Female moths employ their own pheromone blends as a communicational medium in mating behavior. The biosynthesis and release of sex pheromone in female moths are regulated by pheromone biosynthesis activating neuropeptide (PBAN) and the corresponding action of PBAN has been well elucidated in Bombyx mori. However, very little is known about the molecular mechanism regarding the biosynthesis of sex pheromone precursor. In this study, quantitative proteomics was utilized to comprehensively elucidate the expression dynamics of pheromone glands (PGs) during development. Proteomic analysis revealed a serial of differentially expressed sex pheromone biosynthesis-associated proteins at the different time points of B. mori development. Most interestingly B. mori glycerol-3-phosphate O-acyltransferase (BmGPAT) was found to be expressed during the key periods of sex pheromone biosynthesis. RNAi knockdown of BmGPAT confirmed the important function of this protein in the biosynthesis of sex pheromone precursor, triacylglcerol (TAG), and subsequently PBAN-induced production of sex pheromone, bombykol. Behavioral analysis showed that RNAi knockdown of GPAT significantly impaired the ability of females to attract males. Our findings indicate that GPAT acts to regulate the biosynthesis of sex pheromone precursor, TAG, thus influencing PBAN-induced sex pheromone production and subsequent mating behavior.

  15. Glyphosate-resistant goosegrass. Identification of a mutation in the target enzyme 5-enolpyruvylshikimate-3-phosphate synthase.

    PubMed

    Baerson, Scott R; Rodriguez, Damian J; Tran, Minhtien; Feng, Yongmei; Biest, Nancy A; Dill, Gerald M

    2002-07-01

    The spontaneous occurrence of resistance to the herbicide glyphosate in weed species has been an extremely infrequent event, despite over 20 years of extensive use. Recently, a glyphosate-resistant biotype of goosegrass (Eleusine indica) was identified in Malaysia exhibiting an LD(50) value approximately 2- to 4-fold greater than the sensitive biotype collected from the same region. A comparison of the inhibition of 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) activity by glyphosate in extracts prepared from the resistant (R) and sensitive (S) biotypes revealed an approximately 5-fold higher IC(50)(glyphosate) for the (R) biotype. Sequence comparisons of the predicted EPSPS mature protein coding regions from both biotypes revealed four single-nucleotide differences, two of which result in amino acid changes. One of these changes, a proline to serine substitution at position 106 in the (R) biotype, corresponds to a substitution previously identified in a glyphosate-insensitive EPSPS enzyme from Salmonella typhimurium. Kinetic data generated for the recombinant enzymes suggests that the second substitution identified in the (R) EPSPS does not contribute significantly to its reduced glyphosate sensitivity. Escherichia coli aroA- (EPSPS deficient) strains expressing the mature EPSPS enzyme from the (R) biotype exhibited an approximately 3-fold increase in glyphosate tolerance relative to strains expressing the mature EPSPS from the (S) biotype. These results provide the first evidence for an altered EPSPS enzyme as an underlying component of evolved glyphosate resistance in any plant species.

  16. Glyceraldehyde 3-phosphate dehydrogenase protein and mRNA are both differentially expressed in adult chickens but not chick embryos.

    PubMed Central

    Milner, R J; Brow, M D; Cleveland, D W; Shinnick, T M; Sutcliffe, J G

    1983-01-01

    We have determined the 679 nucleotide sequence of a cDNA clone which, by hybridization-translation experiments, corresponds to a 36K chick brain protein. Our studies provide a partial amino acid sequence for this protein, identifying it as chicken glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Antisera raised against purified chicken GAPDH reacted with a 36K protein present in chick brain extracts and estimated to be the fourth most prevalent protein, as determined by either Coomassie Blue staining or by in vitro translation of chick brain mRNA. The amounts of GAPDH mRNA in chick brain, liver and muscle and adult chicken brain are similar, whereas the relative amount of adult chicken muscle GPDH mRNA is greatly elevated and that of adult liver lowered. The GAPDH protein levels showed a similar variation between tissues, suggesting that the levels of GAPDH protein are largely regulated by the amount of available GAPDH mRNA. The chicken GAPDH clone does not hybridize to rat mRNA, even though GAPDH is one of the most evolutionarily conserved proteins, indicating that selection pressures are heavier at the primary protein sequence level than at the nucleic acid sequence level for this gene, a situation contrasting to that of the tubulins. Images PMID:6687938

  17. SIRT1 interacts with and protects glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from nuclear translocation: Implications for cell survival after irradiation

    SciTech Connect

    Joo, Hyun-Yoo; Woo, Seon Rang; Shen, Yan-Nan; Yun, Mi Yong; Shin, Hyun-Jin; Park, Eun-Ran; Kim, Su-Hyeon; Park, Jeong-Eun; Ju, Yeun-Jin; Hong, Sung Hee; Hwang, Sang-Gu; Cho, Myung-Haing; Kim, Joon; Lee, Kee-Ho

    2012-08-10

    Highlights: Black-Right-Pointing-Pointer SIRT1 serves to retain GAPDH in the cytosol, preventing GAPDH nuclear translocation. Black-Right-Pointing-Pointer When SIRT1 is depleted, GAPDH translocation occurs even in the absence of stress. Black-Right-Pointing-Pointer Upon irradiation, SIRT1 interacts with GAPDH. Black-Right-Pointing-Pointer SIRT1 prevents irradiation-induced nuclear translocation of GAPDH. Black-Right-Pointing-Pointer SIRT1 presence rather than activity is essential for inhibiting GAPDH translocation. -- Abstract: Upon apoptotic stimulation, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a cytosolic enzyme normally active in glycolysis, translocates into the nucleus and activates an apoptotic cascade therein. In the present work, we show that SIRT1 prevents nuclear translocation of GAPDH via interaction with GAPDH. SIRT1 depletion triggered nuclear translocation of cytosolic GAPDH even in the absence of apoptotic stress. Such translocation was not, however, observed when SIRT1 enzymatic activity was inhibited, indicating that SIRT1 protein per se, rather than the deacetylase activity of the protein, is required to inhibit GAPDH translocation. Upon irradiation, SIRT1 prevented irradiation-induced nuclear translocation of GAPDH, accompanied by interaction of SIRT1 and GAPDH. Thus, SIRT1 functions to retain GAPDH in the cytosol, protecting the enzyme from nuclear translocation via interaction with these two proteins. This serves as a mechanism whereby SIRT1 regulates cell survival upon induction of apoptotic stress by means that include irradiation.

  18. Autophagy and endosomal trafficking inhibition by Vibrio cholerae MARTX toxin phosphatidylinositol-3-phosphate-specific phospholipase A1 activity

    PubMed Central

    Agarwal, Shivani; Kim, Hyunjin; Chan, Robin B.; Agarwal, Shivangi; Williamson, Rebecca; Cho, Wonhwa; Paolo, Gilbert D.; Satchell, Karla J. F.

    2015-01-01

    Vibrio cholerae, responsible for acute gastroenteritis secretes a large multifunctional-autoprocessing repeat-in-toxin (MARTX) toxin linked to evasion of host immune system, facilitating colonization of small intestine. Unlike other effector domains of the multifunctional toxin that target cytoskeleton, the function of alpha-beta hydrolase (ABH) remained elusive. This study demonstrates that ABH is an esterase/lipase with catalytic Ser–His–Asp triad. ABH binds with high affinity to phosphatidylinositol-3-phosphate (PtdIns3P) and cleaves the fatty acid in PtdIns3P at the sn1 position in vitro making it the first PtdIns3P-specific phospholipase A1 (PLA1). Expression of ABH in vivo reduces intracellular PtdIns3P levels and its PtdIns3P-specific PLA1 activity blocks endosomal and autophagic pathways. In accordance with recent studies acknowledging the potential of extracellular pathogens to evade or exploit autophagy to prevent their clearance and facilitate survival, this is the first report highlighting the role of ABH in inhibiting autophagy and endosomal trafficking induced by extracellular V. cholerae. PMID:26498860

  19. The glyceraldehyde-3-phosphate dehydrogenase homologue is differentially regulated in phases of Paracoccidioides brasiliensis: molecular and phylogenetic analysis.

    PubMed

    Barbosa, Mônica S; Cunha Passos, Daniela A; Felipe, M Sueli S; Jesuíno, Rosália S A; Pereira, Maristela; de Almeida Soares, Célia M

    2004-07-01

    Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) plays important roles in various cellular processes. Here we report the sequence and analysis of a novel developmentally regulated gene and cDNA (Pbgadph), encoding a GAPDH homologue (PbGAPDH), of the pathogenic dimorphic fungus Paracoccidioides brasiliensis. We have analyzed the protein, the cDNA and genomic sequences to provide insights into the structure, function, and potential regulation of PbGAPDH. That Pbgapdh encodes PbGAPDH was demonstrated by micro-sequencing of the native protein homologue isolated from the fungus proteome. The deduced amino acid sequence of Pbgapdh showed identity to those of from other species (88-76%). Phylogenetic analysis indicated that GAPDH could be useful for the determination of evolutionary relationships. Expression of the Pbgapdh gene and the cognate protein were developmentally regulated in phases of P. brasiliensis, with a higher expression in the yeast parasitic phase and was induced during the transition from mycelium to yeast and decreased during the reverse process, transition from yeast to mycelium.

  20. The influence of oxygen on radiation-induced structural and functional changes in glyceraldehyde-3-phosphate dehydrogenase and lactate dehydrogenase

    NASA Astrophysics Data System (ADS)

    Rodacka, Aleksandra; Serafin, Eligiusz; Bubinski, Michal; Krokosz, Anita; Puchala, Mieczyslaw

    2012-07-01

    Proteins are major targets for oxidative damage due to their abundance in cells and high reactivity with free radicals. In the present study we examined the influence of oxygen on radiation-induced inactivation and structural changes of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and lactate dehydrogenase (LDH). We chose these two enzymes because they occur at high concentrations and participate in the most important processes in organisms; furthermore, they show considerable similarity in their structure. Protein solutions were irradiated with X-rays in doses ranging from 0.1 to 0.7 kGy, in air and N2O. The much higher radiation inactivation of GAPDH as compared to LDH is correlated with substantially greater structural changes in this protein, mainly involving the loss of free thiol groups (-SH). Of lesser importance in the differentiation of the radiosensitivity of the studied enzymes are tryptophan residues. Molecular oxygen, present during irradiation, increased to a significantly greater extent the inactivation and structural changes of GAPDH than that of LDH. The results suggest that the greater effect of oxygen on GAPDH is due to the higher efficiency of the superoxide radical, the higher amount of hydroperoxides generated, and the higher degree of unfolding of this protein.

  1. Enhanced resistance in Theobroma cacao against oomycete and fungal pathogens by secretion of phosphatidylinositol-3-phosphate-binding proteins.

    PubMed

    Helliwell, Emily E; Vega-Arreguín, Julio; Shi, Zi; Bailey, Bryan; Xiao, Shunyuan; Maximova, Siela N; Tyler, Brett M; Guiltinan, Mark J

    2016-03-01

    The internalization of some oomycete and fungal pathogen effectors into host plant cells has been reported to be blocked by proteins that bind to the effectors' cell entry receptor, phosphatidylinositol-3-phosphate (PI3P). This finding suggested a novel strategy for disease control by engineering plants to secrete PI3P-binding proteins. In this study, we tested this strategy using the chocolate tree Theobroma cacao. Transient expression and secretion of four different PI3P-binding proteins in detached leaves of T. cacao greatly reduced infection by two oomycete pathogens, Phytophthora tropicalis and Phytophthora palmivora, which cause black pod disease. Lesion size and pathogen growth were reduced by up to 85%. Resistance was not conferred by proteins lacking a secretory leader, by proteins with mutations in their PI3P-binding site, or by a secreted PI4P-binding protein. Stably transformed, transgenic T. cacao plants expressing two different PI3P-binding proteins showed substantially enhanced resistance to both P. tropicalis and P. palmivora, as well as to the fungal pathogen Colletotrichum theobromicola. These results demonstrate that secretion of PI3P-binding proteins is an effective way to increase disease resistance in T. cacao, and potentially in other plants, against a broad spectrum of pathogens. © 2015 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.

  2. In silico peptide prediction for antibody generation to recognize 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) in genetically modified organisms.

    PubMed

    Marani, Mariela M; Costa, Joana; Mafra, Isabel; Oliveira, Maria Beatriz P P; Camperi, Silvia A; Leite, José Roberto de Souza Almeida

    2015-03-01

    For the prospective immunorecognition of 5-enolpyruvylshikimate-3-phosphate synthase (CP4-EPSPS) as a biomarker protein expressed by transgenic soybean, an extensive in silico evaluation of the referred protein was performed. The main objective of this study was the selection of a set of peptides that could function as potential immunogens for the production of novel antibodies against CP4-EPSPS protein. For this purpose, the protein was in silico cleaved with trypsin/chymotrypsin and the resultant peptides were extensively analyzed for further selection of the best candidates for antibody production. The analysis enabled the successful proposal of four peptides with potential immunogenicity for their future use as screening biomarkers of genetically modified organisms. To our knowledge, this is the first attempt to select and define potential linear epitopes for the immunization of animals and, subsequently, to generate adequate antibodies for CP4-EPSPS recognition. The present work will be followed by the synthesis of the candidate peptides to be incubated in animals for antibody generation and potential applicability for the development of an immunosensor for CP4-EPSPS detection.

  3. Molecular basis for covalent inhibition of glyceraldehyde-3-phosphate dehydrogenase by a 2-phenoxy-1,4-naphthoquinone small molecule.

    PubMed

    Bruno, Stefano; Uliassi, Elisa; Zaffagnini, Mirko; Prati, Federica; Bergamini, Christian; Amorati, Riccardo; Paredi, Gianluca; Margiotta, Marilena; Conti, Paola; Costi, Maria Paola; Kaiser, Marcel; Cavalli, Andrea; Fato, Romana; Bolognesi, Maria Laura

    2017-01-12

    Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) has recently gained attention as an antiprotozoan and anticancer drug target. We have previously identified 2-phenoxy-1,4-naphthoquinone as an inhibitor of both Trypanosoma brucei and human GAPDH. Herein, through multiple chemical, biochemical, and biological studies, and through the design of analogs, we confirmed the formation of a covalent adduct, we clarified the inhibition mechanism, and we demonstrated antitrypanosomal, antiplasmodial, and cytotoxic activities in cell cultures. The overall results lent support to the hypothesis that 2-phenoxy-1,4-naphthoquinone binds the GAPDH catalytic cysteine covalently through a phenolate displacement mechanism. By investigating the reactivity of 2-phenoxy-1,4-naphthoquinone and its analogs with four GAPDH homologs, we showed that the covalent inhibition is not preceded by the formation of a strong non-covalent complex. However, an up to fivefold difference in inactivation rates among homologs hinted at structural or electrostatic differences of their active sites that could be exploited to further design kinetically selective inhibitors. Moreover, we preliminarily showed that 2-phenoxy-1,4-naphthoquinone displays selectivity for GAPDHs over two other cysteine-dependent enzymes, supporting its suitability as a warhead starting fragment for the design of novel inhibitors.

  4. Identification of a mammalian glycerol-3-phosphate phosphatase: Role in metabolism and signaling in pancreatic β-cells and hepatocytes.

    PubMed

    Mugabo, Yves; Zhao, Shangang; Seifried, Annegrit; Gezzar, Sari; Al-Mass, Anfal; Zhang, Dongwei; Lamontagne, Julien; Attane, Camille; Poursharifi, Pegah; Iglesias, José; Joly, Erik; Peyot, Marie-Line; Gohla, Antje; Madiraju, S R Murthy; Prentki, Marc

    2016-01-26

    Obesity, and the associated disturbed glycerolipid/fatty acid (GL/FA) cycle, contribute to insulin resistance, islet β-cell failure, and type 2 diabetes. Flux through the GL/FA cycle is regulated by the availability of glycerol-3-phosphate (Gro3P) and fatty acyl-CoA. We describe here a mammalian Gro3P phosphatase (G3PP), which was not known to exist in mammalian cells, that can directly hydrolyze Gro3P to glycerol. We identified that mammalian phosphoglycolate phosphatase, with an uncertain function, acts in fact as a G3PP. We found that G3PP, by controlling Gro3P levels, regulates glycolysis and glucose oxidation, cellular redox and ATP production, gluconeogenesis, glycerolipid synthesis, and fatty acid oxidation in pancreatic islet β-cells and hepatocytes, and that glucose stimulated insulin secretion and the response to metabolic stress, e.g., glucolipotoxicity, in β-cells. In vivo overexpression of G3PP in rat liver lowers body weight gain and hepatic glucose production from glycerol and elevates plasma HDL levels. G3PP is expressed at various levels in different tissues, and its expression varies according to the nutritional state in some tissues. As Gro3P lies at the crossroads of glucose, lipid, and energy metabolism, control of its availability by G3PP adds a key level of metabolic regulation in mammalian cells, and G3PP offers a potential target for type 2 diabetes and cardiometabolic disorders.

  5. Deficiency of glycerol-3-phosphate acyltransferase 1 decreases triacylglycerol storage and induces fatty acid oxidation in insect fat body.

    PubMed

    Alves-Bezerra, Michele; Ramos, Isabela B; De Paula, Iron F; Maya-Monteiro, Clarissa M; Klett, Eric L; Coleman, Rosalind A; Gondim, Katia C

    2017-03-01

    Glycerol-3-phosphate acyltransferases (GPAT) catalyze the initial and rate-limiting step for the de novo synthesis of triacylglycerol (TAG). Four mammalian GPAT isoforms have been identified: the mitochondria-associated GPAT1 and 2, and the endoplasmic reticulum (ER)-associated GPAT3 and 4. In the insect Rhodnius prolixus, a vector of Chagas' disease, we previously predicted a mitochondrial-like isoform (RhoprGPAT1) from genomic data. In the current study, we clone the RhoprGPAT1 coding sequence and identify an ER-associated GPAT (RhoprGPAT4) as the second isoform in the insect. RhoprGPAT1 contributes 15% of the total GPAT activity in anterior midgut, 50% in posterior midgut and fat body, and 70% in the ovary. The RhoprGpat1 gene is the predominant transcript in the midgut and fat body. To evaluate the physiological relevance of RhoprGPAT1, we generate RhoprGPAT1-deficient insects. The knockdown of RhoprGpat1 results in 50% and 65% decrease in TAG content in the posterior midgut and fat body, respectively. RhoprGpat1-deficient insects also exhibits impaired lipid droplet expansion and a 2-fold increase in fatty acid β-oxidation rates in the fat body. We propose that the RhoprGPAT1 mitochondrial-like isoform is required to channel fatty acyl chains towards TAG synthesis and away from β-oxidation. Such a process is crucial for the insect lipid homeostasis.

  6. Tandem amplification of a chromosomal segment harboring 5-enolpyruvylshikimate-3-phosphate synthase locus confers glyphosate resistance in Kochia scoparia.

    PubMed

    Jugulam, Mithila; Niehues, Kindsey; Godar, Amar S; Koo, Dal-Hoe; Danilova, Tatiana; Friebe, Bernd; Sehgal, Sunish; Varanasi, Vijay K; Wiersma, Andrew; Westra, Philip; Stahlman, Phillip W; Gill, Bikram S

    2014-11-01

    Recent rapid evolution and spread of resistance to the most extensively used herbicide, glyphosate, is a major threat to global crop production. Genetic mechanisms by which weeds evolve resistance to herbicides largely determine the level of resistance and the rate of evolution of resistance. In a previous study, we determined that glyphosate resistance in Kochia scoparia is due to the amplification of the 5-Enolpyruvylshikimate-3-Phosphate Synthase (EPSPS) gene, the enzyme target of glyphosate. Here, we investigated the genomic organization of the amplified EPSPS copies using fluorescence in situ hybridization (FISH) and extended DNA fiber (Fiber FISH) on K. scoparia chromosomes. In both glyphosate-resistant K. scoparia populations tested (GR1 and GR2), FISH results displayed a single and prominent hybridization site of the EPSPS gene localized on the distal end of one pair of homologous metaphase chromosomes compared with a faint hybridization site in glyphosate-susceptible samples (GS1 and GS2). Fiber FISH displayed 10 copies of the EPSPS gene (approximately 5 kb) arranged in tandem configuration approximately 40 to 70 kb apart, with one copy in an inverted orientation in GR2. In agreement with FISH results, segregation of EPSPS copies followed single-locus inheritance in GR1 population. This is the first report of tandem target gene amplification conferring field-evolved herbicide resistance in weed populations. © 2014 American Society of Plant Biologists. All Rights Reserved.

  7. Mutation by DNA shuffling of 5-enolpyruvylshikimate-3-phosphate synthase from Malus domestica for improved glyphosate resistance.

    PubMed

    Tian, Yong-Sheng; Xu, Jing; Peng, Ri-He; Xiong, Ai-Sheng; Xu, Hu; Zhao, Wei; Fu, Xiao-Yan; Han, Hong-Juan; Yao, Quan-Hong

    2013-09-01

    A new 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene from Malus domestica (MdEPSPS) was cloned and characterized by rapid amplification of cDNA ends to identify an EPSPS gene appropriate for the development of transgenic glyphosate-tolerant plants. However, wild-type MdEPSPS is not suitable for the development of transgenic glyphosate-tolerant plants because of its poor glyphosate resistance. Thus, we performed DNA shuffling on MdEPSPS, and one highly glyphosate-resistant mutant with mutations in eight amino acids (N63D, N86S, T101A, A187T, D230G, H317R, Y399R and C413A.) was identified after five rounds of DNA shuffling and screening. Among the eight amino acid substitutions on this mutant, only two residue changes (T101A and A187T) were identified by site-directed mutagenesis as essential and additive in altering glyphosate resistance, which was further confirmed by kinetic analyses. The single-site A187T mutation has also never been previously reported as an important residue for glyphosate resistance. Furthermore, transgenic rice was used to confirm the potential of MdEPSPS mutant in developing glyphosate-resistant crops.

  8. Insulin regulation of protein biosynthesis in differentiated 3T3 adipocytes. Regulation of glyceraldehyde-3-phosphate dehydrogenase

    SciTech Connect

    Alexander, M.; Curtis, G.; Avruch, J.; Goodman, H.M.

    1985-10-05

    The effect of insulin on protein biosynthesis was examined in differentiated 3T3-L1 and 3T3-F442A adipocytes. Insulin altered the relative rate of synthesis of specific proteins independent of its ability to hasten conversion of the fibroblast (preadipocyte) phenotype to the adipocyte phenotype. Although more than one pattern of response to insulin was observed, the authors focused on the induction of a Mr 33,000 protein which was identified as the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Exposure of 3T3 adipocytes to insulin throughout differentiation specifically increased GAPDH activity and protein content by 2- to 3-fold as compared to 3T3 adipocytes differentiated in the absence of insulin. These changes in enzyme activity and content could be accounted for by a 4-fold increase in the relative rate of synthesis of GAPDH and a 9-fold increase in hybridizable mRNA levels. Within 2 h of insulin addition to 3T3 adipocytes differentiated in the absence of hormone, hybridizable GAPDH mRNA levels increased 3-fold, and within 24 h GAPDH mRNA levels increased 8-fold, and (TVS) methionine incorporation into GAPDH protein increased 5-fold. These studies demonstrate that insulin, as the sole hormonal perturbant, can increase the synthesis of certain 3T3 adipocyte proteins by altering the cellular content of a specific mRNA.

  9. Altered chloroplast structure and function in a mutant of Arabidopsis deficient in plastid glycerol-3-phosphate acyltransferase activity

    SciTech Connect

    Kunst, L.; Somerville, C. ); Browse, J. )

    1989-07-01

    Mutants of Arabidopsis thaliana deficient in plastid glycerol-3-phosphate acyltransferase activity have altered chloroplast membrane lipid composition. This caused an increase in the number of regions of appressed membrane per chloroplast and a decrease in the average number of thylakoid membranes in the appressed regions. The net effect was a significant decrease in the ratio of appressed to nonappressed membranes. A comparison of 77 K fluorescence emission spectra of thylakoid membranes from the mutant and wild type indicated that the ultrastructural changes were associated with an altered distribution of excitation energy transfer from antenna chlorophyll to photosystem II and photosystem I in the mutant. The changes in leaf lipid composition did not significantly affect growth or development of the mutant under standard conditions. However, at temperatures above 28{degree}C the mutant grew slightly more rapidly than the wild type, and measurements of temperature-induced fluorescence yield enhancement suggested an increased thermal stability of the photosynthetic apparatus of the mutant. These effects are consistent with other evidence suggesting that membrane lipid composition is an important determinant of chloroplast structure but has relatively minor direct effects on the function of the membrane proteins associated with photosynthetic electron transport.

  10. The glyceraldehyde-3-phosphate dehydrogenase gene of Moniliophthoraperniciosa, the causal agent of witches' broom disease of Theobroma cacao

    PubMed Central

    2009-01-01

    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. PMID:21637692

  11. Nuclear translocation and accumulation of glyceraldehyde-3-phosphate dehydrogenase involved in diclazuril-induced apoptosis in Eimeria tenella (E. tenella).

    PubMed

    Wang, Congcong; Han, Chunzhou; Li, Tao; Yang, Dehao; Shen, Xiaojiong; Fan, Yinxin; Xu, Yang; Zheng, Wenli; Fei, Chenzhong; Zhang, Lifang; Xue, Feiqun

    2013-05-07

    In mammalian cells, GAPDH (glyceraldehyde-3-phosphate dehydrogenase) has recently been shown to be implicated in numerous apoptotic paradigms, especially in neuronal apoptosis, and has been demonstrated to play a vital role in some neurodegenerative disorders. However, this phenomenon has not been reported in protists. In the present study, we report for the first time that such a mechanism is involved in diclazuril-induced apoptosis in Eimeria tenella (E. tenella). We found that upon treatment of parasites with diclazuril, the expression levels of GAPDH transcript and protein were significantly increased in second-generation merozoites. Then, we examined the subcellular localization of GAPDH by fluorescence microscopy and Western blot analysis. The results show that a considerable amount of GAPDH protein appeared in the nucleus within diclazuril-treated second-generation merozoites; in contrast, the control group had very low levels of GAPDH in the nucleus. The glycolytic activity of GAPDH was kinetically analyzed in different subcellular fractions. A substantial decrease (48.5%) in glycolytic activity of GAPDH in the nucleus was displayed. Moreover, the activities of caspases-3, -9, and -8 were measured in cell extracts using specific caspase substrates. The data show significant increases in caspase-3 and caspase-9 activities in the diclazuril-treated group.

  12. Nuclear translocation and accumulation of glyceraldehyde-3-phosphate dehydrogenase involved in diclazuril-induced apoptosis in Eimeria tenella (E. tenella)

    PubMed Central

    2013-01-01

    In mammalian cells, GAPDH (glyceraldehyde-3-phosphate dehydrogenase) has recently been shown to be implicated in numerous apoptotic paradigms, especially in neuronal apoptosis, and has been demonstrated to play a vital role in some neurodegenerative disorders. However, this phenomenon has not been reported in protists. In the present study, we report for the first time that such a mechanism is involved in diclazuril-induced apoptosis in Eimeria tenella (E. tenella). We found that upon treatment of parasites with diclazuril, the expression levels of GAPDH transcript and protein were significantly increased in second-generation merozoites. Then, we examined the subcellular localization of GAPDH by fluorescence microscopy and Western blot analysis. The results show that a considerable amount of GAPDH protein appeared in the nucleus within diclazuril-treated second-generation merozoites; in contrast, the control group had very low levels of GAPDH in the nucleus. The glycolytic activity of GAPDH was kinetically analyzed in different subcellular fractions. A substantial decrease (48.5%) in glycolytic activity of GAPDH in the nucleus was displayed. Moreover, the activities of caspases-3, -9, and −8 were measured in cell extracts using specific caspase substrates. The data show significant increases in caspase-3 and caspase-9 activities in the diclazuril-treated group. PMID:23651214

  13. Autophagosome formation from membrane compartments enriched in phosphatidylinositol 3-phosphate and dynamically connected to the endoplasmic reticulum.

    PubMed

    Axe, Elizabeth L; Walker, Simon A; Manifava, Maria; Chandra, Priya; Roderick, H Llewelyn; Habermann, Anja; Griffiths, Gareth; Ktistakis, Nicholas T

    2008-08-25

    Autophagy is the engulfment of cytosol and organelles by double-membrane vesicles termed autophagosomes. Autophagosome formation is known to require phosphatidylinositol 3-phosphate (PI(3)P) and occurs near the endoplasmic reticulum (ER), but the exact mechanisms are unknown. We show that double FYVE domain-containing protein 1, a PI(3)P-binding protein with unusual localization on ER and Golgi membranes, translocates in response to amino acid starvation to a punctate compartment partially colocalized with autophagosomal proteins. Translocation is dependent on Vps34 and beclin function. Other PI(3)P-binding probes targeted to the ER show the same starvation-induced translocation that is dependent on PI(3)P formation and recognition. Live imaging experiments show that this punctate compartment forms near Vps34-containing vesicles, is in dynamic equilibrium with the ER, and provides a membrane platform for accumulation of autophagosomal proteins, expansion of autophagosomal membranes, and emergence of fully formed autophagosomes. This PI(3)P-enriched compartment may be involved in autophagosome biogenesis. Its dynamic relationship with the ER is consistent with the idea that the ER may provide important components for autophagosome formation.

  14. Fatty acid synthesis and generation of glycerol-3-phosphate in brown adipose tissue from rats fed a cafeteria diet.

    PubMed

    Chaves, Valéria E; Frasson, Danúbia; Martins-Santos, Maria E S; Navegantes, Luiz C C; Galban, Victor D; Garófalo, Maria A R; Kettelhut, Isis C; Migliorini, Renato H

    2008-07-01

    In vivo fatty acid synthesis and the pathways of glycerol-3-phosphate (G3P) production were investigated in brown adipose tissue (BAT) from rats fed a cafeteria diet for 3 weeks. In spite of BAT activation, the diet promoted an increase in the carcass fatty acid content. Plasma insulin levels were markedly increased in cafeteria diet-fed rats. Two insulin-sensitive processes, in vivo fatty acid synthesis and in vivo glucose uptake (which was used to evaluate G3P generation via glycolysis) were increased in BAT from rats fed the cafeteria diet. Direct glycerol phosphorylation, evaluated by glycerokinase (GyK) activity and incorporation of [U-14C]glycerol into triacylglycerol (TAG)-glycerol, was also markedly increased in BAT from these rats. In contrast, the cafeteria diet induced a marked reduction of BAT glyceroneogenesis, evaluated by phosphoenolpyruvate carboxykinase-C activity and incorporation of [1-14C]pyruvate into TAG-glycerol. BAT denervation resulted in an approximately 50% reduction of GyK activity, but did not significantly affect BAT in vivo fatty acid synthesis, in vivo glucose uptake, or glyceroneogenesis. The data suggest that the supply of G3P for BAT TAG synthesis can be adjusted independently from the sympathetic nervous system and solely by reciprocal changes in the generation of G3P via glycolysis and via glyceroneogenesis, with no participation of direct phosphorylation of glycerol by GyK.

  15. A Novel 5-Enolpyruvylshikimate-3-Phosphate Synthase Shows High Glyphosate Tolerance in Escherichia coli and Tobacco Plants

    PubMed Central

    Zhang, Shengxue; Yang, Xuewen; Chen, Rongrong; Zhang, Yuwen; Lu, Wei; Liu, Yan; Wang, Jianhua; Lin, Min; Wang, Guoying

    2012-01-01

    A key enzyme in the shikimate pathway, 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) is the primary target of the broad-spectrum herbicide glyphosate. Identification of new aroA genes coding for EPSPS with a high level of glyphosate tolerance is essential for the development of glyphosate-tolerant crops. In the present study, the glyphosate tolerance of five bacterial aroA genes was evaluated in the E. coli aroA-defective strain ER2799 and in transgenic tobacco plants. All five aroA genes could complement the aroA-defective strain ER2799, and AM79 aroA showed the highest glyphosate tolerance. Although glyphosate treatment inhibited the growth of both WT and transgenic tobacco plants, transgenic plants expressing AM79 aroA tolerated higher concentration of glyphosate and had a higher fresh weight and survival rate than plants expressing other aroA genes. When treated with high concentration of glyphosate, lower shikimate content was detected in the leaves of transgenic plants expressing AM79 aroA than transgenic plants expressing other aroA genes. These results suggest that AM79 aroA could be a good candidate for the development of transgenic glyphosate-tolerant crops. PMID:22715408

  16. Improvement of Glyphosate Resistance through Concurrent Mutations in Three Amino Acids of the Ochrobactrum 5-Enopyruvylshikimate-3-Phosphate Synthase ▿

    PubMed Central

    Tian, Yong-Sheng; Xu, Jing; Xiong, Ai-Sheng; Zhao, Wei; Fu, Xiao-Yan; Peng, Ri-He; Yao, Quan-Hong

    2011-01-01

    A mutant of 5-enopyruvylshikimate-3-phosphate synthase from Ochrobactrum anthropi was identified after four rounds of DNA shuffling and screening. Its ability to restore the growth of the mutant ER2799 cell on an M9 minimal medium containing 300 mM glyphosate led to its identification. The mutant had mutations in seven amino acids: E145G, N163H, N267S, P318R, M377V, M425T, and P438L. Among these mutations, N267S, P318R, and M425T have never been previously reported as important residues for glyphosate resistance. However, in the present study they were found by site-directed mutagenesis to collectively contribute to the improvement of glyphosate tolerance. Kinetic analyses of these three mutants demonstrated that the effectiveness of these three individual amino acid alterations on glyphosate tolerance was in the order P318R > M425T > N267S. The results of the kinetic analyses combined with a three-dimensional structure modeling of the location of P318R and M425T demonstrate that the lower hemisphere's upper surface is possibly another important region for glyphosate resistance. Furthermore, the transgenic Arabidopsis was obtained to confirm the potential of the mutant in developing glyphosate-resistant crops. PMID:21948846

  17. Glycerol-3-phosphate O-acyltransferase is required for PBAN-induced sex pheromone biosynthesis in Bombyx mori

    PubMed Central

    Du, Mengfang; Liu, Xiaoguang; Liu, Xiaoming; Yin, Xinming; Han, Shuangyin; Song, Qisheng; An, Shiheng

    2015-01-01

    Female moths employ their own pheromone blends as a communicational medium in mating behavior. The biosynthesis and release of sex pheromone in female moths are regulated by pheromone biosynthesis activating neuropeptide (PBAN) and the corresponding action of PBAN has been well elucidated in Bombyx mori. However, very little is known about the molecular mechanism regarding the biosynthesis of sex pheromone precursor. In this study, quantitative proteomics was utilized to comprehensively elucidate the expression dynamics of pheromone glands (PGs) during development. Proteomic analysis revealed a serial of differentially expressed sex pheromone biosynthesis-associated proteins at the different time points of B. mori development. Most interestingly B. mori glycerol-3-phosphate O-acyltransferase (BmGPAT) was found to be expressed during the key periods of sex pheromone biosynthesis. RNAi knockdown of BmGPAT confirmed the important function of this protein in the biosynthesis of sex pheromone precursor, triacylglcerol (TAG), and subsequently PBAN-induced production of sex pheromone, bombykol. Behavioral analysis showed that RNAi knockdown of GPAT significantly impaired the ability of females to attract males. Our findings indicate that GPAT acts to regulate the biosynthesis of sex pheromone precursor, TAG, thus influencing PBAN-induced sex pheromone production and subsequent mating behavior. PMID:25630665

  18. Glyceraldehyde-3-phosphate dehydrogenase aggregation inhibitor peptide: A potential therapeutic strategy against oxidative stress-induced cell death.

    PubMed

    Itakura, Masanori; Nakajima, Hidemitsu; Semi, Yuko; Higashida, Shusaku; Azuma, Yasu-Taka; Takeuchi, Tadayoshi

    2015-11-13

    The glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) has multiple functions, including mediating oxidative stress-induced neuronal cell death. This process is associated with disulfide-bonded GAPDH aggregation. Some reports suggest a link between GAPDH and the pathogenesis of several oxidative stress-related diseases. However, the pathological significance of GAPDH aggregation in disease pathogenesis remains unclear due to the lack of an effective GAPDH aggregation inhibitor. In this study, we identified a GAPDH aggregation inhibitor (GAI) peptide and evaluated its biological profile. The decapeptide GAI specifically inhibited GAPDH aggregation in a concentration-dependent manner. Additionally, the GAI peptide did not affect GAPDH glycolytic activity or cell viability. The GAI peptide also exerted a protective effect against oxidative stress-induced cell death in SH-SY5Y cells. This peptide could potentially serve as a tool to investigate GAPDH aggregation-related neurodegenerative and neuropsychiatric disorders and as a possible therapy for diseases associated with oxidative stress-induced cell death. Copyright © 2015 Elsevier Inc. All rights reserved.

  19. Improvement of glyphosate resistance through concurrent mutations in three amino acids of the Ochrobactrum 5-enopyruvylshikimate-3-phosphate synthase.

    PubMed

    Tian, Yong-Sheng; Xu, Jing; Xiong, Ai-Sheng; Zhao, Wei; Fu, Xiao-Yan; Peng, Ri-He; Yao, Quan-Hong

    2011-12-01

    A mutant of 5-enopyruvylshikimate-3-phosphate synthase from Ochrobactrum anthropi was identified after four rounds of DNA shuffling and screening. Its ability to restore the growth of the mutant ER2799 cell on an M9 minimal medium containing 300 mM glyphosate led to its identification. The mutant had mutations in seven amino acids: E145G, N163H, N267S, P318R, M377V, M425T, and P438L. Among these mutations, N267S, P318R, and M425T have never been previously reported as important residues for glyphosate resistance. However, in the present study they were found by site-directed mutagenesis to collectively contribute to the improvement of glyphosate tolerance. Kinetic analyses of these three mutants demonstrated that the effectiveness of these three individual amino acid alterations on glyphosate tolerance was in the order P318R > M425T > N267S. The results of the kinetic analyses combined with a three-dimensional structure modeling of the location of P318R and M425T demonstrate that the lower hemisphere's upper surface is possibly another important region for glyphosate resistance. Furthermore, the transgenic Arabidopsis was obtained to confirm the potential of the mutant in developing glyphosate-resistant crops.

  20. [Cloning of the glyceraldehydes 3-phosphate dehydrogenase gene of porphyromonas gingivalis and its expression in E. coli].

    PubMed

    Li, Ang; Xu, Hong-Yan; Shi, Jian-Feng; Zhu, Chun-Hui; Rao, Guo-Zhou; Gou, Jian-Zhong

    2011-04-01

    To clone the glyceraldehydes 3-phosphate dehydrogenase (GAPDH) gene of Porphyromonas gingivalis (P. gingivalis) and to induce its fusion expression in E. coli. GAPDH was obtained by PCR and was inserted into cloning vector pMD-18-T to construct clone recon. Double enzymes digest the recon pMD18-T-GAPDH and the prokaryotic expression vector pET-32a and then connect to get the expressing recon pET-32a-GAPDH. The recombinant expression plasmid which had been confirmed by enzymes digestion was transformed to E. coli competent cells BL21 and induced the expression of GAPDH with isopropyl beta-D-1-thiogalactopyranoside (IPTG) of different density. DNA sequencing showed that the fragment was 99.802% the same to the sequence published in NCBI. Under the best density, IPTG could be highly expressed. The GAPDH had been successfully cloned and expressed in E. coli which gets ready for the following experiment to study the immunity of GAPDH and the homologues antibody preparation.

  1. Identification of a mammalian glycerol-3-phosphate phosphatase: Role in metabolism and signaling in pancreatic β-cells and hepatocytes

    PubMed Central

    Mugabo, Yves; Zhao, Shangang; Seifried, Annegrit; Gezzar, Sari; Al-Mass, Anfal; Zhang, Dongwei; Lamontagne, Julien; Attane, Camille; Poursharifi, Pegah; Iglesias, José; Joly, Erik; Peyot, Marie-Line; Gohla, Antje; Madiraju, S. R. Murthy; Prentki, Marc

    2016-01-01

    Obesity, and the associated disturbed glycerolipid/fatty acid (GL/FA) cycle, contribute to insulin resistance, islet β-cell failure, and type 2 diabetes. Flux through the GL/FA cycle is regulated by the availability of glycerol-3-phosphate (Gro3P) and fatty acyl-CoA. We describe here a mammalian Gro3P phosphatase (G3PP), which was not known to exist in mammalian cells, that can directly hydrolyze Gro3P to glycerol. We identified that mammalian phosphoglycolate phosphatase, with an uncertain function, acts in fact as a G3PP. We found that G3PP, by controlling Gro3P levels, regulates glycolysis and glucose oxidation, cellular redox and ATP production, gluconeogenesis, glycerolipid synthesis, and fatty acid oxidation in pancreatic islet β-cells and hepatocytes, and that glucose stimulated insulin secretion and the response to metabolic stress, e.g., glucolipotoxicity, in β-cells. In vivo overexpression of G3PP in rat liver lowers body weight gain and hepatic glucose production from glycerol and elevates plasma HDL levels. G3PP is expressed at various levels in different tissues, and its expression varies according to the nutritional state in some tissues. As Gro3P lies at the crossroads of glucose, lipid, and energy metabolism, control of its availability by G3PP adds a key level of metabolic regulation in mammalian cells, and G3PP offers a potential target for type 2 diabetes and cardiometabolic disorders. PMID:26755581

  2. A Dimer Interface Mutation in Glyceraldehyde-3-Phosphate Dehydrogenase Regulates Its Binding to AU-rich RNA*

    PubMed Central

    White, Michael R.; Khan, Mohd M.; Deredge, Daniel; Ross, Christina R.; Quintyn, Royston; Zucconi, Beth E.; Wysocki, Vicki H.; Wintrode, Patrick L.; Wilson, Gerald M.; Garcin, Elsa D.

    2015-01-01

    Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is an enzyme best known for its role in glycolysis. However, extra-glycolytic functions of GAPDH have been described, including regulation of protein expression via RNA binding. GAPDH binds to numerous adenine-uridine rich elements (AREs) from various mRNA 3′-untranslated regions in vitro and in vivo despite its lack of a canonical RNA binding motif. How GAPDH binds to these AREs is still unknown. Here we discovered that GAPDH binds with high affinity to the core ARE from tumor necrosis factor-α mRNA via a two-step binding mechanism. We demonstrate that a mutation at the GAPDH dimer interface impairs formation of the second RNA-GAPDH complex and leads to changes in the RNA structure. We investigated the effect of this interfacial mutation on GAPDH oligomerization by crystallography, small-angle x-ray scattering, nano-electrospray ionization native mass spectrometry, and hydrogen-deuterium exchange mass spectrometry. We show that the mutation does not significantly affect GAPDH tetramerization as previously proposed. Instead, the mutation promotes short-range and long-range dynamic changes in regions located at the dimer and tetramer interface and in the NAD+ binding site. These dynamic changes are localized along the P axis of the GAPDH tetramer, suggesting that this region is important for RNA binding. Based on our results, we propose a model for sequential GAPDH binding to RNA via residues located at the dimer and tetramer interfaces. PMID:25451934

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

    PubMed

    Lima, Juliana O; Pereira, Jorge F; Rincones, Johana; Barau, Joan G; Araújo, Elza F; Pereira, Gonçalo A G; Queiroz, Marisa V

    2009-04-01

    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.

  4. Structure of holo-glyceraldehyde-3-phosphate dehydrogenase from Bacillus stearothermophilus at 1.8 A resolution.

    PubMed

    Skarzyński, T; Moody, P C; Wonacott, A J

    1987-01-05

    The structure of holo-glyceraldehyde-3-phosphate dehydrogenase from Bacillus stearothermophilus has been crystallographically refined at 1.8 A resolution using restrained least-squares refinement methods. The final crystallographic R-factor for 93,120 reflexions with F greater than 3 sigma (F) is 0.177. The asymmetric unit of the crystal contains a complete tetramer, the final model of which incorporates a total of 10,272 unique protein and coenzyme atoms together with 677 bound solvent molecules. The structure has been analysed with respect to molecular symmetry, intersubunit contacts, coenzyme binding and active site geometry. The refined model shows the four independent subunits to be remarkable similar apart from local deviations due to intermolecular contacts within the crystal lattice. A number of features are revealed that had previously been misinterpreted from an earlier 2.7 A electron density map. Arginine at position 195 (previously thought to be a glycine) contributes to the formation of the anion binding sites in the active site pocket, which are involved in binding of the substrate and inorganic phosphates during catalysis. This residue seems to be structurally equivalent to the conserved Arg194 in the enzyme from other sources. In the crystal both of the anion binding sites are occupied by sulphate ions. The ND atom of the catalytically important His176 is hydrogen-bonded to the main-chain carbonyl oxygen of Ser177, thus fixing the plane of the histidine imidazole ring and preventing rotation. The analysis has revealed the presence of several internal salt-bridges stabilizing the tertiary and quaternary structure. A significant number of buried water molecules have been found that play an important role in the structural integrity of the molecule.

  5. Tri- and tetra-substituted cyclen based lanthanide(III) ion complexes as ribonuclease mimics: a study into the effect of log Ka, hydration and hydrophobicity on phosphodiester hydrolysis of the RNA-model 2-hydroxypropyl-4-nitrophenyl phosphate (HPNP).

    PubMed

    Fanning, Ann-Marie; Plush, Sally E; Gunnlaugsson, Thorfinnur

    2015-05-28

    A series of tetra-substituted 'pseudo' dipeptide ligands of cyclen (1,4,7,10,-tetraazacyclododecane) and a tri-substituted 3'-pyridine ligand of cyclen, and the corresponding lanthanide(III) complexes were synthesised and characterised as metallo-ribonuclease mimics. All complexes were shown to promote hydrolysis of the phosphodiester bond of 2-hydroxypropyl-4-nitrophenyl phosphate (HPNP, τ1/2 = 5.87 × 10(3) h), a well known RNA mimic. The La(III) and Eu(III) tri-substituted 3'-pyridine lanthanide(III) complexes being the most efficient in promoting such hydrolysis at pH 7.4 and at 37 °C; with τ1/2 = 1.67 h for La(III) and 1.74 h for Eu(III). The series was developed to provide the opportunity to investigate the consequences of altering the lanthanide(III) ion, coordination ability and hydrophobicity of a metallo-cavity on the rate of hydrolysis using the model phosphodiester, HPNP, at 37 °C. To further provide information on the role that the log Ka of the metal bound water plays in phosphodiester hydrolysis the protonation constants and the metal ion stability constants of both a tri and tetra-substituted 3'pyridine complex were determined. Our results highlighted several key features for the design of lanthanide(III) ribonucelase mimics; the presence of two metal bound water molecules are vital for pH dependent rate constants for Eu(III) complexes, optimal pH activity approximating physiological pH (∼7.4) may be achieved if the log Ka values for both MLOH and ML(OH)2 species occur in this region, small changes to hydrophobicity within the metallo cavity influence the rate of hydrolysis greatly and an amide adjacent to the metal ion capable of forming hydrogen bonds with the substrate is required for achieving fast hydrolysis.

  6. The mitochondrial FAD-dependent glycerol-3-phosphate dehydrogenase of Trypanosomatidae and the glycosomal redox balance of insect stages of Trypanosoma brucei and Leishmania spp.

    PubMed

    Guerra, Daniel G; Decottignies, Anabelle; Bakker, Barbara M; Michels, Paul A M

    2006-10-01

    The genes for the mitochondrial FAD-dependent glycerol-3-phosphate dehydrogenase were identified in Trypanosoma brucei and Leishmania major genomes. We have expressed the L. major gene in Saccharomyces cerevisiae and confirmed the subcellular localization and activity of the produced enzyme. Using cultured T. brucei procyclic and Leishmania mexicana promastigote cells with a permeabilized plasma membrane and containing intact glycosomes, it was shown that dihydroxyacetone phosphate is converted into pyruvate, and stimulates oxygen consumption, indicating that all components of the glycerol 3-phosphate/dihydoxyacetone phosphate shuttle between glycosomes and mitochondrion are present in these insect stages of both organisms. A computer model has been prepared for the energy and carbohydrate metabolism of these cells. It was used in an elementary mode analysis to get insight into the metabolic role of the shuttle in these insect-stage parasites. Our analysis suggests that the shuttle fulfils important roles for these organisms, albeit different from its well-known function in the T. brucei bloodstream form. It allows (1) a high yield of further metabolizable glycolytic products by decreasing the need to produce a secreted end product of glycosomal metabolism, succinate; (2) the consumption of glycerol and glycerol 3-phosphate derived from lipids; and (3) to keep the redox balance of the glycosome finely tuned due to a highly flexible and redundant system.

  7. The acylation of sn-glycerol 3-phosphate and the metabolism of phosphatidate in microsomal preparations from the developing cotyledons of safflower (Carthamus tinctorius L.) seed.

    PubMed

    Griffiths, G; Stobart, A K; Stymne, S

    1985-09-01

    Microsomal preparations from the developing cotyledons of safflower (Carthamus tinctorius) catalysed the acylation of sn-glycerol 3-phosphate in the presence of acyl-CoA. The resulting phosphatidate was further utilized in the synthesis of diacyl- and tri-acylglycerol by the reactions of the so-called 'Kennedy pathway' [Kennedy (1961) Fed. Proc. Fed. Am. Soc. Exp. Biol. 20, 934-940]. Diacylglycerol equilibrated with the phosphatidylcholine pool when glycerol backbone, with the associated acyl groups, flowed from phosphatidate to triacylglycerol. The formation of diacylglycerol from phosphatidate through the action of a phosphatidate phosphohydrolase (phosphatidase) was substantially inhibited by EDTA and, under these conditions, phosphatidate accumulated in the microsomal membranes. The inhibition of the phosphatidase by EDTA was alleviated by Mg2+. The presence of Mg2+ in all incubation mixtures stimulated quite considerably the synthesis of triacylglycerol in vitro. Microsomal preparations incubated with acyl-CoA, sn-glycerol 3-phosphate and EDTA synthesized sufficient phosphatidate for the reliable analysis of its intramolecular fatty acid distribution. In the presence of mixed acyl-CoA substrates the sn-glycerol 3-phosphate was acylated exclusively in position 1 with the saturated fatty acids, palmitate and stearate. The polyunsaturated fatty acid linoleate was, however, utilized largely in the acylation of position 2 of sn-glycerol 3-phosphate. The affinity of the enzymes involved in the acylation of positions 1 and 2 of sn-glycerol 3-phosphate for specific species of acyl-CoA therefore governs the non-random distribution of the different acyl groups in the seed triacylglycerols. The acylation of sn-glycerol 3-phosphate in position 1 with saturated acyl components also accounts for the presence of these groups in position 1 of sn-phosphatidylcholine through the equilibration of diacylglycerol with the phosphatidylcholine pool, which occurs when phosphatidate

  8. The acylation of sn-glycerol 3-phosphate and the metabolism of phosphatidate in microsomal preparations from the developing cotyledons of safflower (Carthamus tinctorius L.) seed.

    PubMed Central

    Griffiths, G; Stobart, A K; Stymne, S

    1985-01-01

    Microsomal preparations from the developing cotyledons of safflower (Carthamus tinctorius) catalysed the acylation of sn-glycerol 3-phosphate in the presence of acyl-CoA. The resulting phosphatidate was further utilized in the synthesis of diacyl- and tri-acylglycerol by the reactions of the so-called 'Kennedy pathway' [Kennedy (1961) Fed. Proc. Fed. Am. Soc. Exp. Biol. 20, 934-940]. Diacylglycerol equilibrated with the phosphatidylcholine pool when glycerol backbone, with the associated acyl groups, flowed from phosphatidate to triacylglycerol. The formation of diacylglycerol from phosphatidate through the action of a phosphatidate phosphohydrolase (phosphatidase) was substantially inhibited by EDTA and, under these conditions, phosphatidate accumulated in the microsomal membranes. The inhibition of the phosphatidase by EDTA was alleviated by Mg2+. The presence of Mg2+ in all incubation mixtures stimulated quite considerably the synthesis of triacylglycerol in vitro. Microsomal preparations incubated with acyl-CoA, sn-glycerol 3-phosphate and EDTA synthesized sufficient phosphatidate for the reliable analysis of its intramolecular fatty acid distribution. In the presence of mixed acyl-CoA substrates the sn-glycerol 3-phosphate was acylated exclusively in position 1 with the saturated fatty acids, palmitate and stearate. The polyunsaturated fatty acid linoleate was, however, utilized largely in the acylation of position 2 of sn-glycerol 3-phosphate. The affinity of the enzymes involved in the acylation of positions 1 and 2 of sn-glycerol 3-phosphate for specific species of acyl-CoA therefore governs the non-random distribution of the different acyl groups in the seed triacylglycerols. The acylation of sn-glycerol 3-phosphate in position 1 with saturated acyl components also accounts for the presence of these groups in position 1 of sn-phosphatidylcholine through the equilibration of diacylglycerol with the phosphatidylcholine pool, which occurs when phosphatidate

  9. Photo-oxidation of 5-enolpyruvoylshikimate-3-phosphate synthase from Escherichia coli: evidence for a reactive imidazole group (His385) at the herbicide glyphosate-binding site.

    PubMed

    Huynh, Q K

    1993-03-01

    Photo-oxidation of Escherichia coli 5-enolpyruvoylshikimate-3-phosphate synthase, a target for the non-selective herbicide glyphosate (N-phosphonomethylglycine), in the presence of pyridoxal 5'-phosphate resulted in irreversible inactivation of the enzyme. The inactivation followed pseudo-first-order and saturation kinetics with a Kinact. of 50 microM. The inactivation is specifically prevented by preincubation of the enzyme with the combination of shikimate 3-phosphate and glyphosate. Increasing glyphosate concentration during preincubation resulted in a decreasing rate of inactivation. On 95% inactivation, approximately one histidine per molecule of enzyme was oxidized. Tryptic mapping of the enzyme modified in the absence and presence of shikimate 3-phosphate and glyphosate as well as analyses of the histidine content in the isolated peptides indicated that His385, in the peptide Asn383-Asp-His-Arg386, was the site of oxidation. These results suggest that His385 is the most accessible reactive imidazole group under these conditions and is located close to the glyphosate-binding site.

  10. Crystal Structure of CTP: Glycerol-3-Phosphate Cytidylyl Tranferase from Staphylococcus Aurues: Examination of Structural Basis for Kinetic Mechanism

    SciTech Connect

    Fong,D.; Yim, V.; D'elia, M.; Brown, E.; Berghuis, A.

    2006-01-01

    Integrity of the cell wall is essential for bacterial survival, and as a consequence components involved in its biosynthesis can potentially be exploited as targets for antibiotics. One such potential target is CTP:glycerol-3-phosphate cytidylyltransferase. This enzyme (TarD{sub Sa} in Staphylococcus aureus and TagD{sub Bs} in Bacillus subtilis) catalyzes the formation of CDP-glycerol, which is used for the assembly of linkages between peptidoglycan and teichoic acid polymer in Gram-positive bacteria. Intriguingly, despite the high sequence identity between TarD{sub Sa} and TagD{sub Bs} (69% identity), kinetic studies show that these two enzymes differ markedly in their kinetic mechanism and activity. To examine the basis for the disparate enzymological properties, we have determined the crystal structure of TarD{sub Sa} in the apo state to 3 Angstroms resolution, and performed equilibrium sedimentation analysis. Comparison of the structure with that of CTP- and CDP-glycerol-bound TagD{sub Bs} crystal structures reveals that the overall structure of TarD{sub Sa} is essentially the same as that of TagD{sub Bs}, except in the C-terminus, where it forms a helix in TagD{sub Bs} but is disordered in the apo TarDSa structure. In addition, TarD{sub Sa} can exist both as a tetramer and as a dimer, unlike TagD{sub Bs}, which is a dimer. These observations shed light on the structural basis for the differing kinetic characteristics between TarD{sub Sa} and TagD{sub Bs}.

  11. Structure and topological symmetry of the glyphosate target 5-enolpyruvylshikimate-3-phosphate synthase: a distinctive protein fold.

    PubMed Central

    Stallings, W C; Abdel-Meguid, S S; Lim, L W; Shieh, H S; Dayringer, H E; Leimgruber, N K; Stegeman, R A; Anderson, K S; Sikorski, J A; Padgette, S R; Kishore, G M

    1991-01-01

    5-enol-Pyruvylshikimate-3-phosphate synthase (EPSP synthase; phosphoenolpyruvate:3-phosphoshikimate 1-carboxyvinyltransferase, EC 2.5.1.19) is an enzyme on the pathway toward the synthesis of aromatic amino acids in plants, fungi, and bacteria and is the target of the broad-spectrum herbicide glyphosate. The three-dimensional structure of the enzyme from Escherichia coli has been determined by crystallographic techniques. The polypeptide backbone chain was traced by examination of an electron density map calculated at 3-A resolution. The two-domain structure has a distinctive fold and appears to be formed by 6-fold replication of a protein folding unit comprising two parallel helices and a four-stranded sheet. Each domain is formed from three of these units, which are related by an approximate threefold symmetry axis; in each domain three of the helices are completely buried by a surface formed from the three beta-sheets and solvent-accessible faces of the other three helices. The domains are related by an approximate dyad, but in the present crystals the molecule does not display pseudo-symmetry related to the symmetry of point group 32 because its approximate threefold axes are almost normal. A possible relation between the three-dimensional structure of the protein and the linear sequence of its gene will be described. The topological threefold symmetry and orientation of each of the two observed globular domains may direct the binding of substrates and inhibitors by a helix macrodipole effect and implies that the active site is located near the interdomain crossover segments. The structure also suggests a rationale for the glyphosate tolerance conferred by sequence alterations. Images PMID:11607190

  12. Active site cysteine-null glyceraldehyde-3-phosphate dehydrogenase (GAPDH) rescues nitric oxide-induced cell death.

    PubMed

    Kubo, Takeya; Nakajima, Hidemitsu; Nakatsuji, Masatoshi; Itakura, Masanori; Kaneshige, Akihiro; Azuma, Yasu-Taka; Inui, Takashi; Takeuchi, Tadayoshi

    2016-02-29

    Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a homotetrameric enzyme involved in a key step of glycolysis, also has a role in mediating cell death under nitrosative stress. Our previous reports suggest that nitric oxide-induced intramolecular disulfide-bonding GAPDH aggregation, which occurs through oxidation of the active site cysteine (Cys-152), participates in a mechanism to account for nitric oxide-induced death signaling in some neurodegenerative/neuropsychiatric disorders. Here, we demonstrate a rescue strategy for nitric oxide-induced cell death accompanied by GAPDH aggregation in a mutant with a substitution of Cys-152 to alanine (C152A-GAPDH). Pre-incubation of purified wild-type GAPDH with C152A-GAPDH under exposure to nitric oxide inhibited wild-type GAPDH aggregation in a concentration-dependent manner in vitro. Several lines of structural analysis revealed that C152A-GAPDH extensively interfered with nitric oxide-induced GAPDH-amyloidogenesis. Overexpression of doxycycline-inducible C152A-GAPDH in SH-SY5Y neuroblastoma significantly rescued nitric oxide-induced death, concomitant with the decreased formation of GAPDH aggregates. Further, both co-immunoprecipitation assays and simulation models revealed a heterotetramer composed of one dimer each of wild-type GAPDH and C152A-GAPDH. These results suggest that the C152A-GAPDH mutant acts as a dominant-negative molecule against GAPDH aggregation via the formation of this GAPDH heterotetramer. This study may contribute to a new therapeutic approach utilizing C152A-GAPDH against brain damage in nitrosative stress-related disorders.

  13. DIRECT BINDING OF GLYCERALDEHYDE 3-PHOSPHATE DEHYDROGENASE TO TELOMERIC DNA PROTECTS TELOMERES AGAINST CHEMOTHERAPY-INDUCED RAPID DEGRADATION

    PubMed Central

    Demarse, Neil A.; Ponnusamy, Suriyan; Spicer, Eleanor K.; Apohan, Elif; Baatz, John E.; Ogretmen, Besim; Davies, Christopher

    2009-01-01

    GAPDH (glyceraldehyde 3-phosphate dehydrogenase) is a glycolytic enzyme that displays several non-glycolytic activities, including the maintenance and/or protection of telomeres. In this study, we determined the molecular mechanism and biological role of the interaction between GAPDH and human telomeric DNA. Using gel shift assays, we show that recombinant GAPDH binds directly with high affinity (Kd = 45 nM) to a single-stranded oligonucleotide comprising three telomeric DNA repeats and that nucleotides T1, G5 and G6 of the TTAGGG repeat are essential for binding. The stoichiometry of the interaction is 2:1 (DNA: GAPDH), and GAPDH appears to form a high-molecular weight complex when bound to the oligonucleotide. Mutation of Asp32 and Cys149, which are localized to the NAD-binding site and the active site center of GAPDH, respectively, produced mutants that almost completely lost their telomere-binding functions both in vitro and in situ (in A549 human lung cancer cells). Treatment of A549 cells with the chemotherapeutic agents gemcitabine and doxorubicin resulted in increased nuclear localization of expressed wild-type GAPDH, where it protected telomeres against rapid degradation, concomitant with increased resistance to the growth inhibitory effects of these drugs. The non-DNA-binding mutants of GAPDH also localized to the nucleus when expressed in A549 cells, but did not confer any significant protection of telomeres against chemotherapy-induced degradation or growth inhibition, and this occurred without the involvement of caspase activation or apoptosis regulation. Overall, these data demonstrate that GAPDH binds telomeric DNA directly in vitro and may have a biological role in the protection of telomeres against rapid degradation in response to chemotherapeutic agents in A549 human lung cancer cells. PMID:19800890

  14. Functional divergence and convergent evolution in the plastid-targeted glyceraldehyde-3-phosphate dehydrogenases of diverse eukaryotic algae.

    PubMed

    Gaston, Daniel; Roger, Andrew J

    2013-01-01

    Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a key enzyme of the glycolytic pathway, reversibly catalyzing the sixth step of glycolysis and concurrently reducing the coenzyme NAD(+) to NADH. In photosynthetic organisms a GAPDH paralog (Gap2 in Cyanobacteria, GapA in most photosynthetic eukaryotes) functions in the Calvin cycle, performing the reverse of the glycolytic reaction and using the coenzyme NADPH preferentially. In a number of photosynthetic eukaryotes that acquired their plastid by the secondary endosymbiosis of a eukaryotic red alga (Alveolates, haptophytes, cryptomonads and stramenopiles) GapA has been apparently replaced with a paralog of the host's own cytosolic GAPDH (GapC1). Plastid GapC1 and GapA therefore represent two independent cases of functional divergence and adaptations to the Calvin cycle entailing a shift in subcellular targeting and a shift in binding preference from NAD(+) to NADPH. We used the programs FunDi, GroupSim, and Difference Evolutionary-Trace to detect sites involved in the functional divergence of these two groups of GAPDH sequences and to identify potential cases of convergent evolution in the Calvin-cycle adapted GapA and GapC1 families. Sites identified as being functionally divergent by all or some of these programs were then investigated with respect to their possible roles in the structure and function of both glycolytic and plastid-targeted GAPDH isoforms. In this work we found substantial evidence for convergent evolution in GapA/B and GapC1. In many cases sites in GAPDHs of these groups converged on identical amino acid residues in specific positions of the protein known to play a role in the function and regulation of plastid-functioning enzymes relative to their cytosolic counterparts. In addition, we demonstrate that bioinformatic software like FunDi are important tools for the generation of meaningful biological hypotheses that can then be tested with direct experimental techniques.

  15. Glyceraldehyde 3-phosphate dehydrogenase and galectin from Dirofilaria immitis participate in heartworm disease endarteritis via plasminogen/plasmin system.

    PubMed

    González-Miguel, Javier; Larrazabal, Carmen; Loa-Mesón, Diana; Siles-Lucas, Mar; Simón, Fernando; Morchón, Rodrigo

    2016-06-15

    The interaction between parasitic protozoa and helminths, both in the blood and in tissues and the fibrinolytic system of their hosts is usually considered as a survival parasite mechanism since this system is the physiological route responsible for degrading fibrin clots. The broad-range proteolytic activity of plasmin, the final enzyme of the route, implies that its recruitment by these parasites is an important mechanism that mediates their invasion and establishment in the hosts. However, recent studies have proposed a dual role for plasmin by linking its over-production with pathological mechanisms at vascular level. Most of these studies have been conducted in Dirofilaria immitis, a blood-borne parasite that survives in the pulmonary arteries of its host for years while it produces a chronic inflammatory disease, whose main pathogenic mechanism is the appearance of proliferative endarteritis. Recently, the participation of two proteins from D. immitis, glyceraldehyde 3-phosphate dehydrogenase (DiGAPDH) and galectin (DiGAL), in the activation of the fibrinolytic system of its host has been demonstrated, which has been a priori associated with parasite survival mechanisms. The aim of the present paper was to study the role of plasmin generated by these proteins in the emergence of proliferative endarteritis. An in vitro model of canine endothelial and smooth muscle cells, as well as the two parasitic recombinant proteins were employed. The results show that DiGAPDH and DiGAL stimulate the proliferation and migration of both cell types, as well as the degradation of the extracellular matrix (ECM) via plasminogen (PLG)/plasmin system, being all of these mechanisms related to the appearance of proliferative endarteritis. Due to the high degree of evolutionary conservation of these antigens, these data support the hypothesis of the survival/pathology ambivalence in the interactions between parasites and the fibrinolytic system of their hosts and represent an

  16. Sperm-specific glyceraldehyde-3-phosphate dehydrogenase is stabilized by additional proline residues and an interdomain salt bridge.

    PubMed

    Kuravsky, Mikhail; Barinova, Kseniya; Marakhovskaya, Aleksandra; Eldarov, Mikhail; Semenyuk, Pavel; Muronetz, Vladimir; Schmalhausen, Elena

    2014-10-01

    Sperm-specific glyceraldehyde-3-phosphate dehydrogenase (GAPDS) exhibits enhanced stability compared to the somatic isoenzyme (GAPD). A comparative analysis of the structures of these isoenzymes revealed characteristic features, which could be important for the stability of GAPDS: six specific proline residues and three buried salt bridges. To evaluate the impact of these structural elements into the stability of this isoenzyme, we obtained two series of mutant GAPDS: 1) six mutants each containing a substitution of one of the specific prolines by alanine, and 2) three mutants each containing a mutation breaking one of the salt bridges. Stability of the mutants was evaluated by differential scanning calorimetry and by their resistance towards guanidine hydrochloride (GdnHCl). The most effect on thermostability was observed for the mutants P326A and P164A: the Tm values of the heat-absorption curves decreased by 6.0 and 3.3°C compared to the wild type protein, respectively. The resistance towards GdnHCl was affected most by the mutation D311N breaking the salt bridge between the catalytic and NAD(+)-binding domains: the inactivation rate constant in the presence of GdnHCl increased six-fold, and the value of GdnHCl concentration corresponding to the protein half-denaturation decreased from 1.83 to 1.35M. Besides, the mutation D311N enhanced the enzymatic activity of the protein two-fold. The results suggest that the residues P164 (β-turn), P326 (first position of α-helix), and the interdomain salt bridge D311-H124 are significant for the enhanced stability of GAPDS. The salt bridge D311-H124 enhances stability of the active site of GAPDS at the expense of the catalytic activity.

  17. Identification of glyceraldehyde-3-phosphate dehydrogenase of epithelial cells as a second molecule that binds to Porphyromonas gingivalis fimbriae.

    PubMed

    Sojar, Hakimuddin T; Genco, Robert J

    2005-07-01

    Binding of Porphyromonas gingivalis to the host cells is an essential step in the pathogenesis of periodontal disease. P. gingivalis binds to and invades epithelial cells, and fimbriae are thought to be involved in this process. In our earlier studies, two major epithelial cell components of 40 and 50 kDa were identified as potential fimbrial receptors. Sequencing of a cyanogen bromide digestion fragment of the 50-kDa component resulted in an internal sequence identical to keratin I molecules, and hence this cytokeratin represents one of the epithelial cell receptors for P. gingivalis fimbriae. In this study, the 40-kDa component of KB cells was isolated and its amino-terminal sequence determined. The N-terminal amino sequence was found to be GKVKVGVNGF and showed perfect homology with human glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Furthermore, purified P. gingivalis fimbriae were found to bind to rabbit muscle GAPDH. Antibodies directed against internal peptide 49-68 and 69-90 of fimbrillin were shown to inhibit the binding of P. gingivalis and of fimbriae to epithelial cells. Antibodies against these peptides also inhibited the binding of fimbriae to GAPDH. Our results confirmed that the amino-terminal domain corresponding to amino residues 49-68 of the fimbrillin protein is the major GAPDH binding domain. These studies point to GAPDH as a major receptor for P. gingivalis major fimbriae and, as such, GAPDH likely plays a role in P. gingivalis adherence and colonization of the oral cavity, as well as triggering host cell processes involved in the pathogenesis of P. gingivalis infections.

  18. Nuclear Accumulation of Cytosolic Glyceraldehyde-3-Phosphate Dehydrogenase in Cadmium-Stressed Arabidopsis Roots1[C][W

    PubMed Central

    Vescovi, Marco; Zaffagnini, Mirko; Festa, Margherita; Trost, Paolo; Lo Schiavo, Fiorella; Costa, Alex

    2013-01-01

    NAD-dependent glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a ubiquitous enzyme involved in the glycolytic pathway. It has been widely demonstrated that mammalian GAPDH, in addition to its role in glycolysis, fulfills alternative functions mainly linked to its susceptibility to oxidative posttranslational modifications. Here, we investigated the responses of Arabidopsis (Arabidopsis thaliana) cytosolic GAPDH isoenzymes GAPC1 and GAPC2 to cadmium-induced stress in seedlings roots. GAPC1 was more responsive to cadmium than GAPC2 at the transcriptional level. In vivo, cadmium treatments induced different concomitant effects, including (1) nitric oxide accumulation, (2) cytosolic oxidation (e.g. oxidation of the redox-sensitive Green fluorescent protein2 probe), (3) activation of the GAPC1 promoter, (4) GAPC1 protein accumulation in enzymatically inactive form, and (5) strong relocalization of GAPC1 to the nucleus. All these effects were detected in the same zone of the root tip. In vitro, GAPC1 was inactivated by either nitric oxide donors or hydrogen peroxide, but no inhibition was directly provided by cadmium. Interestingly, nuclear relocalization of GAPC1 under cadmium-induced oxidative stress was stimulated, rather than inhibited, by mutating into serine the catalytic cysteine of GAPC1 (C155S), excluding an essential role of GAPC1 nitrosylation in the mechanism of nuclear relocalization, as found in mammalian cells. Although the function of GAPC1 in the nucleus is unknown, our results suggest that glycolytic GAPC1, through its high sensitivity to the cellular redox state, may play a role in oxidative stress signaling or protection in plants. PMID:23569110

  19. Glyceraldehyde 3-Phosphate Dehydrogenase Negatively Regulates the Replication of Bamboo Mosaic Virus and Its Associated Satellite RNA▿†

    PubMed Central

    Prasanth, K. Reddisiva; Huang, Ying-Wen; Liou, Ming-Ru; Wang, Robert Yung-Liang; Hu, Chung-Chi; Tsai, Ching-Hsiu; Meng, Menghsiao; Lin, Na-Sheng; Hsu, Yau-Heiu

    2011-01-01

    The identification of cellular proteins associated with virus replicase complexes is crucial to our understanding of virus-host interactions, influencing the host range, replication, and virulence of viruses. A previous in vitro study has demonstrated that partially purified Bamboo mosaic virus (BaMV) replicase complexes can be employed for the replication of both BaMV genomic and satellite BaMV (satBaMV) RNAs. In this study, we investigated the BaMV and satBaMV 3′ untranslated region (UTR) binding proteins associated with these replicase complexes. Two cellular proteins with molecular masses of ∼35 and ∼55 kDa were specifically cross-linked with RNA elements, whereupon the ∼35-kDa protein was identified as the glycolytic enzyme glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Gel mobility shift assays confirmed the direct interaction of GAPDH with the 3′ UTR sequences, and competition gel shift analysis revealed that GAPDH binds preferentially to the positive-strand BaMV and satBaMV RNAs over the negative-strand RNAs. It was observed that the GAPDH protein binds to the pseudoknot poly(A) tail of BaMV and stem-loop-C poly(A) tail of satBaMV 3′ UTR RNAs. It is important to note that knockdown of GAPDH in Nicotiana benthamiana enhances the accumulation of BaMV and satBaMV RNA; conversely, transient overexpression of GAPDH reduces the accumulation of BaMV and satBaMV RNA. The recombinant GAPDH principally inhibits the synthesis of negative-strand RNA in exogenous RdRp assays. These observations support the contention that cytosolic GAPDH participates in the negative regulation of BaMV and satBaMV RNA replication. PMID:21715476

  20. A land-plant-specific glycerol-3-phosphate acyltransferase family in Arabidopsis: substrate specificity, sn-2 preference, and evolution.

    PubMed

    Yang, Weili; Simpson, Jeffrey P; Li-Beisson, Yonghua; Beisson, Fred; Pollard, Mike; Ohlrogge, John B

    2012-10-01

    Arabidopsis (Arabidopsis thaliana) has eight glycerol-3-phosphate acyltransferase (GPAT) genes that are members of a plant-specific family with three distinct clades. Several of these GPATs are required for the synthesis of cutin or suberin. Unlike GPATs with sn-1 regiospecificity involved in membrane or storage lipid synthesis, GPAT4 and -6 are unique bifunctional enzymes with both sn-2 acyltransferase and phosphatase activity resulting in 2-monoacylglycerol products. We present enzymology, pathway organization, and evolutionary analysis of this GPAT family. Within the cutin-associated clade, GPAT8 is demonstrated as a bifunctional sn-2 acyltransferase/phosphatase. GPAT4, -6, and -8 strongly prefer C16:0 and C18:1 ω-oxidized acyl-coenzyme As (CoAs) over unmodified or longer acyl chain substrates. In contrast, suberin-associated GPAT5 can accommodate a broad chain length range of ω-oxidized and unsubstituted acyl-CoAs. These substrate specificities (1) strongly support polyester biosynthetic pathways in which acyl transfer to glycerol occurs after oxidation of the acyl group, (2) implicate GPAT specificities as one major determinant of cutin and suberin composition, and (3) argue against a role of sn-2-GPATs (Enzyme Commission 2.3.1.198) in membrane/storage lipid synthesis. Evidence is presented that GPAT7 is induced by wounding, produces suberin-like monomers when overexpressed, and likely functions in suberin biosynthesis. Within the third clade, we demonstrate that GPAT1 possesses sn-2 acyltransferase but not phosphatase activity and can utilize dicarboxylic acyl-CoA substrates. Thus, sn-2 acyltransferase activity extends to all subbranches of the Arabidopsis GPAT family. Phylogenetic analyses of this family indicate that GPAT4/6/8 arose early in land-plant evolution (bryophytes), whereas the phosphatase-minus GPAT1 to -3 and GPAT5/7 clades diverged later with the appearance of tracheophytes.

  1. Pharmacological glycerol-3-phosphate acyltransferase inhibition decreases food intake and adiposity and increases insulin sensitivity in diet-induced obesity

    PubMed Central

    Kuhajda, Francis P.; Tu, Yajun; Han, Wan Fang; Medghalchi, Susan M.; El Meskini, Rajaa; Landree, Leslie E.; Peterson, Jonathan M.; Daniels, Khadija; Wong, Kody; Wydysh, Edward A.; Townsend, Craig A.; Ronnett, Gabriele V.

    2011-01-01

    Storage of excess calories as triglycerides is central to obesity and its associated disorders. Glycerol-3-phosphate acyltransferases (GPATs) catalyze the initial step in acylglyceride syntheses, including triglyceride synthesis. We utilized a novel small-molecule GPAT inhibitor, FSG67, to investigate metabolic consequences of systemic pharmacological GPAT inhibition in lean and diet-induced obese (DIO) mice. FSG67 administered intraperitoneally decreased body weight and energy intake, without producing conditioned taste aversion. Daily FSG67 (5 mg/kg, 15.3 μmol/kg) produced gradual 12% weight loss in DIO mice beyond that due to transient 9- to 10-day hypophagia (6% weight loss in pair-fed controls). Continued FSG67 maintained the weight loss despite return to baseline energy intake. Weight was lost specifically from fat mass. Indirect calorimetry showed partial protection by FSG67 against decreased rates of oxygen consumption seen with hypophagia. Despite low respiratory exchange ratio due to a high-fat diet, FSG67-treated mice showed further decreased respiratory exchange ratio, beyond pair-fed controls, indicating enhanced fat oxidation. Chronic FSG67 increased glucose tolerance and insulin sensitivity in DIO mice. Chronic FSG67 decreased gene expression for lipogenic enzymes in white adipose tissue and liver and decreased lipid accumulation in white adipose, brown adipose, and liver tissues without signs of damage. RT-PCR showed decreased gene expression for orexigenic hypothalamic neuropeptides AgRP or NPY after acute and chronic systemic FSG67. FSG67 given intracerebroventricularly (100 and 320 nmol icv) produced 24-h weight loss and feeding suppression, indicating contributions from direct central nervous system sites of action. Together, these data point to GPAT as a new potential therapeutic target for the management of obesity and its comorbidities. PMID:21490364

  2. Pharmacological glycerol-3-phosphate acyltransferase inhibition decreases food intake and adiposity and increases insulin sensitivity in diet-induced obesity.

    PubMed

    Kuhajda, Francis P; Aja, Susan; Tu, Yajun; Han, Wan Fang; Medghalchi, Susan M; El Meskini, Rajaa; Landree, Leslie E; Peterson, Jonathan M; Daniels, Khadija; Wong, Kody; Wydysh, Edward A; Townsend, Craig A; Ronnett, Gabriele V

    2011-07-01

    Storage of excess calories as triglycerides is central to obesity and its associated disorders. Glycerol-3-phosphate acyltransferases (GPATs) catalyze the initial step in acylglyceride syntheses, including triglyceride synthesis. We utilized a novel small-molecule GPAT inhibitor, FSG67, to investigate metabolic consequences of systemic pharmacological GPAT inhibition in lean and diet-induced obese (DIO) mice. FSG67 administered intraperitoneally decreased body weight and energy intake, without producing conditioned taste aversion. Daily FSG67 (5 mg/kg, 15.3 μmol/kg) produced gradual 12% weight loss in DIO mice beyond that due to transient 9- to 10-day hypophagia (6% weight loss in pair-fed controls). Continued FSG67 maintained the weight loss despite return to baseline energy intake. Weight was lost specifically from fat mass. Indirect calorimetry showed partial protection by FSG67 against decreased rates of oxygen consumption seen with hypophagia. Despite low respiratory exchange ratio due to a high-fat diet, FSG67-treated mice showed further decreased respiratory exchange ratio, beyond pair-fed controls, indicating enhanced fat oxidation. Chronic FSG67 increased glucose tolerance and insulin sensitivity in DIO mice. Chronic FSG67 decreased gene expression for lipogenic enzymes in white adipose tissue and liver and decreased lipid accumulation in white adipose, brown adipose, and liver tissues without signs of damage. RT-PCR showed decreased gene expression for orexigenic hypothalamic neuropeptides AgRP or NPY after acute and chronic systemic FSG67. FSG67 given intracerebroventricularly (100 and 320 nmol icv) produced 24-h weight loss and feeding suppression, indicating contributions from direct central nervous system sites of action. Together, these data point to GPAT as a new potential therapeutic target for the management of obesity and its comorbidities.

  3. Distinct Requirements for Vacuolar Protein Sorting 34 Downstream Effector Phosphatidylinositol 3-Phosphate 5-Kinase in Podocytes Versus Proximal Tubular Cells

    PubMed Central

    Venkatareddy, Madhusudan; Verma, Rakesh; Kalinowski, Anne; Patel, Sanjeevkumar R.; Shisheva, Assia

    2016-01-01

    The mechanisms by which the glomerular filtration barrier prevents the loss of large macromolecules and simultaneously, maintains the filter remain poorly understood. Recent studies proposed that podocytes have an active role in both the endocytosis of filtered macromolecules and the maintenance of the filtration barrier. Deletion of a key endosomal trafficking regulator, the class 3 phosphatidylinositol (PtdIns) 3-kinase vacuolar protein sorting 34 (Vps34), in podocytes results in aberrant endosomal membrane morphology and podocyte dysfunction. We recently showed that the vacuolation phenotype in cultured Vps34–deficient podocytes is caused by the absence of a substrate for the Vps34 downstream effector PtdIns 3-phosphate 5-kinase (PIKfyve), which phosphorylates Vps34-generated PtdIns(3)P to produce PtdIns (3,5)P2. PIKfyve perturbation and PtdIns(3,5)P2 reduction result in massive membrane vacuolation along the endosomal system, but the cell-specific functions of PIKfyve in vivo remain unclear. We show here that the genetic deletion of PIKfyve in endocytically active proximal tubular cells resulted in the development of large cytoplasmic vacuoles caused by arrested endocytic traffic progression at a late-endosome stage. In contrast, deletion of PIKfyve in glomerular podocytes did not significantly alter the endosomal morphology, even in age 18-month-old mice. However, on culturing, the PIKfyve-deleted podocytes developed massive cytoplasmic vacuoles. In summary, these data suggest that glomerular podocytes and proximal tubules have different requirements for PIKfyve function, likely related to distinct in vivo needs for endocytic flux. PMID:26825532

  4. Conformational change of glyceraldehyde-3-phosphate dehydrogenase induced by acetylleucine chloromethyl ketone is followed by unique enzymatic degradation.

    PubMed

    Yamaguchi, Mitsune; Tsuchiya, Yukihiro; Hishinuma, Kayoko; Chikuma, Toshiyuki; Hojo, Hiroshi

    2003-12-01

    We have previously reported that acetylleucine chloromethyl ketone (ALCK), an inhibitor of acylpeptidehydrolase, induces the inhibition and degradation of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in the U937 cell extract. In the present study, the process of ALCK-induced GAPDH degradation was investigated. A kinetic study revealed that GAPDH was irreversibly inhibited by ALCK. ALCK treatment induced a change in the signal intensity of GAPDH in the near-UV region of the circular dichroism (CD) spectrum, and the fluorescence intensity of GAPDH at 330 nm increased to about 10% when excited at 280 nm, suggesting that a significant conformational change of GAPDH was induced by ALCK. When the U937 cell extract was incubated with ALCK and the products were separated by SDS-polyacrylamide gel electrophoresis (PAGE), a 23-kDa fragment from GAPDH was detected by Western blotting using anti-GAPDH serum. When ALCK-treated GAPDH was incubated with protease fractions from the U937 cell extract, a 17-kDa fragment was also detected. Sequence analysis showed that the N-terminal amino acid sequence of the 23-kDa fragment was GKVKVG and that of 17-kDa fragment was RDGRGAL. Therefore, ALCK-modified GAPDH is deduced to be digested at the peptide bond Trp(195)-Arg(196). The protease activity liberating a 23-kDa fragment from ALCK-treated GAPDH was effective under the basic condition. Results suggested that ALCK binds to GAPDH to modulate the conformation of enzyme, which is susceptible to chymotrypsin-like protease activity.

  5. Degradation of glyceraldehyde-3-phosphate dehydrogenase triggered by 4-hydroxy-2-nonenal and 4-hydroxy-2-hexenal.

    PubMed

    Tsuchiya, Yukihiro; Yamaguchi, Mitsune; Chikuma, Toshiyuki; Hojo, Hiroshi

    2005-06-15

    Lipid peroxidation products such as 4-hydroxy-2-nonenal (HNE) may be responsible for various pathophysiological events under oxidative stress, since they injure cellular components such as proteins and DNA. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which is a key enzyme of glycolysis and has been reported to be a multifunctional enzyme, is one of the enzymes inhibited by HNE. Previous studies showed that GAPDH is degraded when incubated with acetylleucine chloromethyl ketone (ALCK), resulting in the liberation of a 23-kDa fragment. In this study, we examined whether GAPDH incubated with HNE or other aldehydes of lipid peroxidation products are degraded similarly to that with ALCK. The U937 cell extract was incubated with these aldehydes at 37 degrees C and analyzed by Western blotting using anti-GAPDH antibodies. Incubation with HNE or 4-hydroxy-2-hexenal (HHE) decreased GAPDH activity and GAPDH protein level, and increased the 23-kDa fragment, in time- and dose-dependent manners, but that with other aldehydes did not. Gel filtration using the Superose 6 showed that the GAPDH-degrading activity was eluted in higher molecular fractions than proteasome activity. The enzyme activity was detected at the basic range of pH and inhibited by serine protease inhibitors, diisopropyl fluorophosphate and phenylmethylsulfonyl fluoride, but not by other protease inhibitors including a proteasome inhibitor, MG-132, and a tripeptidyl peptidase II (TPP II) inhibitor, AAF-CMK. These results suggest that GAPDH modified by HNE and HHE is degraded by a giant serine protease, releasing the 23-kDa fragment, not by proteasome or TPP II.

  6. Phylogenetic Analysis of Glycerol 3-Phosphate Acyltransferases in Opisthokonts Reveals Unexpected Ancestral Complexity and Novel Modern Biosynthetic Components

    PubMed Central

    Smart, Heather C.; Mast, Fred D.; Chilije, Maxwell F. J.; Tavassoli, Marjan; Dacks, Joel B.; Zaremberg, Vanina

    2014-01-01

    Glycerolipid synthesis represents a central metabolic process of all forms of life. In the last decade multiple genes coding for enzymes responsible for the first step of the pathway, catalyzed by glycerol 3-phosphate acyltransferase (GPAT), have been described, and characterized primarily in model organisms like Saccharomyces cerevisiae and mice. Notoriously, the fungal enzymes share low sequence identity with their known animal counterparts, and the nature of their homology is unclear. Furthermore, two mitochondrial GPAT isoforms have been described in animal cells, while no such enzymes have been identified in Fungi. In order to determine if the yeast and mammalian GPATs are representative of the set of enzymes present in their respective groups, and to test the hypothesis that metazoan orthologues are indeed absent from the fungal clade, a comparative genomic and phylogenetic analysis was performed including organisms spanning the breadth of the Opisthokonta supergroup. Surprisingly, our study unveiled the presence of ‘fungal’ orthologs in the basal taxa of the holozoa and ‘animal’ orthologues in the basal holomycetes. This includes a novel clade of fungal homologues, with putative peroxisomal targeting signals, of the mitochondrial/peroxisomal acyltransferases in Metazoa, thus potentially representing an undescribed metabolic capacity in the Fungi. The overall distribution of GPAT homologues is suggestive of high relative complexity in the ancestors of the opisthokont clade, followed by loss and sculpting of the complement in the descendent lineages. Divergence from a general versatile metabolic model, present in ancestrally deduced GPAT complements, points to distinctive contributions of each GPAT isoform to lipid metabolism and homeostasis in contemporary organisms like humans and their fungal pathogens. PMID:25340523

  7. Phylogenetically-based variation in the regulation of the Calvin cycle enzymes, phosphoribulokinase and glyceraldehyde-3-phosphate dehydrogenase, in algae.

    PubMed

    Maberly, Stephen C; Courcelle, Carine; Groben, Rene; Gontero, Brigitte

    2010-03-01

    Aquatic photosynthesis is responsible for about half of the global production and is undertaken by a huge phylogenetic diversity of algae that are poorly studied. The diversity of redox-regulation of phosphoribulokinase (PRK) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was investigated in a wide range of algal groups under standard conditions. Redox-regulation of PRK was greatest in chlorophytes, low or absent in a red alga and most chromalveolates, and linked to the number of amino acids between two regulatory cysteine residues. GAPDH regulation was not strongly-related to the different forms of this enzyme and was less variable than for PRK. Addition of recombinant CP12, a protein that forms a complex with PRK and GAPDH, to crude extracts inhibited GAPDH and PRK inversely in the Plantae, but in most chromalveolates had little effect on GAPDH and inhibited or stimulated PRK depending on the species. Patterns of enzyme regulation were used to produce a phylogenetic tree in which cryptophytes and haptophytes, at the base of the chromalveolates, formed a distinct clade. A second clade comprised only chromalveolates. A third clade comprised a mixture of Plantae, an excavate and three chromalveolates: a marine diatom and two others (a xanthophyte and eustigmatophyte) that are distinguished by a low content of chlorophyll c and a lack of fucoxanthin. Regulation of both enzymes was greater in freshwater than in marine taxa, possibly because most freshwaters are more dynamic than oceans. This work highlights the importance of understanding enzyme regulation in diverse algae if their ecology and productivity is to be understood.

  8. Construction and immune effect of Haemophilus parasuis DNA vaccine encoding glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in mice.

    PubMed

    Fu, Shulin; Zhang, Minmin; Ou, Jiwen; Liu, Huazhen; Tan, Chen; Liu, Jinlin; Chen, Huanchun; Bei, Weicheng

    2012-11-06

    Haemophilus parasuis, the causative agent of swine polyserositis, polyarthritis, and meningitis, is one of the most important bacterial diseases of pigs worldwide. The development of a vaccine against H. parasuis has been impeded due to the lack of induction of reliable cross-serotype protection. In this study the gapA gene that encodes glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was shown to be present and highly conserved in various serotypes of H. parasuis and we constructed a novel DNA vaccine encoding GAPDH (pCgap) to evaluate the immune response and protective efficacy against infection with H. parasuis MD0322 serovar 4 or SH0165 serovar 5 in mice. A significant antibody response against GAPDH was generated following pCgap intramuscular immunization; moreover, antibodies to the pCgap DNA vaccine were bactericidal, suggesting that it was expressed in vivo. The gapA transcript was detected in muscle, liver, spleen, and kidney of the mice seven days post-vaccination. The IgG subclass (IgG1 and IgG2a) analysis indicated that the DNA vaccine induced both Th1 and Th2 immune responses, but the IgG1 response was greater than the IgG2a response. Moreover, the groups vaccinated with the pCgap vaccine exhibited 83.3% and 50% protective efficacy against the H. parasuis MD0322 serovar 4 or SH0165 serovar 5 challenges, respectively. The pCgap DNA vaccine provided significantly greater protective efficacy compared to the negative control groups or blank control groups (P<0.05 for both). Taken together, these findings indicate that the pCgap DNA vaccine provides a novel strategy against infection of H. parasuis and offer insight concerning the underlying immune mechanisms of a bacterial DNA vaccine.

  9. The Glycerol-3-Phosphate Acyltransferase GPAT6 from Tomato Plays a Central Role in Fruit Cutin Biosynthesis1[OPEN

    PubMed Central

    Petit, Johann; Mauxion, Jean-Philippe; Tai, Fabienne Wong Jun; Fich, Eric A.; Joubès, Jérôme; Rothan, Christophe

    2016-01-01

    The thick cuticle covering and embedding the epidermal cells of tomato (Solanum lycopersicum) fruit acts not only as a protective barrier against pathogens and water loss but also influences quality traits such as brightness and postharvest shelf-life. In a recent study, we screened a mutant collection of the miniature tomato cultivar Micro-Tom and isolated several glossy fruit mutants in which the abundance of cutin, the polyester component of the cuticle, was strongly reduced. We employed a newly developed mapping-by-sequencing strategy to identify the causal mutation underlying the cutin deficiency in a mutant thereafter named gpat6-a (for glycerol-3-phosphate acyltransferase6). To this end, a backcross population (BC1F2) segregating for the glossy trait was phenotyped. Individuals displaying either a wild-type or a glossy fruit trait were then pooled into bulked populations and submitted to whole-genome sequencing prior to mutation frequency analysis. This revealed that the causal point mutation in the gpat6-a mutant introduces a charged amino acid adjacent to the active site of a GPAT6 enzyme. We further showed that this mutation completely abolished the GPAT activity of the recombinant protein. The gpat6-a mutant showed perturbed pollen formation but, unlike a gpat6 mutant of Arabidopsis (Arabidopsis thaliana), was not male sterile. The most striking phenotype was observed in the mutant fruit, where cuticle thickness, composition, and properties were altered. RNA sequencing analysis highlighted the main processes and pathways that were affected by the mutation at the transcriptional level, which included those associated with lipid, secondary metabolite, and cell wall biosynthesis. PMID:27208295

  10. The Glycerol-3-Phosphate Acyltransferase GPAT6 from Tomato Plays a Central Role in Fruit Cutin Biosynthesis.

    PubMed

    Petit, Johann; Bres, Cécile; Mauxion, Jean-Philippe; Tai, Fabienne Wong Jun; Martin, Laetitia B B; Fich, Eric A; Joubès, Jérôme; Rose, Jocelyn K C; Domergue, Frédéric; Rothan, Christophe

    2016-06-01

    The thick cuticle covering and embedding the epidermal cells of tomato (Solanum lycopersicum) fruit acts not only as a protective barrier against pathogens and water loss but also influences quality traits such as brightness and postharvest shelf-life. In a recent study, we screened a mutant collection of the miniature tomato cultivar Micro-Tom and isolated several glossy fruit mutants in which the abundance of cutin, the polyester component of the cuticle, was strongly reduced. We employed a newly developed mapping-by-sequencing strategy to identify the causal mutation underlying the cutin deficiency in a mutant thereafter named gpat6-a (for glycerol-3-phosphate acyltransferase6). To this end, a backcross population (BC1F2) segregating for the glossy trait was phenotyped. Individuals displaying either a wild-type or a glossy fruit trait were then pooled into bulked populations and submitted to whole-genome sequencing prior to mutation frequency analysis. This revealed that the causal point mutation in the gpat6-a mutant introduces a charged amino acid adjacent to the active site of a GPAT6 enzyme. We further showed that this mutation completely abolished the GPAT activity of the recombinant protein. The gpat6-a mutant showed perturbed pollen formation but, unlike a gpat6 mutant of Arabidopsis (Arabidopsis thaliana), was not male sterile. The most striking phenotype was observed in the mutant fruit, where cuticle thickness, composition, and properties were altered. RNA sequencing analysis highlighted the main processes and pathways that were affected by the mutation at the transcriptional level, which included those associated with lipid, secondary metabolite, and cell wall biosynthesis.

  11. Regulation of adenine nucleotide translocase and glycerol 3-phosphate dehydrogenase expression by thyroid hormones in different rat tissues.

    PubMed Central

    Dümmler, K; Müller, S; Seitz, H J

    1996-01-01

    Thyroid hormone (T3)-dependent gene expression of the adenine nucleotide translocase (ANT) and the FAD-linked glycerol 3-phosphate dehydrogenase (mGPDH) was investigated in several rat tissues. Both proteins provide an important link between cytosolic and mitochondrial metabolic pathways and seem to be involved in the stimulation of mitochondrial oxygen consumption in response to T3. Here we show that two ANT isoforms are expressed in rat, the muscle-specific ANT1 form and the ubiquitous ANT2 form. The expression of ANT1 mRNA is not sensitive to T3 whereas the amount of ANT2 mRNA is increased 7-9-fold in liver and heart within 12-48 h after T3 application. Little or no effect of T3 on ANT2 mRNA was observed in kidney and brain. The mRNA changes are paralleled by an increase in ANT protein, thus explaining the accelerated ADP/ATP exchange observed in mitochondria isolated from hyperthyroid rats. The key role of ANT2 in the control of hyperthyroid metabolism is evident because the expression of the mersalyl-sensitive phosphate carrier and the mitochondrial creatine kinase mRNA, which are functionally linked to ANT, did not respond to T3. Similarly to the ADP/ATP exchange, the transfer of cytosolic NADH to the respiratory chain via the glycerophosphate shuttle is very sensitive to T3. Recently we demonstrated the 10-15-fold induction of mGPDH mRNA in rat liver after administration of T3 [Müller and Seitz (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 10581-10585]. Here we show that, in contrast with ANT2, the time course of induction is fast (4-6 h). Furthermore, mGPDH mRNA is induced 6-fold by T3 in heart and 4-fold in kidney. From these results we conclude that the T3-mediated transcriptional induction leading to increased activity of ANT2 and mGPDH contributes considerably to the increase in mitochondrial oxygen consumption in rat tissues. PMID:8760382

  12. Overexpression of the triose phosphate translocator (TPT) complements the abnormal metabolism and development of plastidial glycolytic glyceraldehyde-3-phosphate dehydrogenase mutants.

    PubMed

    Flores-Tornero, María; Anoman, Armand D; Rosa-Téllez, Sara; Toujani, Walid; Weber, Andreas P M; Eisenhut, Marion; Kurz, Samantha; Alseekh, Saleh; Fernie, Alisdair R; Muñoz-Bertomeu, Jesús; Ros, Roc

    2017-03-01

    The presence of two glycolytic pathways working in parallel in plastids and cytosol has complicated the understanding of this essential process in plant cells, especially the integration of the plastidial pathway into the metabolism of heterotrophic and autotrophic organs. It is assumed that this integration is achieved by transport systems, which exchange glycolytic intermediates across plastidial membranes. However, it is unknown whether plastidial and cytosolic pools of 3-phosphoglycerate (3-PGA) can equilibrate in non-photosynthetic tissues. To resolve this question, we employed Arabidopsis mutants of the plastidial glycolytic isoforms of glyceraldehyde-3-phosphate dehydrogenase (GAPCp) that express the triose phosphate translocator (TPT) under the control of the 35S (35S:TPT) or the native GAPCp1 (GAPCp1:TPT) promoters. TPT expression under the control of both promoters complemented the vegetative developmental defects and metabolic disorders of the GAPCp double mutants (gapcp1gapcp2). However, as the 35S is poorly expressed in the tapetum, full vegetative and reproductive complementation of gapcp1gapcp2 was achieved only by transforming this mutant with the GAPCp1:TPT construct. Our results indicate that the main function of GAPCp is to supply 3-PGA for anabolic pathways in plastids of heterotrophic cells and suggest that the plastidial glycolysis may contribute to fatty acid biosynthesis in seeds. They also suggest a 3-PGA deficiency in the plastids of gapcp1gapcp2, and that 3-PGA pools between cytosol and plastid do not equilibrate in heterotrophic cells.

  13. Expression, purification, crystallization and preliminary X-ray analysis of an NAD-dependent glyceraldehyde-3-phosphate dehydrogenase from Helicobacter pylori

    SciTech Connect

    Elliott, Paul R.; Mohammad, Shabaz; Melrose, Helen J.; Moody, Peter C. E.

    2008-08-01

    Glyceraldehyde-3-phosphate dehydrogenase B from H. pylori has been cloned, expressed, purified and crystallized in the presence of NAD. Crystals of GAPDHB diffracted to 2.8 Å resolution and belonged to space group P6{sub 5}22, with unit-cell parameters a = b = 166.1, c = 253.1 Å. Helicobacter pylori is a dangerous human pathogen that resides in the upper gastrointestinal tract. Little is known about its metabolism and with the onset of antibiotic resistance new treatments are required. In this study, the expression, purification, crystallization and preliminary X-ray diffraction of an NAD-dependent glyceraldehyde-3-phosphate dehydrogenase from H. pylori are reported.

  14. Formation of homo- and heterooligomeric supramolecular structures by D-glyceraldehyde-3-phosphate dehydrogenase and lactate dehydrogenase in reversed micelles of aerosol OT in octane.

    PubMed

    Levashov, A V; Ugolnikova, A V; Ivanov, M V; Klyachko, N L

    1997-07-01

    The supramolecular structure of oligomeric enzymes can be specifically regulated by changing the size of an inner cavity of Aerosol OT reversed micelles in octane. Both D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and lactate dehydrogenase (LDH) reveal an ability to exist and function in monomeric, dimeric and tetrameric forms (homooligomers). Various heterooligomeric complexes, in particular, GAPDH monomer--LDH monomer, GAPDH dimer--LDH tetramer were detected in reversed micelles.

  15. Improving ethanol productivity by modification of glycolytic redox factor generation in glycerol-3-phosphate dehydrogenase mutants of an industrial ethanol yeast.

    PubMed

    Guo, Zhong-peng; Zhang, Liang; Ding, Zhong-yang; Wang, Zheng-Xiang; Shi, Gui-Yang

    2011-08-01

    The GPD2 gene, encoding NAD(+)-dependent glycerol-3-phosphate dehydrogenase in an industrial ethanol-producing strain of Saccharomyces cerevisiae, was deleted. And then, either the non-phosphorylating NADP(+)-dependent glyceraldehyde-3-phosphate dehydrogenase (GAPN) from Bacillus cereus, or the NADP(+)-dependent glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from Kluyveromyces lactis, was expressed in the obtained mutant AG2 deletion of GPD2, respectively. The resultant recombinant strain AG2A (gpd2Δ P (PGK)-gapN) exhibited a 48.70 ± 0.34% (relative to the amount of substrate consumed) decrease in glycerol production and a 7.60 ± 0.12% (relative to the amount of substrate consumed) increase in ethanol yield, while recombinant AG2B (gpd2Δ P (PGK)-GAPDH) exhibited a 52.90 ± 0.45% (relative to the amount of substrate consumed) decrease in glycerol production and a 7.34 ± 0.15% (relative to the amount of substrate consumed) increase in ethanol yield compared with the wild-type strain. More importantly, the maximum specific growth rates (μ (max)) of the recombinant AG2A and AG2B were higher than that of the mutant gpd2Δ and were indistinguishable compared with the wild-type strain in anaerobic batch fermentations. The results indicated that the redox imbalance of the mutant could be partially solved by expressing the heterologous genes.

  16. Synthesis of 2-n-(hexadecanoyl)-amino-4-nitrophenyl phosphorylcholine-hydroxide, a chromogenic substrate for assaying sphingomyelinase activity.

    PubMed

    Gal, A E; Fash, F J

    1976-02-01

    2-N-(Hexadecanoyl)-amino-4-nitrophenyl phosphorylcholine-hydroxide a compound resembling sphingomyelin is synthesized. It is cleaved by sphingomyelinase to the chromogenic N-acylaminonitrophenyl moiety. Phospholipase C preparations do not hydrolyze this compound. The starting material is 2-amino-4-nitrophenol which when acylated with palmitoyl chloride yields the hexadecananilide. Reaction with beta-bromoethylphosphoryldichloride gives the phosphate which is quaternized with trimethylamine to give the title compound.

  17. Chemoselective amide formation using O-(4-nitrophenyl)hydroxylamines and pyruvic acid derivatives.

    PubMed

    Kumar, Sonali; Sharma, Rashi; Garcia, Megan; Kamel, Joseph; McCarthy, Caroline; Muth, Aaron; Phanstiel, Otto

    2012-12-07

    A series of O-(4-nitrophenyl)hydroxylamines were synthesized from their respective oximes using a pulsed addition of excess NaBH(3)CN at pH 3 in 65-75% yield. Steric hindrance near the oxime functional group played a key role in both the ease by which the oxime could be reduced and the subsequent reactivity of the respective hydroxylamine. Reaction of the respective hydroxylamines with pyruvic acid derivatives generated the desired amides in good yields. A comparison of phenethylamine systems bearing different leaving groups revealed significant differences in the rates of these systems and suggested that the leaving group ability of the N-OR substituent plays an important role in determining their reactivity with pyruvic acid. Competition experiments (in 68% DMSO/phosphate buffered saline) using 1 equiv of N-phenethyl-O-(4-nitrophenyl)hydroxylamine and 2 equiv of pyruvic acid in the presence of other nucleophiles such as glycine, cysteine, phenol, hexanoic acid, and lysine demonstrated that significant chemoselectivity is present in this reaction. The results suggest that this chemoselective reaction can occur in the presence of excess α-amino acids, phenols, acids, thiols, and amines.

  18. Ablation of Succinate Production from Glucose Metabolism in the Procyclic Trypanosomes Induces Metabolic Switches to the Glycerol 3-Phosphate/Dihydroxyacetone Phosphate Shuttle and to Proline Metabolism*

    PubMed Central

    Ebikeme, Charles; Hubert, Jane; Biran, Marc; Gouspillou, Gilles; Morand, Pauline; Plazolles, Nicolas; Guegan, Fabien; Diolez, Philippe; Franconi, Jean-Michel; Portais, Jean-Charles; Bringaud, Frédéric

    2010-01-01

    Trypanosoma brucei is a parasitic protist that undergoes a complex life cycle during transmission from its mammalian host (bloodstream forms) to the midgut of its insect vector (procyclic form). In both parasitic forms, most glycolytic steps take place within specialized peroxisomes, called glycosomes. Here, we studied metabolic adaptations in procyclic trypanosome mutants affected in their maintenance of the glycosomal redox balance. T. brucei can theoretically use three strategies to maintain the glycosomal NAD+/NADH balance as follows: (i) the glycosomal succinic fermentation branch; (ii) the glycerol 3-phosphate (Gly-3-P)/dihydroxyacetone phosphate (DHAP) shuttle that transfers reducing equivalents to the mitochondrion; and (iii) the glycosomal glycerol production pathway. We showed a hierarchy in the use of these glycosomal NADH-consuming pathways by determining metabolic perturbations and adaptations in single and double mutant cell lines using a combination of NMR, ion chromatography-MS/MS, and HPLC approaches. Although functional, the Gly-3-P/DHAP shuttle is primarily used when the preferred succinate fermentation pathway is abolished in the Δpepck knock-out mutant cell line. In the absence of these two pathways (Δpepck/RNAiFAD-GPDH.i mutant), glycerol production is used but with a 16-fold reduced glycolytic flux. In addition, the Δpepck mutant cell line shows a 3.3-fold reduced glycolytic flux compensated by an increase of proline metabolism. The inability of the Δpepck mutant to maintain a high glycolytic flux demonstrates that the Gly-3-P/DHAP shuttle is not adapted to the procyclic trypanosome context. In contrast, this shuttle was shown earlier to be the only way used by the bloodstream forms of T. brucei to sustain their high glycolytic flux. PMID:20702405

  19. Increasing seed oil content in oil-seed rape (Brassica napus L.) by over-expression of a yeast glycerol-3-phosphate dehydrogenase under the control of a seed-specific promoter.

    PubMed

    Vigeolas, Helene; Waldeck, Peter; Zank, Thorsten; Geigenberger, Peter

    2007-05-01

    Previous attempts to manipulate oil synthesis in plants have mainly concentrated on the genes involved in the biosynthesis and use of fatty acids, neglecting the possible role of glycerol-3-phosphate supply on the rate of triacylglycerol synthesis. In this study, a yeast gene coding for cytosolic glycerol-3-phosphate dehydrogenase (gpd1) was expressed in transgenic oil-seed rape under the control of the seed-specific napin promoter. It was found that a twofold increase in glycerol-3-phosphate dehydrogenase activity led to a three- to fourfold increase in the level of glycerol-3-phosphate in developing seeds, resulting in a 40% increase in the final lipid content of the seed, with the protein content remaining substantially unchanged. This was accompanied by a decrease in the glycolytic intermediate dihydroxyacetone phosphate, the direct precursor of glycerol-3-phosphate dehydrogenase. The levels of sucrose and various metabolites in the pathway from sucrose to fatty acids remained unaltered. The results show that glycerol-3-phosphate supply co-limits oil accumulation in developing seeds. This has important implications for strategies that aim to increase the overall level of oil in commercial oil-seed crops for use as a renewable alternative to petrol.

  20. The initial step of the glycerolipid pathway: identification of glycerol 3-phosphate/dihydroxyacetone phosphate dual substrate acyltransferases in Saccharomyces cerevisiae.

    PubMed

    Zheng, Z; Zou, J

    2001-11-09

    The initial step of phospholipid biosynthesis in yeast is carried out through the acylation of glycerol 3-phosphate (G-3-P) and dihydroxyacetone phosphate by stereospecific sn-1 acyltransferases. Here we report the identification of two key fatty acyltransferases of the glycerolipid biosynthesis pathway in Saccharomyces cerevisiae. Disruption of the open reading frame YBL011w, corresponding to a gene previously identified as a choline transporter suppressor (SCT1), resulted in a substantial decrease of total cellular G-3-P acyltransferase activity. A yeast strain disrupted at the open reading frame YKR067w, which encodes a protein closely related to Sct1p, also exhibited a dramatic reduction in G-3-P acyltransferase activity. Molecular characterizations of the genes revealed that a missense mutation in YKR067w accounted for a defect in the activities of the G-3-P acyltransferase in the yeast mutant strain TTA1. Heterologous expression of YKR067w in Escherichia coli further confirmed its enzyme activity. These results indicate that YKR067w and YBL011w, designated herein as GAT1 and GAT2(SCT1), respectively, are yeast G-3-P acyltransferase genes. Furthermore, biochemical results are presented to show that both Gat1p and Gat2p(Sct1p) are G-3-P/dihydroxyacetone phosphate dual substrate-specific sn-1 acyltransferases. The fatty acyl specificity of Gat1p is similar to that of the mammalian microsomal G-3-P acyltransferase, as it can effectively utilize a broad range of fatty acids as acyl donors. In contrast, Gat2p(Sct1p) displayed preference toward 16-carbon fatty acids. The most notable of the altered phospholipid compositions of the gat1Delta and gat2(sct1)Delta strains are a decreased phosphatidic acid pool and an increased phosphatidylserine/phosphatidylinositol ratio. This did not appear to affect the mutants as no growth defect was found. However, null mutations of both GAT1 and GAT2(SCT1) are synthetically lethal to yeast.

  1. Characterization and site-directed mutagenesis of a novel class II 5-enopyruvylshikimate-3-phosphate (EPSP) synthase from the deep-sea bacterium Alcanivorax sp. L27.

    PubMed

    Zhang, Yi; Yi, Licong; Lin, Yongjun; Zhang, Lili; Shao, Zongze; Liu, Ziduo

    2014-09-01

    The 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) is a key enzyme in the aromatic amino acid biosynthetic pathway in microorganisms and plants, which catalyzes the formation of 5-enolpyruvylshikimate-3-phosphate (EPSP) from shikimate-3-phosphate (S3P) and phosphoenolpyruvate (PEP). In this study, a novel AroA-encoding gene was identified from the deep sea bacterium Alcanivorax sp. L27 through screening the genomic library and termed as AroAA.sp. A phylogenetic analysis revealed that AroAA.sp (1317 bp and 438 amino acids) is a class II AroA. This enzyme exhibited considerable activity between pH 5.5 and pH 8.0 and notable activity at low temperatures. The KM for PEP and IC50 [glyphosate] values (the concentration of glyphosate that inhibited enzyme activity by 50%) of AroAA.sp were 78 μM and 1.5 mM, respectively. Furthermore, site-directed mutagenesis revealed that the G100A mutant had a 30-fold increase in the IC50 [glyphosate] value; while the L105P mutant showed only 20% catalytic activity compared to wild-type AroAA.sp. The specific activity of the wild-type AroAA.sp, the G100A mutant and the L105P mutant were 7.78 U/mg, 7.26 U/mg and 1.76 U/mg, respectively. This is the first report showing that the G100A mutant of AroA displays considerably improved glyphosate resistance and demonstrates that Leu105 is essential for the enzyme's activity.

  2. Glyceraldehyde-3-phosphate dehydrogenase acts as an adhesin in Erysipelothrix rhusiopathiae adhesion to porcine endothelial cells and as a receptor in recruitment of host fibronectin and plasminogen.

    PubMed

    Zhu, Weifeng; Zhang, Qiang; Li, Jingtao; Wei, Yanmin; Cai, Chengzhi; Liu, Liang; Xu, Zhongmin; Jin, Meilin

    2017-03-21

    Erysipelothrix rhusiopathiae is the causative agent of animal erysipelas and human erysipeloid. Previous studies suggested glyceraldehyde 3-phosphate dehydrogenase (GAPDH) plays a role in the pathogenesis of E. rhusiopathiae infection. We studied E. rhusiopathiae GAPDH interactions with pig vascular endothelial cells, fibronectin, and plasminogen. Recombinant GAPDH (rGAPDH) was successfully obtained, and it was shown that it plays a role in E. rhusiopathiae adhesion to pig vascular endothelial cells. Moreover, rGAPDH could bind fibronectin and plasminogen in a dose-dependent manner. To our knowledge, this is the first study demonstrating that a moonlighting protein plays a role in pathogenesis of E. rhusiopathiae infections.

  3. The 2',4'-dihydroxychalcone could be explored to develop new inhibitors against the glycerol-3-phosphate dehydrogenase from Leishmania species.

    PubMed

    Passalacqua, Thais G; Torres, Fábio A E; Nogueira, Camila T; de Almeida, Leticia; Del Cistia, Mayara L; dos Santos, Mariana B; Regasini, Luis O; Graminha, Márcia A S; Marchetto, Reinaldo; Zottis, Aderson

    2015-09-01

    The enzyme glycerol-3-phosphate dehydrogenase (G3PDH) from Leishmania species is considered as an attractive target to design new antileishmanial drugs and a previous in silico study reported on the importance of chalcones to achieve its inhibition. Here, we report the identification of a synthetic chalcone in our in vitro assays with promastigote cells from Leishmania amazonensis, its biological activity in animal models, and docking followed by molecular dynamics simulation to investigate the molecular interactions and structural patterns that are crucial to achieve the inhibition complex between this compound and G3PDH. A molecular fragment of this natural product derivative can provide new inhibitors with increased potency and selectivity.

  4. Over-expression of Arabidopsis thaliana SFD1/GLY1, the gene encoding plastid localized glycerol-3-phosphate dehydrogenase, increases plastidic lipid content in transgenic rice plants

    PubMed Central

    Siddiqui, Adnan; Singh, Subaran; Banday, Zeeshan Zahoor; Nandi, Ashis Kumar

    2016-01-01

    Lipids are the major constituents of all membranous structures in plants. Plants possess two pathways for lipid biosynthesis: the prokaryotic pathway (i.e., plastidic pathway) and the eukaryotic pathway (i.e., endoplasmic-reticulum (ER) pathway). Whereas some plants synthesize galactolipids from diacylglycerol assembled in the plastid, others, including rice, derive their galactolipids from diacylglycerols assembled by the eukaryotic pathway. Arabidopsis thaliana glycerol-3-phosphate dehydrogenase (G3pDH), coded by SUPPRESSOR OF FATTY ACID DESATURASE 1 (SFD1; alias GLY1) gene, catalyzes the formation of glycerol 3-phosphate (G3p), the backbone of many membrane lipids. Here SFD1 was introduced to rice as a transgene. Arabidopsis SFD1 localizes in rice plastids and its over-expression increases plastidic membrane lipid content in transgenic rice plants without any major impact on ER lipids. The results suggest that over-expression of plastidic G3pDH enhances biosynthesis of plastid-localized lipids in rice. Lipid composition in the transgenic plants is consistent with increased phosphatidylglycerol synthesis in the plastid and increased galactolipid synthesis from diacylglycerol produced via the ER pathway. The transgenic plants show a higher photosynthetic assimilation rate, suggesting a possible application of this finding in crop improvement. PMID:26747130

  5. The specific role of plastidial glycolysis in photosynthetic and heterotrophic cells under scrutiny through the study of glyceraldehyde-3-phosphate dehydrogenase.

    PubMed

    Anoman, Armand Djoro; Flores-Tornero, María; Rosa-Telléz, Sara; Muñoz-Bertomeu, Jesús; Segura, Juan; Ros, Roc

    2016-01-01

    The cellular compartmentalization of metabolic processes is an important feature in plants where the same pathways could be simultaneously active in different compartments. Plant glycolysis occurs in the cytosol and plastids of green and non-green cells in which the requirements of energy and precursors may be completely different. Because of this, the relevance of plastidial glycolysis could be very different depending on the cell type. In the associated study, we investigated the function of plastidial glycolysis in photosynthetic and heterotrophic cells by specifically driving the expression of plastidial glyceraldehyde-3-phosphate dehydrogenase (GAPCp) in a glyceraldehyde-3-phosphate dehydrogenase double mutant background (gapcp1gapcp2). We showed that GAPCp is not functionally significant in photosynthetic cells, while it plays a crucial function in heterotrophic cells. We also showed that (i) GAPCp activity expression in root tips is necessary for primary root growth, (ii) its expression in heterotrophic cells of aerial parts and roots is necessary for plant growth and development, and (iii) GAPCp is an important metabolic connector of carbon and nitrogen metabolism through the phosphorylated pathway of serine biosynthesis (PPSB). We discuss here the role that this pathway could play in the control of plant growth and development.

  6. Metabolomic analysis reveals hepatic metabolite perturbations in citrin/mitochondrial glycerol-3-phosphate dehydrogenase double-knockout mice, a model of human citrin deficiency.

    PubMed

    Saheki, Takeyori; Inoue, Kanako; Ono, Hiromi; Tushima, Anmi; Katsura, Natsumi; Yokogawa, Mana; Yoshidumi, Yukari; Kuhara, Tomiko; Ohse, Morimasa; Eto, Kazuhiro; Kadowaki, Takashi; Sinasac, David S; Kobayashi, Keiko

    2011-12-01

    The citrin/mitochondrial glycerol-3-phosphate dehydrogenase (mGPD) double-knockout mouse displays phenotypic attributes of both neonatal intrahepatic cholestasis and adult-onset type II citrullinemia, making it a suitable model of human citrin deficiency. In the present study, we investigated metabolic disturbances in the livers of wild-type, citrin (Ctrn) knockout, mGPD knockout, and Ctrn/mGPD double-knockout mice following oral sucrose versus saline administration using metabolomic approaches. By using gas chromatography/mass spectrometry and capillary electrophoresis/mass spectrometry, we found three general groupings of metabolite changes in the livers of the double-knockout mice following sucrose administration that were subsequently confirmed using liquid chromatography/mass spectrometry or enzymatic methods: a marked increase of hepatic glycerol 3-phosphate, a generalized decrease of hepatic tricarboxylic acid cycle intermediates, and alterations of hepatic amino acid levels related to the urea cycle or lysine catabolism including marked increases in citrulline and lysine. Furthermore, concurrent oral administration of sodium pyruvate with sucrose ameliorated the hyperammonemia induced by sucrose, as had been shown previously, as well as almost completely normalizing the hepatic metabolite perturbations found. Overall, we have identified additional metabolic disturbances in double-KO mice following oral sucrose administration, and provided further evidence for the therapeutic use of sodium pyruvate in our mouse model of citrin deficiency. Copyright © 2011 Elsevier Inc. All rights reserved.

  7. Safety evaluation of the double mutant 5-enol pyruvylshikimate-3-phosphate synthase (2mEPSPS) from maize that confers tolerance to glyphosate herbicide in transgenic plants.

    PubMed

    Herouet-Guicheney, Corinne; Rouquié, David; Freyssinet, Martine; Currier, Thomas; Martone, Aris; Zhou, Junguo; Bates, Elizabeth E M; Ferullo, Jean-Marc; Hendrickx, Koen; Rouan, Dominique

    2009-07-01

    Glyphosate tolerance can be conferred by decreasing the herbicide's ability to inhibit the enzyme 5-enol pyruvylshikimate-3-phosphate synthase, which is essential for the biosynthesis of aromatic amino acids in all plants, fungi, and bacteria. Glyphosate tolerance is based upon the expression of the double mutant 5-enol pyruvylshikimate-3-phosphate synthase (2mEPSPS) protein. The 2mEPSPS protein, with a lower binding affinity for glyphosate, is highly resistant to the inhibition by glyphosate and thus allows sufficient enzyme activity for the plants to grow in the presence of herbicides that contain glyphosate. Based on both a review of published literature and experimental studies, the potential safety concerns related to the transgenic 2mEPSPS protein were assessed. The safety evaluation supports that the expressed protein is innocuous. The 2mEPSPS enzyme does not possess any of the properties associated with known toxins or allergens, including a lack of amino acid sequence similarity to known toxins and allergens, a rapid degradation in simulated gastric and intestinal fluids, and no adverse effects in mice after intravenous or oral administration (at 10 or 2000 mg/kg body weight, respectively). In conclusion, there is a reasonable certainty of no harm resulting from the inclusion of the 2mEPSPS protein in human food or in animal feed.

  8. Over-expression of Arabidopsis thaliana SFD1/GLY1, the gene encoding plastid localized glycerol-3-phosphate dehydrogenase, increases plastidic lipid content in transgenic rice plants.

    PubMed

    Singh, Vijayata; Singh, Praveen Kumar; Siddiqui, Adnan; Singh, Subaran; Banday, Zeeshan Zahoor; Nandi, Ashis Kumar

    2016-03-01

    Lipids are the major constituents of all membranous structures in plants. Plants possess two pathways for lipid biosynthesis: the prokaryotic pathway (i.e., plastidic pathway) and the eukaryotic pathway (i.e., endoplasmic-reticulum (ER) pathway). Whereas some plants synthesize galactolipids from diacylglycerol assembled in the plastid, others, including rice, derive their galactolipids from diacylglycerols assembled by the eukaryotic pathway. Arabidopsis thaliana glycerol-3-phosphate dehydrogenase (G3pDH), coded by SUPPRESSOR OF FATTY ACID DESATURASE 1 (SFD1; alias GLY1) gene, catalyzes the formation of glycerol 3-phosphate (G3p), the backbone of many membrane lipids. Here SFD1 was introduced to rice as a transgene. Arabidopsis SFD1 localizes in rice plastids and its over-expression increases plastidic membrane lipid content in transgenic rice plants without any major impact on ER lipids. The results suggest that over-expression of plastidic G3pDH enhances biosynthesis of plastid-localized lipids in rice. Lipid composition in the transgenic plants is consistent with increased phosphatidylglycerol synthesis in the plastid and increased galactolipid synthesis from diacylglycerol produced via the ER pathway. The transgenic plants show a higher photosynthetic assimilation rate, suggesting a possible application of this finding in crop improvement.

  9. Overexpression of ACC gene from oleaginous yeast Lipomyces starkeyi enhanced the lipid accumulation in Saccharomyces cerevisiae with increased levels of glycerol 3-phosphate substrates.

    PubMed

    Wang, Jiancai; Xu, Ronghua; Wang, Ruling; Haque, Mohammad Enamul; Liu, Aizhong

    2016-06-01

    The conversion of acetyl-CoA to malonyl-CoA by acetyl-CoA carboxylase (ACC) is the rate-limiting step in fatty acid biosynthesis. In this study, a gene coding for ACC was isolated and characterized from an oleaginous yeast, Lipomyces starkeyi. Real-time quantitative PCR (qPCR) analysis of L. starkeyi acetyl-CoA carboxylase gene (LsACC1) showed that the expression levels were upregulated with the fast accumulation of lipids. The LsACC1 was co-overexpressed with the glycerol 3-phosphate dehydrogenase gene (GPD1), which regulates lipids biosynthesis by supplying another substrates glycerol 3-phosphate for storage lipid assembly, in the non-oleaginous yeast Saccharomyces cerevisiae. Further, the S. cerevisiae acetyl-CoA carboxylase (ScACC1) was transferred with GPD1 and its function was analyzed in comparison with LsACC1. The results showed that overexpressed LsACC1 and GPD1 resulted in a 63% increase in S. cerevisiae. This study gives new data in understanding of the molecular mechanisms underlying the regulation of fatty acids and lipid biosynthesis in yeasts.

  10. Metabolism of aspirin and procaine in mice pretreated with O-4-nitrophenyl methyl(phenyl)phosphinate or O-4-nitrophenyl diphenylphosphinate

    SciTech Connect

    Joly, J.M.; Brown, T.M.

    1986-07-01

    Concentrations of (carboxyl-/sup 14/C)procaine in blood of mice were increased threefold for 27 min by exposure to O-4-nitrophenyl diphenylphosphinate 2 hr prior to (carboxyl-/sup 14/C)procaine injection ip, while there was no effect of O-4-nitrophenyl methyl(phenyl)phosphinate pretreatment. There was no effect of either organophosphinate on the primary hydrolysis of (acetyl-l-/sup 14/C)aspirin when assessed by the expiration of (/sup 14/C)carbon dioxide; however, O-4-nitrophenyl diphenylphosphinate pretreatment produced transient increases in blood concentrations of both (carboxyl-/sup 14/C)aspirin and (carboxyl-/sup 14/C)salicylic acid following administration of (carboxyl-/sup 14/C)aspirin. Liver carboxylesterase activity in O-4-nitrophenyl diphenylphosphinate pretreated mice was 11% of control activity. These results indicate the potential for drug interaction with O-4-nitrophenyl diphenylphosphinate but not with O-4-nitrophenyl methyl(phenyl)phosphinate. It appears that liver carboxylesterase activity has a minor role in hydrolysis of aspirin in vivo, but may be more important in procaine metabolism.

  11. A multidomain enzyme, with glycerol-3-phosphate dehydrogenase and phosphatase activities, is involved in a chloroplastic pathway for glycerol synthesis in Chlamydomonas reinhardtii.

    PubMed

    Morales-Sánchez, Daniela; Kim, Yeongho; Terng, Ee Leng; Peterson, Laura; Cerutti, Heriberto

    2017-03-08

    Understanding the unique features of algal metabolism may be necessary to realize the full potential of algae as feedstock for the production of biofuels and biomaterials. Under nitrogen deprivation, the green alga C. reinhardtii showed substantial triacylglycerol (TAG) accumulation and up-regulation of a gene, GPD2, encoding a multidomain enzyme with a putative phosphoserine phosphatase (PSP) motif fused to glycerol-3-phosphate dehydrogenase (GPD) domains. Canonical GPD enzymes catalyze the synthesis of glycerol-3-phosphate (G3P) by reduction of dihydroxyacetone phosphate (DHAP). G3P forms the backbone of TAGs and membrane glycerolipids and it can be dephosphorylated to yield glycerol, an osmotic stabilizer and compatible solute under hypertonic stress. Recombinant Chlamydomonas GPD2 showed both reductase and phosphatase activities in vitro and it can work as a bifunctional enzyme capable of synthesizing glycerol directly from DHAP. In addition, GPD2 and a gene encoding glycerol kinase were up-regulated in Chlamydomonas cells exposed to high salinity. RNA-mediated silencing of GPD2 revealed that the multidomain enzyme was required for TAG accumulation under nitrogen deprivation and for glycerol synthesis under high salinity. Moreover, a GPD2-mCherry fusion protein was found to localize to the chloroplast, supporting the existence of a GPD2-dependent plastid pathway for the rapid synthesis of glycerol in response to hyperosmotic stress. We hypothesize that the reductase and phosphatase activities of PSP-GPD multidomain enzymes may be modulated by post-translational modifications/mechanisms, allowing them to synthesize primarily G3P or glycerol depending on environmental conditions and/or metabolic demands in algal species of the core Chlorophytes. This article is protected by copyright. All rights reserved.

  12. Site-Directed Mutagenesis from Arg195 to His of a Microalgal Putatively Chloroplastidial Glycerol-3-Phosphate Acyltransferase Causes an Increase in Phospholipid Levels in Yeast

    PubMed Central

    Ouyang, Long-Ling; Li, Hui; Yan, Xiao-Jun; Xu, Ji-Lin; Zhou, Zhi-Gang

    2016-01-01

    To analyze the contribution of glycerol-3-phosphate acyltransferase (GPAT) to the first acylation of glycerol-3-phosphate (G-3-P), the present study focused on a functional analysis of the GPAT gene from Lobosphaera incisa (designated as LiGPAT). A full-length cDNA of LiGPAT consisting of a 1,305-bp ORF, a 1,652-bp 5′-UTR, and a 354-bp 3′-UTR, was cloned. The ORF encoded a 434-amino acid peptide, of which 63 residues at the N-terminus defined a chloroplast transit peptide. Multiple sequence alignment and phylogeny analysis of GPAT homologs provided the convincible bioinformatics evidence that LiGPAT was localized to chloroplasts. Considering the conservation of His among the G-3-P binding sites from chloroplastidial GPATs and the substitution of His by Arg at position 195 in the LiGPAT mature protein (designated mLiGPAT), we established the heterologous expression of either mLiGPAT or its mutant (Arg195His) (sdmLiGPAT) in the GPAT-deficient yeast mutant gat1Δ. Lipid profile analyses of these transgenic yeasts not only validated the acylation function of LiGPAT but also indicated that the site-directed mutagenesis from Arg195 to His led to an increase in the phospholipid level in yeast. Semi-quantitative analysis of mLiGPAT and sdmLiGPAT, together with the structural superimposition of their G-3-P binding sites, indicated that the increased enzymatic activity was caused by the enlarged accessible surface of the phosphate group binding pocket when Arg195 was mutated to His. Thus, the potential of genetic manipulation of GPAT to increase the glycerolipid level in L. incisa and other microalgae would be of great interest. PMID:27014309

  13. Differential response of the catalase, superoxide dismutase and glycerol-3-phosphate dehydrogenase to different environmental stresses in Debaryomyces nepalensis NCYC 3413.

    PubMed

    Kumar, Sawan; Kalyanasundaram, Gayathiri T; Gummadi, Sathyanarayana N

    2011-02-01

    The effect of salt, pH, and temperature stress on the cellular level of antioxidant enzymes, catalase and superoxide dismutase (SOD) and glycerol-3-phosphate dehydrogenase (G3PDH) was studied in Debaryomyces nepalensis NCYC 3413, a halotolerant yeast. The catalase activity increased in different phases, while SOD and G3PDH activities declined in late stationary phase. A significant increase in SOD activity was observed under different stress as compared to control. Salt and temperature stress enhanced the catalase activity where as it was suppressed by pH stress. G3PDH level increased with salt stress, however, no significant change was observed under pH and temperature stress. The observations recorded in this investigation suggested that D. nepalensis has an efficient protective mechanism of antioxidant enzymes and G3PDH against salt, pH, and temperature stresses.

  14. Double-Mutated 5-Enol Pyruvylshikimate-3-phosphate Synthase Protein Expressed in MZHG0JG Corn (Zea mays L.) Has No Impact on Toxicological Safety and Nutritional Composition.

    PubMed

    Matthews, Bethany A; Launis, Karen L; Bauman, Patricia A; Juba, Nicole C

    2017-09-27

    MZHG0JG corn will offer growers the flexibility to alternate between herbicides with two different modes of action in their weed-management programs, helping to mitigate and manage the evolution of herbicide resistance in weed populations. The proteins conferring herbicide tolerence in MZHG0JG corn, double-mutated 5-enol pyruvylshikimate-3-phosphate synthase protein (mEPSPS) and phosphinothricin acetyltransferase (PAT), as well as the MZHG0JG corn event, have been assessed by regulatory authorities globally and have been determined to be safe for humans, animals, and the environment. In addition to the safety data available for these proteins, further studies were conducted on MZHG0JG corn to assess levels of mEPSPS as compared to previously registered genetically modified (GM) corn. The results support the conclusion of no impact on toxicological safety or nutritional composition.

  15. Studies of the molecular pathogenesis of hexane neuropathy. I. evaluation of the inhibition of glyceraldehyde-3-phosphate dehydrogenase by 2,5-hexanedione.

    PubMed

    Graham, D G; Abou-Donia, M B

    1980-05-01

    Inhibition of the sulfhydryl enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) by 2,5-hexanedione (2,5-HD) was found to be irreversible, proceeding via a reversible enzyme-inhibitor intermediate, while acetone was a weak reversible inhibitor. Comparison of 2,5-HD and acetone with p-chloromercuribenzoate (PCMB) and N-ethylmaleimide (NEM) demonstrated that the former are not significant sulfhydryl reagents, since they must be present at more than 10(4) times higher concentrations than PCMB or NEM to effect measurable inhibition of this enzyme. Thus it is unlikely that inhibition of GAPDH by 2,5-HD has any significance in the molecular pathogenesis of hexane neuropathy. The irreversibility of 2,5-HD inhibition, on the othe hand, suggests that 2,5-HD reacts with amino groups rather than sulfhydryl groups on proteins. This reaction is proposed as the molecular lesion in hexane neuropathy.

  16. Metabolic and structural evidence for the existence of a third species of polyphosphoinositide in cells: D-phosphatidyl-myo-inositol 3-phosphate.

    PubMed Central

    Stephens, L; Hawkins, P T; Downes, C P

    1989-01-01

    When human 1321 N1 astrocytoma cells were labelled to steady state with [3H]inositol and briefly with [32P]orthophosphate, a compound which contained both radiotracers and which co-migrated with phosphatidylinositol-myo-inositol 4-phosphate during t.l.c. could be extracted in acidic chloroform/methanol. Treatment with methylamine under conditions which lead to deacylation of conventional glycerophospholipids yielded a water-soluble moiety which was labelled with both radioisotopes and was eluted from an anion-exchange h.p.l.c. column with a retention time similar to, but distinct from, that of glycerophosphoinositol 4-phosphate. Experiments using sodium periodate and selective phosphatase enzymes to degrade this compound systematically generated a series of products which suggested the structure of the parent phospholipid was phosphatidyl-myo-inositol 3-phosphate (PtdIns3P). PtdIns3P is metabolically closely related to the pool(s) of inositol phospholipid(s) that serves as substrate(s) for an agonist-sensitive phosphoinositidase C, as the levels of PtdIns3P fell significantly when 1321 N1 cells were stimulated with carbachol. The relative rate of turnover of the inositol moiety of PtdIns3P is similar to that of both of the major polyphosphoinositides and significantly higher than that of total cellular phosphatidyl-myo-inositol. This suggests that all three polyphosphoinositides are synthesized from a common, rapidly metabolized, pool of phosphatidyl-myo-inositol. PMID:2541684

  17. Seasonal freeze resistance of rainbow smelt (Osmerus mordax) is generated by differential expression of glycerol-3-phosphate dehydrogenase, phosphoenolpyruvate carboxykinase, and antifreeze protein genes.

    PubMed

    Liebscher, Ryan S; Richards, Robert C; Lewis, Johanne M; Short, Connie E; Muise, Denise M; Driedzic, William R; Ewart, K Vanya

    2006-01-01

    In winter, rainbow smelt (Osmerus mordax) accumulate glycerol and produce an antifreeze protein (AFP), which both contribute to freeze resistance. The role of differential gene expression in the seasonal pattern of these adaptations was investigated. First, cDNAs encoding smelt and Atlantic salmon (Salmo salar) phosphoenolpyruvate carboxykinase (PEPCK) and smelt glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were cloned so that all sequences required for expression analysis would be available. Using quantitative PCR, expression of beta actin in rainbow smelt liver was compared with that of GAPDH in order to determine its validity as a reference gene. Then, levels of glycerol-3-phosphate dehydrogenase (GPDH), PEPCK, and AFP relative to beta actin were measured in smelt liver over a fall-winter-spring interval. Levels of GPDH mRNA increased in the fall just before plasma glycerol accumulation, implying a driving role in glycerol synthesis. GPDH mRNA levels then declined during winter, well in advance of serum glycerol, suggesting the possibility of GPDH enzyme or glycerol conservation in smelt during the winter months. PEPCK mRNA levels rose in parallel with serum glycerol in the fall, consistent with an increasing requirement for amino acids as metabolic precursors, remained elevated for much of the winter, and then declined in advance of the decline in plasma glycerol. AFP mRNA was elevated at the onset of fall sampling in October and remained elevated until April, implying separate regulation from GPDH and PEPCK. Thus, winter freezing point depression in smelt appears to result from a seasonal cycle of GPDH gene expression, with an ensuing increase in the expression of PEPCK, and a similar but independent cycle of AFP gene expression.

  18. The Class II Phosphatidylinositol 3-Phosphate Kinase PIK3C2A Promotes Shigella flexneri Dissemination through Formation of Vacuole-Like Protrusions

    PubMed Central

    Dragoi, Ana-Maria

    2015-01-01

    Intracellular pathogens such as Shigella flexneri and Listeria monocytogenes achieve dissemination in the intestinal epithelium by displaying actin-based motility in the cytosol of infected cells. As they reach the cell periphery, motile bacteria form plasma membrane protrusions that resolve into vacuoles in adjacent cells, through a poorly understood mechanism. Here, we report on the role of the class II phosphatidylinositol 3-phosphate kinase PIK3C2A in S. flexneri dissemination. Time-lapse microscopy revealed that PIK3C2A was required for the resolution of protrusions into vacuoles through the formation of an intermediate membrane-bound compartment that we refer to as a vacuole-like protrusion (VLP). Genetic rescue of PIK3C2A depletion with RNA interference (RNAi)-resistant cDNA constructs demonstrated that VLP formation required the activity of PIK3C2A in primary infected cells. PIK3C2A expression was required for production of phosphatidylinositol 3-phosphate [PtdIns(3)P] at the plasma membrane surrounding protrusions. PtdIns(3)P production was not observed in the protrusions formed by L. monocytogenes, whose dissemination did not rely on PIK3C2A. PIK3C2A-mediated PtdIns(3)P production in S. flexneri protrusions was regulated by host cell tyrosine kinase signaling and relied on the integrity of the S. flexneri type 3 secretion system (T3SS). We suggest a model of S. flexneri dissemination in which the formation of VLPs is mediated by the PIK3C2A-dependent production of the signaling lipid PtdIns(3)P in the protrusion membrane, which relies on the T3SS-dependent activation of tyrosine kinase signaling in protrusions. PMID:25667265

  19. Glycerol-3-Phosphate Acyltranferase-2 Behaves as a Cancer Testis Gene and Promotes Growth and Tumorigenicity of the Breast Cancer MDA-MB-231 Cell Line

    PubMed Central

    Lacunza, Ezequiel; Garcia-Fabiani, Maria B.; Soler-Gerino, Mercedes C.; Cattaneo, Elizabeth R.; Quiroga, Ivana Y.; Abba, Martin C.; Coleman, Rosalind A.; Gonzalez-Baro, Maria R.

    2014-01-01

    The de novo synthesis of glycerolipids in mammalian cells begins with the acylation of glycerol-3-phosphate, catalyzed by glycerol-3-phosphate acyltransferase (GPAT). GPAT2 is a mitochondrial isoform primarily expressed in testis under physiological conditions. Because it is aberrantly expressed in multiple myeloma, it has been proposed as a novel cancer testis gene. Using a bioinformatics approach, we found that GPAT2 is highly expressed in melanoma, lung, prostate and breast cancer, and we validated GPAT2 expression at the protein level in breast cancer by immunohistochemistry. In this case GPAT2 expression correlated with a higher histological grade. 5-Aza-2′ deoxycytidine treatment of human cells lines induced GPAT2 expression suggesting epigenetic regulation of gene expression. In order to evaluate the contribution of GPAT2 to the tumor phenotype, we silenced its expression in MDA-MB-231 cells. GPAT2 knockdown diminished cell proliferation, anchorage independent growth, migration and tumorigenicity, and increased staurosporine-induced apoptosis. In contrast, GPAT2 over-expression increased cell proliferation rate and resistance to staurosporine-induced apoptosis. To understand the functional role of GPAT2, we performed a co-expression analysis in mouse and human testis and found a significant association with semantic terms involved in cell cycle, DNA integrity maintenance, piRNA biogenesis and epigenetic regulation. Overall, these results indicate the GPAT2 would be directly associated with the control of cell proliferation. In conclusion, we confirm GPAT2 as a cancer testis gene and that its expression contributes to the tumor phenotype of MDA-MB-231 cells. PMID:24967918

  20. High-resolution crystal structures of the photoreceptor glyceraldehyde 3-phosphate dehydrogenase (GAPDH) with three and four-bound NAD molecules.

    PubMed

    Baker, Bo Y; Shi, Wuxian; Wang, Benlian; Palczewski, Krzysztof

    2014-11-01

    Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) catalyzes the oxidative phosphorylation of d-glyceraldehyde 3-phosphate (G3P) into 1,3-diphosphoglycerate (BGP) in the presence of the NAD cofactor. GAPDH is an important drug target because of its central role in glycolysis, and nonglycolytic processes such as nuclear RNA transport, DNA replication/repair, membrane fusion and cellular apoptosis. Recent studies found that GAPDH participates in the development of diabetic retinopathy and its progression after the cessation of hyperglycemia. Here, we report two structures for native bovine photoreceptor GAPDH as a homotetramer with differing occupancy by NAD, bGAPDH(NAD)4 , and bGAPDH(NAD)3 . The bGAPDH(NAD)4 was solved at 1.52 Å, the highest resolution for GAPDH. Structural comparison of the bGAPDH(NAD)4 and bGAPDH(NAD)3 models revealed novel details of conformational changes induced by cofactor binding, including a loop region (residues 54-56). Structure analysis of bGAPDH confirmed the importance of Phe34 in NAD binding, and demonstrated that Phe34 was stabilized in the presence of NAD but displayed greater mobility in its absence. The oxidative state of the active site Cys149 residue is regulated by NAD binding, because this residue was found oxidized in the absence of dinucleotide. The distance between Cys149 and His176 decreased upon NAD binding and Cys149 remained in a reduced state when NAD was bound. These findings provide an important structural step for understanding the mechanism of GAPDH activity in vision and its pathological role in retinopathies.

  1. Studies on the metabolism of diethyl 4-nitrophenyl phosphorothionate (parathion) in vitro.

    PubMed

    Neal, R A

    1967-04-01

    1. The metabolism of the phosphorothionate parathion in vitro was examined by using [(32)P]parathion and microsomes isolated from the livers of various animal species. 2. The major metabolic products of parathion in this system in vitro were identified as diethyl 4-nitrophenyl phosphate (paraoxon), diethyl hydrogen phosphate, diethyl hydrogen phosphorothionate and p-nitrophenol. 3. The reaction leading to the formation of diethyl hydrogen phosphorothionate and p-nitrophenol requires the same cofactors (NADPH and oxygen) required for metabolism of parathion to its active anti-acetylcholinesterase paraoxon. 4. The enzyme activity towards parathion per unit weight of liver is increased some 65-130% by pretreatment of male rats with phenobarbital and 3,4-benzopyrene. 5. The metabolism of parathion is inhibited by incubation in a nitrogen atmosphere and in an atmosphere containing carbon monoxide. Pure oxygen is also inhibitory. These results are discussed in terms of a deficiency of oxygen for maximal activity as well as the lability of some component of the system to oxidation.

  2. The control of fatty acid and triglyceride synthesis in rat epididymal adipose tissue. Roles of coenzyme A derivatives, citrate and l-glycerol 3-phosphate

    PubMed Central

    Denton, R. M.; Halperin, M. L.

    1968-01-01

    1. Methods are described for the extraction and assay of acetyl-CoA and of total acid-soluble and total acid-insoluble CoA derivatives in rat epididymal adipose tissue. 2. The concentration ranges of the CoA derivatives in fat pads incubated in vitro under various conditions were: total acid-soluble CoA, 0·20–0·59mm; total acid-insoluble CoA, 0·08–0·23mm; acetyl-CoA, 0·03–0·14mm. 3. An investigation was made of some postulated mechanisms of control of fatty acid and triglyceride synthesis in rat epididymal fat pads incubated in vitro. The concentrations of intermediates of possible regulatory significance were measured at various rates of fatty acid and triglyceride synthesis produced by the addition to the incubation medium (Krebs bicarbonate buffer containing glucose) of insulin, adrenaline, albumin, palmitate or acetate. 4. The whole-tissue concentrations of glucose 6-phosphate, l-glycerol 3-phosphate, citrate, acetyl-CoA, total acid-soluble CoA and total acid-insoluble CoA were assayed after 30 or 60min. incubation. The rates of fatty acid and triglyceride synthesis, calculated from the incorporation of [U-14C]glucose into fatty acids and glyceride glycerol respectively, and the rates of glucose uptake, lactate plus pyruvate output and glycerol output were measured over a 60min. incubation. 5. The rate of triglyceride synthesis could not be correlated with the concentrations of either l-glycerol 3-phosphate or long-chain fatty acyl-CoA (measured as total acid-insoluble CoA). Factor(s) other than the whole-tissue concentrations of these recognized precursors appear to be involved in the determination of the rate of triglyceride synthesis. 6. No relationship was found between the rate of fatty acid synthesis and the whole-tissue concentrations of the intermediates, citrate or acetyl-CoA, or with the two proposed effectors of acetyl-CoA carboxylase, citrate (as activator) or long-chain fatty acyl-CoA (as inhibitor). The control of fatty acid synthesis

  3. Plastidial Glycolytic Glyceraldehyde-3-Phosphate Dehydrogenase Is an Important Determinant in the Carbon and Nitrogen Metabolism of Heterotrophic Cells in Arabidopsis1

    PubMed Central

    Anoman, Armand D.; Muñoz-Bertomeu, Jesús; Rosa-Téllez, Sara; Flores-Tornero, María; Serrano, Ramón; Bueso, Eduardo; Fernie, Alisdair R.; Segura, Juan; Ros, Roc

    2015-01-01

    This study functionally characterizes the Arabidopsis (Arabidopsis thaliana) plastidial glycolytic isoforms of glyceraldehyde-3-phosphate dehydrogenase (GAPCp) in photosynthetic and heterotrophic cells. We expressed the enzyme in gapcp double mutants (gapcp1gapcp2) under the control of photosynthetic (Rubisco small subunit RBCS2B [RBCS]) or heterotrophic (phosphate transporter PHT1.2 [PHT]) cell-specific promoters. Expression of GAPCp1 under the control of RBCS in gapcp1gapcp2 had no significant effect on the metabolite profile or growth in the aerial part (AP). GAPCp1 expression under the control of the PHT promoter clearly affected Arabidopsis development by increasing the number of lateral roots and having a major effect on AP growth and metabolite profile. Our results indicate that GAPCp1 is not functionally important in photosynthetic cells but plays a fundamental role in roots and in heterotrophic cells of the AP. Specifically, GAPCp activity may be required in root meristems and the root cap for normal primary root growth. Transcriptomic and metabolomic analyses indicate that the lack of GAPCp activity affects nitrogen and carbon metabolism as well as mineral nutrition and that glycerate and glutamine are the main metabolites responding to GAPCp activity. Thus, GAPCp could be an important metabolic connector of glycolysis with other pathways, such as the phosphorylated pathway of serine biosynthesis, the ammonium assimilation pathway, or the metabolism of γ-aminobutyrate, which in turn affect plant development. PMID:26134167

  4. Plastidial Glycolytic Glyceraldehyde-3-Phosphate Dehydrogenase Is an Important Determinant in the Carbon and Nitrogen Metabolism of Heterotrophic Cells in Arabidopsis.

    PubMed

    Anoman, Armand D; Muñoz-Bertomeu, Jesús; Rosa-Téllez, Sara; Flores-Tornero, María; Serrano, Ramón; Bueso, Eduardo; Fernie, Alisdair R; Segura, Juan; Ros, Roc

    2015-11-01

    This study functionally characterizes the Arabidopsis (Arabidopsis thaliana) plastidial glycolytic isoforms of glyceraldehyde-3-phosphate dehydrogenase (GAPCp) in photosynthetic and heterotrophic cells. We expressed the enzyme in gapcp double mutants (gapcp1gapcp2) under the control of photosynthetic (Rubisco small subunit RBCS2B [RBCS]) or heterotrophic (phosphate transporter PHT1.2 [PHT]) cell-specific promoters. Expression of GAPCp1 under the control of RBCS in gapcp1gapcp2 had no significant effect on the metabolite profile or growth in the aerial part (AP). GAPCp1 expression under the control of the PHT promoter clearly affected Arabidopsis development by increasing the number of lateral roots and having a major effect on AP growth and metabolite profile. Our results indicate that GAPCp1 is not functionally important in photosynthetic cells but plays a fundamental role in roots and in heterotrophic cells of the AP. Specifically, GAPCp activity may be required in root meristems and the root cap for normal primary root growth. Transcriptomic and metabolomic analyses indicate that the lack of GAPCp activity affects nitrogen and carbon metabolism as well as mineral nutrition and that glycerate and glutamine are the main metabolites responding to GAPCp activity. Thus, GAPCp could be an important metabolic connector of glycolysis with other pathways, such as the phosphorylated pathway of serine biosynthesis, the ammonium assimilation pathway, or the metabolism of γ-aminobutyrate, which in turn affect plant development.

  5. Improved production of 2,3-butanediol in Bacillus amyloliquefaciens by over-expression of glyceraldehyde-3-phosphate dehydrogenase and 2,3-butanediol dehydrogenase.

    PubMed

    Yang, Taowei; Rao, Zhiming; Zhang, Xian; Xu, Meijuan; Xu, Zhenghong; Yang, Shang-Tian

    2013-01-01

    Previously, a safe strain, Bacillus amyloliquefaciens B10-127 was identified as an excellent candidate for industrial-scale microbial fermentation of 2,3-butanediol (2,3-BD). However, B. amyloliquefaciens fermentation yields large quantities of acetoin, lactate and succinate as by-products, and the 2,3-BD yield remains prohibitively low for commercial production. In the 2,3-butanediol metabolic pathway, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) catalyzes the conversion of 3-phosphate glyceraldehyde to 1,3-bisphosphoglycerate, with concomitant reduction of NAD(+) to NADH. In the same pathway, 2,3-BD dehydrogenase (BDH) catalyzes the conversion of acetoin to 2,3-BD with concomitant oxidation of NADH to NAD(+). In this study, to improve 2,3-BD production, we first over-produced NAD(+)-dependent GAPDH and NADH-dependent BDH in B. amyloliquefaciens. Excess GAPDH reduced the fermentation time, increased the 2,3-BD yield by 12.7%, and decreased the acetoin titer by 44.3%. However, the process also enhanced lactate and succinate production. Excess BDH increased the 2,3-BD yield by 16.6% while decreasing acetoin, lactate and succinate production, but prolonged the fermentation time. When BDH and GAPDH were co-overproduced in B. amyloliquefaciens, the fermentation time was reduced. Furthermore, in the NADH-dependent pathways, the molar yield of 2,3-BD was increased by 22.7%, while those of acetoin, lactate and succinate were reduced by 80.8%, 33.3% and 39.5%, relative to the parent strain. In fed-batch fermentations, the 2,3-BD concentration was maximized at 132.9 g/l after 45 h, with a productivity of 2.95 g/l·h. Co-overexpression of bdh and gapA genes proved an effective method for enhancing 2,3-BD production and inhibiting the accumulation of unwanted by-products (acetoin, lactate and succinate). To our knowledge, we have attained the highest 2,3-BD fermentation yield thus far reported for safe microorganisms.

  6. Improved Production of 2,3-Butanediol in Bacillus amyloliquefaciens by Over-Expression of Glyceraldehyde-3-Phosphate Dehydrogenase and 2,3-butanediol Dehydrogenase

    PubMed Central

    Yang, Taowei; Rao, Zhiming; Zhang, Xian; Xu, Meijuan; Xu, Zhenghong; Yang, Shang-Tian

    2013-01-01

    Background Previously, a safe strain, Bacillus amyloliquefaciens B10-127 was identified as an excellent candidate for industrial-scale microbial fermentation of 2,3-butanediol (2,3-BD). However, B. amyloliquefaciens fermentation yields large quantities of acetoin, lactate and succinate as by-products, and the 2,3-BD yield remains prohibitively low for commercial production. Methodology/Principal Findings In the 2,3-butanediol metabolic pathway, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) catalyzes the conversion of 3-phosphate glyceraldehyde to 1,3-bisphosphoglycerate, with concomitant reduction of NAD+ to NADH. In the same pathway, 2,3-BD dehydrogenase (BDH) catalyzes the conversion of acetoin to 2,3-BD with concomitant oxidation of NADH to NAD+. In this study, to improve 2,3-BD production, we first over-produced NAD+-dependent GAPDH and NADH-dependent BDH in B. amyloliquefaciens. Excess GAPDH reduced the fermentation time, increased the 2,3-BD yield by 12.7%, and decreased the acetoin titer by 44.3%. However, the process also enhanced lactate and succinate production. Excess BDH increased the 2,3-BD yield by 16.6% while decreasing acetoin, lactate and succinate production, but prolonged the fermentation time. When BDH and GAPDH were co-overproduced in B. amyloliquefaciens, the fermentation time was reduced. Furthermore, in the NADH-dependent pathways, the molar yield of 2,3-BD was increased by 22.7%, while those of acetoin, lactate and succinate were reduced by 80.8%, 33.3% and 39.5%, relative to the parent strain. In fed-batch fermentations, the 2,3-BD concentration was maximized at 132.9 g/l after 45 h, with a productivity of 2.95 g/l·h. Conclusions/Significance Co-overexpression of bdh and gapA genes proved an effective method for enhancing 2,3-BD production and inhibiting the accumulation of unwanted by-products (acetoin, lactate and succinate). To our knowledge, we have attained the highest 2,3-BD fermentation yield thus far reported for safe

  7. The E3 ubiquitin-ligase SEVEN IN ABSENTIA like 7 mono-ubiquitinates glyceraldehyde-3-phosphate dehydrogenase 1 isoform in vitro and is required for its nuclear localization in Arabidopsis thaliana.

    PubMed

    Peralta, Diego A; Araya, Alejandro; Busi, Maria V; Gomez-Casati, Diego F

    2016-01-01

    The E3 ubiquitin-protein ligases are associated to various processes such as cell cycle control and diverse developmental pathways. Arabidopsis thaliana SEVEN IN ABSENTIA like 7, which has ubiquitin ligase activity, is located in the nucleus and cytosol and is expressed at several stages in almost all plant tissues suggesting an important role in plant functions. However, the mechanism underlying the regulation of this protein is unknown. Since we found that the SEVEN IN ABSENTIA like 7 gene expression is altered in plants with impaired mitochondria, and in plants deficient in the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase 1, we decided to study the possible interactions between both proteins as potential partners in plant signaling functions. We found that SEVEN IN ABSENTIA like 7 is able to interact in vitro with glyceraldehyde-3-phosphate dehydrogenase and that the Lys231 residue of the last is essential for this function. Following the interaction, a concomitant increase in the glyceraldehyde-3-phosphate dehydrogenase catalytic activity was observed. However, when SEVEN IN ABSENTIA like 7 was supplemented with E1 and E2 proteins to form a complete E1-E2-E3 modifier complex, we observed the mono-ubiquitination of glyceraldehyde-3-phosphate dehydrogenase 1 at the Lys76 residue and a dramatic decrease of its catalytic activity. Moreover, we found that localization of glyceraldehyde-3-phosphate dehydrogenase 1 in the nucleus is dependent on the expression SEVEN IN ABSENTIA like 7. These observations suggest that the association of both proteins might result in different biological consequences in plants either through affecting the glycolytic flux or via cytoplasm-nucleus relocation. Copyright © 2015 Elsevier Ltd. All rights reserved.

  8. Phosphorus-31, sup 15 N, and sup 13 C NMR of glyphosate: Comparison of pH titrations to the herbicidal dead-end complex with 5-enolpyruvoylshikimate-3-phosphate synthase

    SciTech Connect

    Castellino, S.; Leo, G.C.; Sammons, R.D.; Sikorski, J.A. )

    1989-05-02

    The herbicidal dead-end ternary complex (E{sup S3P}{sub Glyph}) of glyphosate (N-(phosphonomethyl)glycine) with 5-enolpyruvoylshikimate-3-phosphate synthase (EPSPS) and the substrate shikimate 3-phosphate (S3P) has been characterized by {sup 31}P, {sup 15}N, and {sup 13}C NMR. The NMR spectra of EPSPS-bound glyphosate show unique chemical shifts ({delta}) for each of the three nuclei. By {sup 31}P NMR, glyphosate in the dead-end complex is a distinct species 3.5 ppm downfield from free glyphosate. The {sup 13}C signal of glyphosate in the dead-end complex is shifted 4 ppm downfield from that of free glyphosate. The {sup 15}N signal for glyphosate (99%) in the dead-end complex is 5 ppm further downfield than that of any free zwitterionic species and 10 ppm downfield from that of the average free species at pH 10.1. The structures of each ionic state of glyphosate are modeled with force field calculations by using MacroModel. A correlation is made for the {sup 31}P {delta} and the C-P-O bond angle, and the {sup 13}C and {sup 15}N {delta} values are postulated to be related to C-C-O and C-N-C bond angles, respectively. The downfield {sup 31}P chemical shift perturbation for S3P in the EPSPS binary complex is consistent with ionization of the 3-phosphate of S3P upon binding. Comparison with the S3P {sup 31}P {delta} vs pH titration curve specifies predominantly the dianion of the 3-phosphate in the E{sup S3P} binary complex, while the E{sup S3P}{sub Glyph} complex indicates net protonation at the 3-phosphate. Chemical shift perturbations of this latter type may be explained by changes in the O-P-O bond angle.

  9. The Expression of Glyceraldehyde-3-Phosphate Dehydrogenase Associated Cell Cycle (GACC) Genes Correlates with Cancer Stage and Poor Survival in Patients with Solid Tumors

    PubMed Central

    Wang, Dunrui; Moothart, Daniel R.; Lowy, Douglas R.; Qian, Xiaolan

    2013-01-01

    Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is often used as a stable housekeeping marker for constant gene expression. However, the transcriptional levels of GAPDH may be highly up-regulated in some cancers, including non-small cell lung cancers (NSCLC). Using a publically available microarray database, we identified a group of genes whose expression levels in some cancers are highly correlated with GAPDH up-regulation. The majority of the identified genes are cell cycle-dependent (GAPDH Associated Cell Cycle, or GACC). The up-regulation pattern of GAPDH positively associated genes in NSCLC is similar to that observed in cultured fibroblasts grown under conditions that induce anti-senescence. Data analysis demonstrated that up-regulated GAPDH levels are correlated with aberrant gene expression related to both glycolysis and gluconeogenesis pathways. Down-regulation of fructose-1,6-bisphosphatase (FBP1) in gluconeogenesis in conjunction with up-regulation of most glycolytic genes is closely related to high expression of GAPDH in the tumors. The data presented demonstrate that up-regulation of GAPDH positively associated genes is proportional to the malignant stage of various tumors and is associated with an unfavourable prognosis. Thus, this work suggests that GACC genes represent a potential new signature for cancer stage identification and disease prognosis. PMID:23620736

  10. Apicoplast-Localized Lysophosphatidic Acid Precursor Assembly Is Required for Bulk Phospholipid Synthesis in Toxoplasma gondii and Relies on an Algal/Plant-Like Glycerol 3-Phosphate Acyltransferase

    PubMed Central

    Callahan, Damien L.; Dubois, David; van Dooren, Giel G.; Shears, Melanie J.; Cesbron-Delauw, Marie-France; Maréchal, Eric; McConville, Malcolm J.; McFadden, Geoffrey I.; Yamaryo-Botté, Yoshiki; Botté, Cyrille Y.

    2016-01-01

    Most apicomplexan parasites possess a non-photosynthetic plastid (the apicoplast), which harbors enzymes for a number of metabolic pathways, including a prokaryotic type II fatty acid synthesis (FASII) pathway. In Toxoplasma gondii, the causative agent of toxoplasmosis, the FASII pathway is essential for parasite growth and infectivity. However, little is known about the fate of fatty acids synthesized by FASII. In this study, we have investigated the function of a plant-like glycerol 3-phosphate acyltransferase (TgATS1) that localizes to the T. gondii apicoplast. Knock-down of TgATS1 resulted in significantly reduced incorporation of FASII-synthesized fatty acids into phosphatidic acid and downstream phospholipids and a severe defect in intracellular parasite replication and survival. Lipidomic analysis demonstrated that lipid precursors are made in, and exported from, the apicoplast for de novo biosynthesis of bulk phospholipids. This study reveals that the apicoplast-located FASII and ATS1, which are primarily used to generate plastid galactolipids in plants and algae, instead generate bulk phospholipids for membrane biogenesis in T. gondii. PMID:27490259

  11. Determination of Cellular Phosphatidylinositol-3-phosphate (PI3P) Levels Using a Fluorescently Labelled Selective PI3P Binding Domain (PX)

    PubMed Central

    Munson, Michael J.; Ganley, Ian G.

    2017-01-01

    The lipid Phosphatidylinositol-3-phosphate [PtdIns3P or PI(3)P] plays many membrane trafficking roles and is primarily produced by the Class III PI3K, VPS34. Determining the level of cellular PI(3)P however can be complex. Extraction of cellular lipids by methanol/chloroform can struggle to separate and identify distinct phospholipid species. Alternately mass spectrometry may be utilised but this requires significant set up of specialised equipment and time to utilise. Use of a PI(3)P-binding-specific recombinant protein domain is a quick method for ascertaining cellular PI(3)P levels and can also allow visualisation of sub-cellular localisation. The PX domain of p40phox (herein referred to as PX) is very specific for PI(3)P over other phospholipid species (Kanai et al., 2001). However, expressing PX directly in cells can be problematic, as it will act in a dominant negative manner to bind and sequester PI(3)P with greater affinity than endogenous proteins, thus disturbing cellular pathways and the normal balance of PI(3)P levels. Using fluorescently labelled PX following cell fixation is therefore more suitable, as it is able to highlight PI(3)P rich structures without risk of perturbing the system. PMID:28127574

  12. Protein recognition of the S23906-1-DNA adduct by nuclear proteins: direct involvement of glyceraldehyde-3-phosphate dehydrogenase (GAPDH).

    PubMed

    Lenglet, Gaëlle; Depauw, Sabine; Mendy, Denise; David-Cordonnier, Marie-Hélène

    2013-05-15

    In a view to develop new DNA alkylating antitumour drugs, evaluating the precise mechanism of action and the molecular/cellular consequences of the alkylation is a point of major interest. The benzo-b-acronycine derivative S23906-1 alkylates guanine nucleobases in the minor groove of the DNA helix and presents an original ability to locally open the double helix of DNA, which appears to be associated with its cytotoxic activity. However, the molecular mechanism linking adduct formation to cellular consequences is not precisely known. The objective of the present study was to identify proteins involved in the recognition and mechanism of action of S23906-DNA adducts. We found that GAPDH (glyceraldehyde-3-phosphate dehydrogenase) is a protein that binds to S23906-alkylated single-stranded, double-stranded and telomeric sequences in a drug-dependent and DNA sequence/structure-dependent manner. We used the CASTing (cyclic amplification of sequence targeting) method to identify GAPDH DNA-binding selectivity and then evaluated its binding to such selected S23906-alkylated sequences. At the cellular level, alkylation of S23906-1 results in an increase in the binding of GAPDH and its protein partner HMG (high-mobility group) B1 to the chromatin. Regarding the multiple roles of GAPDH in apoptosis and DNA repair, the cytotoxic and apoptotic activities of GAPDH were evaluated and present opposite effects in two different cellular models.

  13. Evolutionary engineering of a glycerol-3-phosphate dehydrogenase-negative, acetate-reducing Saccharomyces cerevisiae strain enables anaerobic growth at high glucose concentrations.

    PubMed

    Guadalupe-Medina, Víctor; Metz, Benjamin; Oud, Bart; van Der Graaf, Charlotte M; Mans, Robert; Pronk, Jack T; van Maris, Antonius J A

    2014-01-01

    Glycerol production by Saccharomyces cerevisiae, which is required for redox-cofactor balancing in anaerobic cultures, causes yield reduction in industrial bioethanol production. Recently, glycerol formation in anaerobic S. cerevisiae cultures was eliminated by expressing Escherichia coli (acetylating) acetaldehyde dehydrogenase (encoded by mhpF) and simultaneously deleting the GPD1 and GPD2 genes encoding glycerol-3-phosphate dehydrogenase, thus coupling NADH reoxidation to reduction of acetate to ethanol. Gpd⁻ strains are, however, sensitive to high sugar concentrations, which complicates industrial implementation of this metabolic engineering concept. In this study, laboratory evolution was used to improve osmotolerance of a Gpd⁻ mhpF-expressing S. cerevisiae strain. Serial batch cultivation at increasing osmotic pressure enabled isolation of an evolved strain that grew anaerobically at 1 M glucose, at a specific growth rate of 0.12 h⁻¹. The evolved strain produced glycerol at low concentrations (0.64 ± 0.33 g l⁻¹). However, these glycerol concentrations were below 10% of those observed with a Gpd⁺ reference strain. Consequently, the ethanol yield on sugar increased from 79% of the theoretical maximum in the reference strain to 92% for the evolved strains. Genetic analysis indicated that osmotolerance under aerobic conditions required a single dominant chromosomal mutation, and one further mutation in the plasmid-borne mhpF gene for anaerobic growth.

  14. Modulation of glycerol and ethanol yields during alcoholic fermentation in Saccharomyces cerevisiae strains overexpressed or disrupted for GPD1 encoding glycerol 3-phosphate dehydrogenase.

    PubMed

    Michnick, S; Roustan, J L; Remize, F; Barre, P; Dequin, S

    1997-07-01

    The possibility of the diversion of carbon flux from ethanol towards glycerol in Saccharomyces cerevisiae during alcoholic fermentation was investigated. Variations in the glycerol 3-phosphate dehydrogenase (GPDH) level and similar trends for alcohol dehydrogenase (ADH), pyruvate decarboxylase and glycerol-3-phosphatase were found when low and high glycerol-forming wine yeast strains were compared. GPDH is thus a limiting enzyme for glycerol production. Wine yeast strains with modulated GPD1 (encoding one of the two GPDH isoenzymes) expression were constructed and characterized during fermentation on glucose-rich medium. Engineered strains fermented glucose with a strongly modified [glycerol] : [ethanol] ratio. gpd1delta mutants exhibited a 50% decrease in glycerol production and increased ethanol yield. Overexpression of GPD1 on synthetic must (200 g/l glucose) resulted in a substantial increase in glycerol production ( x 4) at the expense of ethanol. Acetaldehyde accumulated through the competitive regeneration of NADH via GPDH. Accumulation of by-products such as pyruvate, acetate, acetoin, 2,3 butane-diol and succinate was observed, with a marked increase in acetoin production.

  15. Detection of a mutation in the intron of Sperm-specific glyceraldehyde-3-phosphate dehydrogenase gene in patients with fibrous sheath dysplasia of the sperm flagellum.

    PubMed

    Elkina, Y L; Kuravsky, M L; Bragina, E E; Kurilo, L F; Khayat, S S; Sukhomlinova, M Y; Schmalhausen, E V

    2017-03-01

    The fibrous sheath is a unique cytoskeletal structure surrounding the axoneme and outer dense fibres of the sperm flagellum. Dysplasia of the fibrous sheath (DFS) is a defect of spermatozoa observed in severe asthenozoospermic patients and characterised by morphologically abnormal flagella with distorted fibrous sheaths. Sperm-specific glyceraldehyde-3-phosphate dehydrogenase (GAPDS) is a glycolytic enzyme that is tightly associated with the fibrous sheath of the sperm flagellum. The enzymatic activity of GAPDS was investigated in sperm samples of seven patients with DFS and compared to that of normal spermatozoa (n = 10). The difference in GAPDS activity in DFS and normal spermatozoa was statistically significant (0.19 ± 0.11 and 0.75 ± 0.11 μmol NADH per min per mg protein respectively). Immunochemical staining revealed irregular distribution of GAPDS in the flagellum of DFS spermatozoa. Other five samples with typical alterations in the fibrous sheath were assayed for mutations within human GAPDS gene. In all five cases, a replacement of guanine by adenine was revealed in the intron region between the sixth and the seventh exons of GAPDS. It is assumed that the deficiency in GAPDS observed in most DFS sperm samples is ascribable to a disorder in the regulation of GAPDS expression caused by the mutation in the intron region of GAPDS gene. © 2016 Blackwell Verlag GmbH.

  16. A H2 very high frequency capacitively coupled plasma inactivates glyceraldehyde 3-phosphate dehydrogenase(GapDH) more efficiently than UV photons and heat combined

    NASA Astrophysics Data System (ADS)

    Stapelmann, Katharina; Lackmann, Jan-Wilm; Buerger, Ines; Bandow, Julia Elisabeth; Awakowicz, Peter

    2014-02-01

    Plasma sterilization is a promising alternative to commonly used sterilization techniques, because the conventional methods suffer from certain limitations, e.g. incompatibility with heat-sensitive materials, or use of toxic agents. However, plasma-based sterilization mechanisms are not fully understood yet. A low-pressure very high frequency capacitively coupled plasma is used to investigate the impact of a hydrogen discharge on the protein glyceraldehyde 3-phosphate dehydrogenase (GapDH). GapDH is an enzyme of glycolysis. As a part of the central metabolism, it occurs in nearly all organisms from bacteria to humans. The plasma is investigated with absolutely calibrated optical emission spectroscopy in order to identify and to quantify plasma components that can contribute to enzyme inactivation. The contribution of UV photons and heat to GapDH inactivation is investigated separately, and neither seems to be a major factor. In order to investigate the mechanisms of GapDH inactivation by the hydrogen discharge, samples are investigated for etching, induction of amino acid backbone breaks, and chemical modifications. While neither etching nor strand breaks are observed, chemical modifications occur at different amino acid residues of GapDH. Deamidations of asparagines as well as methionine and cysteine oxidations are detected after VHF-CCP treatment. In particular, oxidation of the cysteine in the active centre is known to lead to GapDH inactivation.

  17. Use of 5-Enolpyruvylshikmate-3-Phosphate Synthase Encoding Gene for Typing of Staphylococcus aureus Isolated from Skin and Urinary Tract Infections of Human

    PubMed Central

    Talebi-Satlou, Reza; Ahmadi, Malahat; Ghavam, Farokh; Saei, Habib Dastmalchi

    2012-01-01

    Objective(s): Staphylococcus aureus is both a successful human commensal and a major pathogen. In this study we investigated the genetic diversity of 26 S. aureus isolates recovered from human skin and urinary tract infections. Materials and Methods:Typing procedure for the studied S. aureus isolates was performed based on PCR amplification of the aroA gene, which encodes the enzyme 5-enolpyruvylshikmate-3-phosphate synthase (EPSPS) that involves in aromatic amino acid biosynthesis, and restriction fragment length polymorphism (RFLP) analysis of the product. Results:All S. aureus isolates produced a single PCR amplification product of 1,153 bp. Digestion of the PCR products with the TaqI endonuclease revealed four different aroA gene patterns designated as A, B, N and H according to the nomenclature system of previous studies. In general, 80.77% of the studied isolates displayed type N, 7.69% were type B, 7.69% were type H and 3.85% displayed type A. Conclusion:Divergent aroA types were detected among S. aureus isolates from skin and urinary tract infections. The results showed that urinary tract infections were contaminated by S. aureus isolates with identical banding patterns (A), while isolates recovered from skin infections had different aroA types. This study also indicates that aroA genotypes vary not only from region to region, but also in individual hosts within a region. PMID:23495360

  18. Taurine chloramine is more selective than hypochlorous acid at targeting critical cysteines and inactivating creatine kinase and glyceraldehyde-3-phosphate dehydrogenase.

    PubMed

    Peskin, Alexander V; Winterbourn, Christine C

    2006-01-01

    Hypochlorous acid (HOCl) and chloramines are produced by the neutrophil enzyme, myeloperoxidase. Both react readily with thiols, although chloramines differ from HOCl in discriminating between low molecular weight thiols on the basis of their pKa. Here, we have compared the reactivity of HOCl and taurine chloramine with thiol proteins by examining inactivation of creatine kinase (CK) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). With both enzymes, loss of activity paralleled thiol loss. For CK both were complete at a 1:1 taurine chloramine:thiol mole ratio. For GAPDH each chloramine oxidized two thiols. Three times more HOCl than taurine chloramine was required for inactivation, indicating that HOCl is less thiol specific. Competition studies showed that thiols of CK were 4 times more reactive with taurine chloramine than thiols of GAPDH (rate constants of 1200 and 300 M-1s-1 respectively). These compare with 205 M-1s-1 for cysteine and are consistent with their lower pKa's. Both enzymes were equally susceptible to HOCl. GSH competed directly with the enzyme thiols for taurine chloramine and protected against oxidative inactivation. At lower GSH concentrations, mixed disulfides were formed. We propose that chloramines should preferentially attack proteins with low pKa thiols and this could be important in regulatory processes.

  19. Development and Implementation of a High Throughput Screen for the Human Sperm-Specific Isoform of Glyceraldehyde 3-Phosphate Dehydrogenase (GAPDHS)

    PubMed Central

    Sexton, Jonathan Z; Danshina, Polina V; Lamson, David R; Hughes, Mark; House, Alan J; Yeh, Li-An; O’Brien, Deborah A; Williams, Kevin P

    2011-01-01

    Glycolytic isozymes that are restricted to the male germline are potential targets for the development of reversible, non-hormonal male contraceptives. GAPDHS, the sperm-specific isoform of glyceraldehyde-3-phosphate dehydrogenase, is an essential enzyme for glycolysis making it an attractive target for rational drug design. Toward this goal, we have optimized and validated a high-throughput spectrophotometric assay for GAPDHS in 384-well format. The assay was stable over time and tolerant to DMSO. Whole plate validation experiments yielded Z’ values >0.8 indicating a robust assay for HTS. Two compounds were identified and confirmed from a test screen of the Prestwick collection. This assay was used to screen a diverse chemical library and identified fourteen small molecules that modulated the activity of recombinant purified GAPDHS with confirmed IC50 values ranging from 1.8 to 42 µM. These compounds may provide useful scaffolds as molecular tools to probe the role of GAPDHS in sperm motility and long term to develop potent and selective GAPDHS inhibitors leading to novel contraceptive agents. PMID:21760877

  20. Crystal Structure of Glyceraldehyde-3-Phosphate Dehydrogenase from the Gram-Positive Bacterial Pathogen A. vaginae, an Immunoevasive Factor that Interacts with the Human C5a Anaphylatoxin

    PubMed Central

    Querol-García, Javier; Fernández, Francisco J.; Marin, Ana V.; Gómez, Sara; Fullà, Daniel; Melchor-Tafur, Cecilia; Franco-Hidalgo, Virginia; Albertí, Sebastián; Juanhuix, Jordi; Rodríguez de Córdoba, Santiago; Regueiro, José R.; Vega, M. Cristina

    2017-01-01

    The Gram-positive anaerobic human pathogenic bacterium Atopobium vaginae causes most diagnosed cases of bacterial vaginosis as well as opportunistic infections in immunocompromised patients. In addition to its well-established role in carbohydrate metabolism, D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from Streptococcus pyogenes and S. pneumoniae have been reported to act as extracellular virulence factors during streptococcal infections. Here, we report the crystal structure of GAPDH from A. vaginae (AvGAPDH) at 2.19 Å resolution. The refined model has a crystallographic Rfree of 22.6%. AvGAPDH is a homotetramer wherein each subunit is bound to a nicotinamide adenine dinucleotide (NAD+) molecule. The AvGAPDH enzyme fulfills essential glycolytic as well as moonlight (non-glycolytic) functions, both of which might be targets of chemotherapeutic intervention. We report that AvGAPDH interacts in vitro with the human C5a anaphylatoxin and inhibits C5a-specific granulocyte chemotaxis, thereby suggesting the participation of AvGAPDH in complement-targeted immunoevasion in a context of infection. The availability of high-quality structures of AvGAPDH and other homologous virulence factors from Gram-positive pathogens is critical for drug discovery programs. In this study, sequence and structural differences between AvGAPDH and related bacterial and eukaryotic GAPDH enzymes are reported in an effort to understand how to subvert the immunoevasive properties of GAPDH and evaluate the potential of AvGAPDH as a druggable target. PMID:28443070

  1. Proteome profiling of the dimorphic fungus Penicillium marneffei extracellular proteins and identification of glyceraldehyde-3-phosphate dehydrogenase as an important adhesion factor for conidial attachment.

    PubMed

    Lau, Susanna K P; Tse, Herman; Chan, Joanna S Y; Zhou, Anna C; Curreem, Shirly O T; Lau, Candy C Y; Yuen, Kwok-Yung; Woo, Patrick C Y

    2013-12-01

    Despite being the most important thermal dimorphic fungus causing systemic mycosis in Southeast Asia, the pathogenic mechanisms of Penicillium marneffei remain largely unknown. By comparing the extracellular proteomes of P. marneffei in mycelial and yeast phases, we identified 12 differentially expressed proteins among which glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and heat shock protein 60 (HSP60) were found to be upregulated in mycelial and yeast phases respectively. Based on previous findings in other pathogens, we hypothesized that these two extracellular proteins may be involved in adherence during P. marneffei-host interaction. Using inhibition assays with recombinant GAPDH (rGAPDH) proteins and anti-rGAPDH sera, we demonstrated that adhesion of P. marneffei conidia to fibronectin and laminin was inhibited by rGAPDH or rabbit anti-rGAPDH serum in a dose-dependent manner. Similarly, a dose-dependent inhibition of conidial adherence to A549 pneumocytes by rGAPDH or rabbit anti-rGAPDH serum was observed, suggesting that P. marneffei GAPDH can mediate binding of conidia to human extracellular matrix proteins and pneumocytes. However, HSP60 did not exhibit similar inhibition on conidia adherence, and neither GAPDH norHSP60 exhibited inhibition on adherence to J774 or THP-1 macrophage cell lines. This report demonstrates GAPDH as an adherence factor in P. marneffei by mediating conidia adherence to host bronchoalveolar epithelium during the early establishment phase of infection.

  2. Reconstructed Ancestral Myo-Inositol-3-Phosphate Synthases Indicate That Ancestors of the Thermococcales and Thermotoga Species Were More Thermophilic than Their Descendants

    PubMed Central

    Butzin, Nicholas C.; Lapierre, Pascal; Green, Anna G.; Swithers, Kristen S.; Gogarten, J. Peter; Noll, Kenneth M.

    2013-01-01

    The bacterial genomes of Thermotoga species show evidence of significant interdomain horizontal gene transfer from the Archaea. Members of this genus acquired many genes from the Thermococcales, which grow at higher temperatures than Thermotoga species. In order to study the functional history of an interdomain horizontally acquired gene we used ancestral sequence reconstruction to examine the thermal characteristics of reconstructed ancestral proteins of the Thermotoga lineage and its archaeal donors. Several ancestral sequence reconstruction methods were used to determine the possible sequences of the ancestral Thermotoga and Archaea myo-inositol-3-phosphate synthase (MIPS). These sequences were predicted to be more thermostable than the extant proteins using an established sequence composition method. We verified these computational predictions by measuring the activities and thermostabilities of purified proteins from the Thermotoga and the Thermococcales species, and eight ancestral reconstructed proteins. We found that the ancestral proteins from both the archaeal donor and the Thermotoga most recent common ancestor recipient were more thermostable than their descendants. We show that there is a correlation between the thermostability of MIPS protein and the optimal growth temperature (OGT) of its host, which suggests that the OGT of the ancestors of these species of Archaea and the Thermotoga grew at higher OGTs than their descendants. PMID:24391933

  3. Reconstructed ancestral Myo-inositol-3-phosphate synthases indicate that ancestors of the Thermococcales and Thermotoga species were more thermophilic than their descendants.

    PubMed

    Butzin, Nicholas C; Lapierre, Pascal; Green, Anna G; Swithers, Kristen S; Gogarten, J Peter; Noll, Kenneth M

    2013-01-01

    The bacterial genomes of Thermotoga species show evidence of significant interdomain horizontal gene transfer from the Archaea. Members of this genus acquired many genes from the Thermococcales, which grow at higher temperatures than Thermotoga species. In order to study the functional history of an interdomain horizontally acquired gene we used ancestral sequence reconstruction to examine the thermal characteristics of reconstructed ancestral proteins of the Thermotoga lineage and its archaeal donors. Several ancestral sequence reconstruction methods were used to determine the possible sequences of the ancestral Thermotoga and Archaea myo-inositol-3-phosphate synthase (MIPS). These sequences were predicted to be more thermostable than the extant proteins using an established sequence composition method. We verified these computational predictions by measuring the activities and thermostabilities of purified proteins from the Thermotoga and the Thermococcales species, and eight ancestral reconstructed proteins. We found that the ancestral proteins from both the archaeal donor and the Thermotoga most recent common ancestor recipient were more thermostable than their descendants. We show that there is a correlation between the thermostability of MIPS protein and the optimal growth temperature (OGT) of its host, which suggests that the OGT of the ancestors of these species of Archaea and the Thermotoga grew at higher OGTs than their descendants.

  4. Iron starvation causes release from the group A streptococcus of the ADP-ribosylating protein called plasmin receptor or surface glyceraldehyde-3-phosphate-dehydrogenase.

    PubMed Central

    Eichenbaum, Z; Green, B D; Scott, J R

    1996-01-01

    In many pathogenic bacteria, iron starvation serves as an environmental signal that triggers the expression of virulence factors, many of which are found on the cell surface or secreted into the culture supernatant. Using the chelating agent nitrilotriacetic acid, we have established conditions for iron starvation of the important human pathogen Streptococcus pyogenes (the group A streptococcus) and determined that iron limitation results in the specific appearance of several new proteins in the culture supernatant. One of these supernatant proteins is the ADP-ribosylating protein known as streptococcal plasmin receptor (Plr) or as the streptococcal surface glyceraldehyde-3-phosphate-dehydrogenase because of its other activities. Upon iron starvation, Plr is specifically released into the culture supernatant in a time-dependent manner, and its appearance in the supernatant is not accompanied by induction of plr mRNA synthesis. Release of Plr from the bacteria may be important for the virulence of group A streptococci and the manifestation of diseases. PMID:8675293

  5. Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of glyceraldehyde-3-phosphate dehydrogenase from Streptococcus agalactiae NEM316

    PubMed Central

    Nagarajan, Revathi; Ponnuraj, Karthe

    2014-01-01

    Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is an essential enzyme involved in glycolysis. Despite lacking the secretory signal sequence, this cytosolic enzyme has been found localized at the surface of several bacteria and fungi. As a surface protein, GAPDH exhibits various adhesive functions, thereby facilitating colonization and invasion of host tissues. Streptococcus agalactiae, also known as group B streptococcus (GBS), binds onto the host using its surface adhesins and causes sepsis and pneumonia in neonates. GAPDH is one of the surface adhesins of GBS binding to human plasminogen and is a virulent factor associated with host colonization. Although the surface-associated GAPDH has been shown to bind to a variety of host extracellular matrix (ECM) molecules in various bacteria, the molecular mechanism underlying their interaction is not fully understood. To investigate this, structural studies on GAPDH of S. agalactiae were initiated. The gapC gene of S. agalactiae NEM316 encoding GAPDH protein was cloned into pET-28a vector, overexpressed in Escherichia coli BL21(DE3) cells and purified to homogeneity. The purified protein was crystallized using the hanging-drop vapour-diffusion method. The GAPDH crystals obtained in two different crystallization conditions diffracted to 2.8 and 2.6 Å resolution, belonging to two different space groups P21 and P212121, respectively. The structure was solved by molecular replacement and structure refinement is now in progress. PMID:25005093

  6. Targeted gene disruption of glycerol-3-phosphate dehydrogenase in Colletotrichum gloeosporioides reveals evidence that glycerol is a significant transferred nutrient from host plant to fungal pathogen.

    PubMed

    Wei, Yangdou; Shen, Wenyun; Dauk, Melanie; Wang, Feng; Selvaraj, Gopalan; Zou, Jitao

    2004-01-02

    Unidirectional transfer of nutrients from plant host to pathogen represents a most revealing aspect of the parasitic lifestyle of plant pathogens. Whereas much effort has been focused on sugars and amino acids, the identification of other significant metabolites is equally important for comprehensive characterization of metabolic interactions between plants and biotrophic fungal pathogens. Employing a strategy of targeted gene disruption, we generated a mutant strain (gpdhDelta) defective in glycerol-3-phosphate dehydrogenase in a hemibiotrophic plant pathogen, Colletotrichum gloeosporioides f.sp. malvae. The gpdhDelta strain had severe defects in carbon utilization as it could use neither glucose nor amino acids for sustained growth. Although the mutant mycelia were able to grow on potato dextrose agar medium, they displayed arrhythmicity in growth and failure to conidiate. The metabolic defect of gpdhDelta could be entirely ameliorated by glycerol in chemically defined minimal medium. Furthermore, glycerol was the one and only metabolite that could restore rhythmic growth and conidiation of gpdhDelta. Despite the profound defects in carbon source utilization, in planta the gpdhDelta strain exhibited normal pathogenicity, proceeded normally in its life cycle, and produced abundant conidia. Analysis of plant tissues at the peripheral zone of fungal infection sites revealed a time-dependent reduction in glycerol content. This study provides strong evidence for a role of glycerol as a significant transferred metabolite from plant to fungal pathogen.

  7. Cloning of a coconut endosperm cDNA encoding a 1-acyl-sn-glycerol-3-phosphate acyltransferase that accepts medium-chain-length substrates.

    PubMed Central

    Knutzon, D S; Lardizabal, K D; Nelsen, J S; Bleibaum, J L; Davies, H M; Metz, J G

    1995-01-01

    Immature coconut (Cocos nucifera) endosperm contains a 1-acyl-sn-glycerol-3-phosphate acyltransferase (LPAAT) activity that shows a preference for medium-chain-length fatty acyl-coenzyme A substrates (H.M. Davies, D.J. Hawkins, J.S. Nelsen [1995] Phytochemistry 39:989-996). Beginning with solubilized membrane preparations, we have used chromatographic separations to identify a polypeptide with an apparent molecular mass of 29 kD, whose presence in various column fractions correlates with the acyltransferase activity detected in those same fractions. Amino acid sequence data obtained from several peptides generated from this protein were used to isolate a full-length clone from a coconut endosperm cDNA library. Clone pCGN5503 contains a 1325-bp cDNA insert with an open reading frame encoding a 308-amino acid protein with a calculated molecular mass of 34.8 kD. Comparison of the deduced amino acid sequence of pCGN5503 to sequences in the data banks revealed significant homology to other putative LPAAT sequences. Expression of the coconut cDNA in Escherichia coli conferred upon those cells a novel LPAAT activity whose substrate activity profile matched that of the coconut enzyme. PMID:8552723

  8. Comparison of the regulation, metabolic functions, and roles in virulence of the glyceraldehyde-3-phosphate dehydrogenase homologues gapA and gapB in Staphylococcus aureus.

    PubMed

    Purves, Joanne; Cockayne, Alan; Moody, Peter C E; Morrissey, Julie A

    2010-12-01

    The Gram-positive bacterium Staphylococcus aureus contains two glyceraldehyde-3-phosphate dehydrogenase (GAPDH) homologues known as GapA and GapB. GapA has been characterized as a functional GAPDH protein, but currently there is no biological evidence for the role of GapB in metabolism in S. aureus. In this study we show through a number of complementary methods that S. aureus GapA is essential for glycolysis while GapB is essential in gluconeogenesis. These proteins are reciprocally regulated in response to glucose concentrations, and both are influenced by the glycolysis regulator protein GapR, which is the first demonstration of the role of this regulator in S. aureus and the first indication that GapR homologues control genes other than those within the glycolytic operon. Furthermore, we show that both GapA and GapB are important in the pathogenesis of S. aureus in a Galleria mellonella model of infection, showing for the first time in any bacteria that both glycolysis and gluconeogenesis have important roles in virulence.

  9. Ste12/Fab1 phosphatidylinositol-3-phosphate 5-kinase is required for nitrogen-regulated mitotic commitment and cell size control.

    PubMed

    Cobley, David; Hálová, Lenka; Schauries, Marie; Kaczmarek, Adrian; Franz-Wachtel, Mirita; Du, Wei; Krug, Karsten; Maček, Boris; Petersen, Janni

    2017-01-01

    Tight coupling of cell growth and cell cycle progression enable cells to adjust their rate of division, and therefore size, to the demands of proliferation in varying nutritional environments. Nutrient stress promotes inhibition of Target Of Rapamycin Complex 1 (TORC1) activity. In fission yeast, reduced TORC1 activity advances mitotic onset and switches growth to a sustained proliferation at reduced cell size. A screen for mutants, that failed to advance mitosis upon nitrogen stress, identified a mutant in the PIKFYVE 1-phosphatidylinositol-3-phosphate 5-kinase fission yeast homolog Ste12. Ste12PIKFYVE deficient mutants were unable to advance the cell cycle to reduce cell size after a nitrogen downshift to poor nitrogen (proline) growth conditions. While it is well established that PI(3,5)P2 signalling is required for autophagy and that Ste12PIKFYVE mutants have enlarged vacuoles (yeast lysosomes), neither a block to autophagy or mutants that independently have enlarged vacuoles had any impact upon nitrogen control of mitotic commitment. The addition of rapamycin to Ste12PIKFYVE deficient mutants reduced cell size at division to suggest that Ste12PIKFYVE possibly functions upstream of TORC1. ste12 mutants display increased Torin1 (TOR inhibitor) sensitivity. However, no major impact on TORC1 or TORC2 activity was observed in the ste12 deficient mutants. In summary, Ste12PIKFYVE is required for nitrogen-stress mediated advancement of mitosis to reduce cell size at division.

  10. A novel 5-enolpyruvoylshikimate-3-phosphate (EPSP) synthase transgene for glyphosate resistance stimulates growth and fecundity in weedy rice (Oryza sativa) without herbicide

    PubMed Central

    Wang, Wei; Xia, Hui; Yang, Xiao; Xu, Ting; Si, Hong Jiang; Cai, Xing Xing; Wang, Feng; Su, Jun; Snow, Allison A; Lu, Bao-Rong

    2014-01-01

    Understanding evolutionary interactions among crops and weeds can facilitate effective weed management. For example, gene flow from crops to their wild or weedy relatives can lead to rapid evolution in recipient populations. In rice (Oryza sativa), transgenic herbicide resistance is expected to spread to conspecific weedy rice (Oryza sativa f. spontanea) via hybridization. Here, we studied fitness effects of transgenic over-expression of a native 5-enolpyruvoylshikimate-3-phosphate synthase (epsps) gene developed to confer glyphosate resistance in rice. Controlling for genetic background, we examined physiological traits and field performance of crop–weed hybrid lineages that segregated for the presence or absence of this novel epsps transgene. Surprisingly, we found that transgenic F2 crop–weed hybrids produced 48–125% more seeds per plant than nontransgenic controls in monoculture- and mixed-planting designs without glyphosate application. Transgenic plants also had greater EPSPS protein levels, tryptophan concentrations, photosynthetic rates, and per cent seed germination compared with nontransgenic controls. Our findings suggest that over-expression of a native rice epsps gene can lead to fitness advantages, even without exposure to glyphosate. We hypothesize that over-expressed epsps may be useful to breeders and, if deployed, could result in fitness benefits in weedy relatives following transgene introgression. PMID:23905647

  11. Tandem Amplification of a Chromosomal Segment Harboring 5-Enolpyruvylshikimate-3-Phosphate Synthase Locus Confers Glyphosate Resistance in Kochia scoparia1[W][OPEN

    PubMed Central

    Jugulam, Mithila; Niehues, Kindsey; Godar, Amar S.; Koo, Dal-Hoe; Danilova, Tatiana; Friebe, Bernd; Sehgal, Sunish; Varanasi, Vijay K.; Wiersma, Andrew; Westra, Philip; Stahlman, Phillip W.; Gill, Bikram S.

    2014-01-01

    Recent rapid evolution and spread of resistance to the most extensively used herbicide, glyphosate, is a major threat to global crop production. Genetic mechanisms by which weeds evolve resistance to herbicides largely determine the level of resistance and the rate of evolution of resistance. In a previous study, we determined that glyphosate resistance in Kochia scoparia is due to the amplification of the 5-Enolpyruvylshikimate-3-Phosphate Synthase (EPSPS) gene, the enzyme target of glyphosate. Here, we investigated the genomic organization of the amplified EPSPS copies using fluorescence in situ hybridization (FISH) and extended DNA fiber (Fiber FISH) on K. scoparia chromosomes. In both glyphosate-resistant K. scoparia populations tested (GR1 and GR2), FISH results displayed a single and prominent hybridization site of the EPSPS gene localized on the distal end of one pair of homologous metaphase chromosomes compared with a faint hybridization site in glyphosate-susceptible samples (GS1 and GS2). Fiber FISH displayed 10 copies of the EPSPS gene (approximately 5 kb) arranged in tandem configuration approximately 40 to 70 kb apart, with one copy in an inverted orientation in GR2. In agreement with FISH results, segregation of EPSPS copies followed single-locus inheritance in GR1 population. This is the first report of tandem target gene amplification conferring field-evolved herbicide resistance in weed populations. PMID:25037215

  12. Re-evaluation of the glycerol-3-phosphate dehydrogenase/L-lactate dehydrogenase enzyme system. Evidence against the direct transfer of NADH between active sites.

    PubMed Central

    Brooks, S P; Storey, K B

    1991-01-01

    An investigation of the direct transfer of metabolites from rabbit muscle L-lactate dehydrogenase (LDH, EC 1.1.1.27) to glycerol-3-phosphate dehydrogenase (GPDH, EC 1.1.1.8) revealed discrepancies between theoretical predictions and experimental results. Measurements of the GPDH reaction rate at a fixed NADH concentration and in the presence of increasing LDH concentrations gave experimental results similar to those previously obtained by Srivastava, Smolen, Betts, Fukushima, Spivey & Bernhard [(1989) Proc. Natl. Acad. Sci. U.S.A. 86, 6464-6468]. However, a mathematical solution of the direct-transfer-mechanism equations as described by Srivastava et al. (1989) showed that the direct-transfer model did not adequately describe the experimental behaviour of the reaction rate at increasing LDH concentrations. In addition, experiments designed to measure the formation of an LDH4.NADH.GPDH2 complex, predicted by the direct-transfer model, indicated that no significant formation of tertiary complex occurred. An examination of other kinetic models, developed to describe the LDH/GPDH/NADH system better, revealed that the experimental results may be best explained by assuming that free NADH, and not E1.NADH, is the sole substrate for GPDH. These results suggest that direct transfer of NADH between rabbit muscle LDH and GPDH does not occur in vitro. PMID:1898374

  13. The complete sequence of a full length cDNA for human liver glyceraldehyde-3-phosphate dehydrogenase: evidence for multiple mRNA species.

    PubMed Central

    Arcari, P; Martinelli, R; Salvatore, F

    1984-01-01

    A recombinant M13 clone (O42) containing a 65 b.p. cDNA fragment from human fetal liver mRNA coding for glyceraldehyde-3-phosphate dehydrogenase has been identified and it has been used to isolate from a full-length human adult liver cDNA library a recombinant clone, pG1, which has been subcloned in M13 phage and completely sequenced with the chain terminator method. Besides the coding region of 1008 b.p., the cDNA sequence includes 60 nucleotides at the 5'-end and 204 nucleotides at the 3'-end up to the polyA tail. Hybridization of pG1 to human liver total RNA shows only one band about the size of pG1 cDNA. A much stronger hybridization signal was observed using RNA derived from human hepatocarcinoma and kidney carcinoma cell lines. Sequence homology between clone 042 and the homologous region of clone pG1 is 86%. On the other hand, homology among the translated sequences and the known human muscle protein sequence ranges between 77 and 90%; these data demonstrate the existence of more than one gene coding for G3PD. Southern blot of human DNA, digested with several restriction enzymes, also indicate that several homologous sequences are present in the human genome. Images PMID:6096821

  14. Transgenic tobacco simultaneously overexpressing glyphosate N-acetyltransferase and 5-enolpyruvylshikimate-3-phosphate synthase are more resistant to glyphosate than those containing one gene.

    PubMed

    Liu, Yunjun; Cao, Gaoyi; Chen, Rongrong; Zhang, Shengxue; Ren, Yuan; Lu, Wei; Wang, Jianhua; Wang, Guoying

    2015-08-01

    5-Enolpyruvylshikimate-3-phosphate synthase (EPSPS) and glyphosate N-acetyltransferase (GAT) can detoxify glyphosate by alleviating the suppression of shikimate pathway. In this study, we obtained transgenic tobacco plants overexpressing AM79 aroA, GAT, and both of them, respectively, to evaluate whether overexpression of both genes could confer transgenic plants with higher glyphosate resistance. The transgenic plants harboring GAT or AM79 aroA, respectively, showed good glyphosate resistance. As expected, the hybrid plants containing both GAT and AM79 aroA exhibited improved glyphosate resistance than the transgenic plants overexpressing only a single gene. When grown on media with high concentration of glyphosate, seedlings containing a single gene were severely inhibited, whereas plants expressing both genes were affected less. When transgenic plants grown in the greenhouse were sprayed with glyphosate, less damage was observed for the plants containing both genes. Metabolomics analysis showed that transgenic plants containing two genes could maintain the metabolism balance better than those containing one gene after glyphosate treatment. Glyphosate treatment did not lead to a huge increase of shikimate contents of tobacco leaves in transgenic plants overexpressing two genes, whereas significant increase of shikimate contents in transgenic plants containing only a single gene was observed. These results demonstrated that pyramiding both aroA and GAT in transgenic plants can enhance glyphosate resistance, and this strategy can be used for the development of transgenic glyphosate-resistant crops.

  15. Isolation and characterization of rat and human glyceraldehyde-3-phosphate dehydrogenase cDNAs: genomic complexity and molecular evolution of the gene.

    PubMed Central

    Tso, J Y; Sun, X H; Kao, T H; Reece, K S; Wu, R

    1985-01-01

    Full length cDNAs encoding the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from rat and man have been isolated and sequenced. Many GAPDH gene-related sequences have been found in both genomes based on genomic blot hybridization analysis. Only one functional gene product is known. Results from genomic library screenings suggest that there are 300-400 copies of these sequences in the rat genome and approximately 100 in the human genome. Some of these related sequences have been shown to be processed pseudogenes. We have isolated several rat cDNA clones corresponding to these pseudogenes indicating that some pseudogenes are transcribed. Rat and human cDNAs are 89% homologous in the coding region, and 76% homologous in the first 100 base pairs of the 3'-noncoding region. Comparison of these two cDNA sequences with those of the chicken, Drosophila and yeast genes allows the analysis of the evolution of the GAPDH genes in detail. Images PMID:2987855

  16. Increased levels of glycerol-3-phosphate lead to a stimulation of flux into triacylglycerol synthesis after supplying glycerol to developing seeds of Brassica napus L. in planta.

    PubMed

    Vigeolas, Helene; Geigenberger, Peter

    2004-09-01

    Glycerol-3-phosphate (glycerol-3P) is a primary substrate for triacylglycerol synthesis. In the present study, changes in the levels of glycerol-3P during rape (Brassica napus L.) seed development and the influence of manipulating glycerol-3P levels on triacylglycerol synthesis were investigated. (i) Glycerol-3P levels were high in young seeds and decreased during seed development at 30 and 40 days after flowering (DAF), when lipid accumulation was maximal. (ii) To manipulate glycerol-3P levels in planta, various concentrations of glycerol were injected directly into 30-DAF seeds, which remained otherwise intact within their siliques and attached to the plant. Injection of 0-10 nmol glycerol led to a progressive increase in seed glycerol-3P levels within 28 h. (iii). Increased levels of glycerol-3P were accompanied by an increase in the flux of injected [14C]sucrose into total lipids and triacylglycerol, whereas fluxes to organic acids, amino acids, starch, protein and cell walls were not affected. (iv) When [14C]acetate was injected into seeds, label incorporation into total lipids and triacylglycerol increased progressively with increasing glycerol-3P levels. (v) There was a strong correlation between the level of glycerol-3P and the incorporation of injected [14C]acetate and [14C]sucrose into triacylglycerol. (v) The results provide evidence that the prevailing levels of glycerol-3P co-limit triacylglycerol synthesis in developing rape seeds.

  17. Heterologous expression of Zygosaccharomyces rouxii glycerol 3-phosphate dehydrogenase gene (ZrGPD1) and glycerol dehydrogenase gene (ZrGCY1) in Saccharomyces cerevisiae.

    PubMed

    Watanabe, Yasuo; Tsuchimoto, Syoko; Tamai, Youichi

    2004-01-01

    We examined the effects of heterologous expression of the open reading frames (ORF) of two genes on salt tolerance and glycerol production in a Saccharomyces cerevisiae strain deficient in glycerol synthesis (gpd1Deltagpd2Delta). When the ORF of the Zygosaccharomyces rouxii glycerol 3-phosphate dehydrogenase gene (ZrGPD1) was expressed under the control of the GAL10 promoter, salt tolerance and glycerol production increased; when the ORF of the glycerol dehydrogenase gene (ZrGCY1) was expressed under the control of the GAL1 promoter, no such changes were observed. Zrgcy1p had a weak effect on glycerol production. These results suggest that Zrgpd1p is the primary enzyme involved in Z. rouxii glycerol production, following a mechanism similar to that of S. cerevisiae (Gpd1p). When the ORFs of the S. cerevisiae glycerol 3-phosphatase gene (GPP2) and ZrGPD1 were simultaneously expressed, glycerol production increased, compared with that in yeast expressing only ZrGPD1.

  18. Phosphatidylinositol 3-phosphate-binding protein AtPH1 controls the localization of the metal transporter NRAMP1 in Arabidopsis.

    PubMed

    Agorio, Astrid; Giraudat, Jérôme; Bianchi, Michele Wolfe; Marion, Jessica; Espagne, Christelle; Castaings, Loren; Lelièvre, Françoise; Curie, Catherine; Thomine, Sébastien; Merlot, Sylvain

    2017-04-03

    "Too much of a good thing" perfectly describes the dilemma that living organisms face with metals. The tight control of metal homeostasis in cells depends on the trafficking of metal transporters between membranes of different compartments. However, the mechanisms regulating the location of transport proteins are still largely unknown. Developing Arabidopsis thaliana seedlings require the natural resistance-associated macrophage proteins (NRAMP3 and NRAMP4) transporters to remobilize iron from seed vacuolar stores and thereby acquire photosynthetic competence. Here, we report that mutations in the pleckstrin homology (PH) domain-containing protein AtPH1 rescue the iron-deficient phenotype of nramp3nramp4 Our results indicate that AtPH1 binds phosphatidylinositol 3-phosphate (PI3P) in vivo and acts in the late endosome compartment. We further show that loss of AtPH1 function leads to the mislocalization of the metal uptake transporter NRAMP1 to the vacuole, providing a rationale for the reversion of nramp3nramp4 phenotypes. This work identifies a PH domain protein as a regulator of plant metal transporter localization, providing evidence that PH domain proteins may be effectors of PI3P for protein sorting.

  19. Characterization of glyceraldehyde-3-phosphate dehydrogenase gene RtGPD1 and development of genetic transformation method by dominant selection in oleaginous yeast Rhodosporidium toruloides.

    PubMed

    Liu, Yanbin; Koh, Chong Mei John; Sun, Longhua; Hlaing, Mya Myintzu; Du, Minge; Peng, Ni; Ji, Lianghui

    2013-01-01

    The oleaginous yeast Rhodosporidium toruloides, which belongs to the Pucciniomycotina subphylum in the Basidiomycota, has attracted strong interest in the biofuel community recently due to its ability to accumulate more than 60% of dry biomass as lipid under high-density fermentation. A 3,543-nucleotide (nt) DNA fragment of the glyceraldehyde-3-phosphate dehydrogenase gene (GPD1) was isolated from R. toruloides ATCC 10657 and characterized in details. The 1,038-nt mRNA derived from seven exons encodes an open reading frame (ORF) of 345 amino acids that shows high identity (80%) to the Ustilago maydis homolog. Notably, the ORF is composed of codons strongly biased towards cytosine at the Wobble position. GPD1 is transcriptionally regulated by temperature shock, osmotic stress, and carbon source. Nested deletion analysis of the GPD1 promoter by GFP reporter assay revealed that two regions, -975 to -1,270 and -1,270 to -1,429, upstream from the translational start site of GPD1 were important for responses to various stress stimuli. Interestingly, a 176-bp short fragment maintained 42.2% promoter activity of the 795-bp version in U. maydis whereas it was reduced to 17.4% in R. toruloides. The GPD1 promoter drove strong expression of a codon-optimized enhanced green fluorescent protein gene (RtGFP) and a codon-optimized hygromycin phosphotransferase gene (hpt-3), which was critical for Agrobacterium tumefaciens-mediated transformation in R. toruloides.

  20. Glycerate-3-phosphate, produced by CO2 fixation in the Calvin cycle, is critical for the synthesis of the D1 protein of photosystem II.

    PubMed

    Takahashi, Shunichi; Murata, Norio

    2006-03-01

    We demonstrated recently that, in intact cells of Chlamydomonas reinhardtii, interruption of CO2 fixation via the Calvin cycle inhibits the synthesis of proteins in photosystem II (PSII), in particular, synthesis of the D1 protein, during the repair of PSII after photodamage. In the present study, we investigated the mechanism responsible for this phenomenon using intact chloroplasts isolated from spinach leaves. When CO2 fixation was inhibited by exogenous glycolaldehyde, which inhibits the phosphoribulokinase that synthesizes ribulose-1,5-bisphosphate, the synthesis de novo of the D1 protein was inhibited. However, when glycerate-3-phosphate (3-PGA), which is a product of CO2 fixation in the Calvin cycle, was supplied exogenously, the inhibitory effect of glycolaldehyde was abolished. A reduced supply of CO2 also suppressed the synthesis of the D1 protein, and this inhibitory effect was also abolished by exogenous 3-PGA. These findings suggest that the supply of 3-PGA, generated by CO2 fixation, is important for the synthesis of the D1 Protein. It is likely that 3-PGA accepts electrons from NADPH and decreases the level of reactive oxygen species, which inhibit the synthesis of proteins, such as the D1 protein.

  1. Evolutionary conserved light regulation of Calvin cycle activity by NADPH-mediated reversible phosphoribulokinase/CP12/ glyceraldehyde-3-phosphate dehydrogenase complex dissociation.

    PubMed

    Wedel, N; Soll, J

    1998-08-04

    For higher plant chloroplasts, two key enzymes of the Calvin cycle, phosphoribulokinase (EC 2.7.1.19) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, EC 1.2.1.13), have recently been shown to be oligomerized onto the nonenzymatic peptide CP12. Enzymatic activity depends on complex dissociation, mediated by NADPH. The discovery of genes for CP12 in mosses, green algae, and cyanobacteria, together with the analysis of equivalent multiprotein complexes of Chlamydomonas and Synechocystis suggests that light regulation of Calvin cycle activity via NADPH-mediated reversible phosphoribulokinase/CP12/GAPDH complex dissociation is conserved in all photosynthetic organisms, prokaryotes and eukaryotes. In vitro complex reconstitution assays with heterologously expressed Synechocystis wild-type and mutagenized CP12 demonstrate a conserved subunit composition, stoichiometry, and topology in this complex. Further finding of genes, coding for chimeric proteins, carrying CP12 or parts of it as genetic fusions, indicates that evolution has used the peptide loops of CP12 as universal modules to keep various enzymatic activities under the control of NADP(H). These fusion events occurred at least twice in evolution. First was the fusion of the duplicated genes for CP12 and the ORF4 protein of Anabaena variabilis to the chimeric gene for the heterocyst-specific expressed ORF3 protein, most probably involved in N2 fixation. A second gene fusion, which led to the higher plant chloroplast-specific GAPDH subunit, GAPB, has taken place during the transition from water- to land plants.

  2. Ste12/Fab1 phosphatidylinositol-3-phosphate 5-kinase is required for nitrogen-regulated mitotic commitment and cell size control

    PubMed Central

    Schauries, Marie; Kaczmarek, Adrian; Franz-Wachtel, Mirita; Du, Wei; Krug, Karsten; Maček, Boris; Petersen, Janni

    2017-01-01

    Tight coupling of cell growth and cell cycle progression enable cells to adjust their rate of division, and therefore size, to the demands of proliferation in varying nutritional environments. Nutrient stress promotes inhibition of Target Of Rapamycin Complex 1 (TORC1) activity. In fission yeast, reduced TORC1 activity advances mitotic onset and switches growth to a sustained proliferation at reduced cell size. A screen for mutants, that failed to advance mitosis upon nitrogen stress, identified a mutant in the PIKFYVE 1-phosphatidylinositol-3-phosphate 5-kinase fission yeast homolog Ste12. Ste12PIKFYVE deficient mutants were unable to advance the cell cycle to reduce cell size after a nitrogen downshift to poor nitrogen (proline) growth conditions. While it is well established that PI(3,5)P2 signalling is required for autophagy and that Ste12PIKFYVE mutants have enlarged vacuoles (yeast lysosomes), neither a block to autophagy or mutants that independently have enlarged vacuoles had any impact upon nitrogen control of mitotic commitment. The addition of rapamycin to Ste12PIKFYVE deficient mutants reduced cell size at division to suggest that Ste12PIKFYVE possibly functions upstream of TORC1. ste12 mutants display increased Torin1 (TOR inhibitor) sensitivity. However, no major impact on TORC1 or TORC2 activity was observed in the ste12 deficient mutants. In summary, Ste12PIKFYVE is required for nitrogen-stress mediated advancement of mitosis to reduce cell size at division. PMID:28273166

  3. The complex of band 3 protein of the human erythrocyte membrane and glyceraldehyde-3-phosphate dehydrogenase: stoichiometry and competition by aldolase.

    PubMed

    von Rückmann, Bogdan; Schubert, Dieter

    2002-02-10

    The cytoplasmic domain of band 3, the main intrinsic protein of the erythrocyte membrane, possesses binding sites for a variety of other proteins of the membrane and the cytoplasm, including the glycolytic enzymes glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and aldolase. We have studied the stoichiometry of the complexes of human band 3 protein and GAPDH and the competition by aldolase for the binding sites. In addition, we have tried to verify the existence of mixed band 3/GAPDH/aldolase complexes, which could represent the nucleus of a putative glycolytic multienzyme complex on the erythrocyte membrane. The technique applied was analytical ultracentrifugation, in particular sedimentation equilibrium analysis, on mixtures of detergent-solubilized band 3 and dye-labelled GAPDH, in part of the experiments supplemented by aldolase. The results obtained were analogous to those reported for the binding of hemoglobin, aldolase and band 4.1 to band 3: (1) the predominant or even sole band 3 oligomer forming the binding site is the tetramer. (2) The band 3 tetramer can bind up to four tetramers of GAPDH. (3) The band 3/GAPDH complexes are unstable. (4) Artificially stabilized band 3 dimers also represent GAPDH binding sites. In addition it was found that aldolase competes with GAPDH for binding to the band 3 tetramer, and that ternary complexes of band 3 tetramers, GAPDH and aldolase do exist.

  4. Effects of deletion of glycerol-3-phosphate dehydrogenase and glutamate dehydrogenase genes on glycerol and ethanol metabolism in recombinant Saccharomyces cerevisiae.

    PubMed

    Kim, Jin-Woo; Chin, Young-Wook; Park, Yong-Cheol; Seo, Jin-Ho

    2012-01-01

    Bioethanol is currently used as an alternative fuel for gasoline worldwide. For economic production of bioethanol by Saccharomyces cerevisiae, formation of a main by-product, glycerol, should be prevented or minimized in order to reduce a separation cost of ethanol from fermentation broth. In this study, S. cerevisiae was engineered to investigate the effects of the sole and double disruption of NADH-dependent glycerol-3-phosphate dehydrogenase 1 (GPD1) and NADPH-requiring glutamate dehydrogenase 1 (GDH1) on the production of glycerol and ethanol from glucose. Even though sole deletion of GPD1 or GDH1 reduced glycerol production, double deletion of GPD1 and GDH1 resulted in the lowest glycerol concentration of 2.31 g/L, which was 46.4% lower than the wild-type strain. Interestingly, the recombinant S. cerevisiae ∆GPD1∆GDH1 strain showed a slight improvement in ethanol yield (0.414 g/g) compared with the wild-type strain (0.406 g/g). Genetic engineering of the glycerol and glutamate metabolic pathways modified NAD(P)H-requiring metabolic pathways and exerted a positive effect on glycerol reduction without affecting ethanol production.

  5. A novel 5-enolpyruvoylshikimate-3-phosphate (EPSP) synthase transgene for glyphosate resistance stimulates growth and fecundity in weedy rice (Oryza sativa) without herbicide.

    PubMed

    Wang, Wei; Xia, Hui; Yang, Xiao; Xu, Ting; Si, Hong Jiang; Cai, Xing Xing; Wang, Feng; Su, Jun; Snow, Allison A; Lu, Bao-Rong

    2014-04-01

    Understanding evolutionary interactions among crops and weeds can facilitate effective weed management. For example, gene flow from crops to their wild or weedy relatives can lead to rapid evolution in recipient populations. In rice (Oryza sativa), transgenic herbicide resistance is expected to spread to conspecific weedy rice (Oryza sativa f. spontanea) via hybridization. Here, we studied fitness effects of transgenic over-expression of a native 5-enolpyruvoylshikimate-3-phosphate synthase (epsps) gene developed to confer glyphosate resistance in rice. Controlling for genetic background, we examined physiological traits and field performance of crop-weed hybrid lineages that segregated for the presence or absence of this novel epsps transgene. Surprisingly, we found that transgenic F2 crop-weed hybrids produced 48-125% more seeds per plant than nontransgenic controls in monoculture- and mixed-planting designs without glyphosate application. Transgenic plants also had greater EPSPS protein levels, tryptophan concentrations, photosynthetic rates, and per cent seed germination compared with nontransgenic controls. Our findings suggest that over-expression of a native rice epsps gene can lead to fitness advantages, even without exposure to glyphosate. We hypothesize that over-expressed epsps may be useful to breeders and, if deployed, could result in fitness benefits in weedy relatives following transgene introgression. © 2013 The Authors. New Phytologist © 2013 New Phytologist Trust.

  6. Cloning and molecular characterization of a glycerol-3-phosphate O-acyltransferase (GPAT) gene from Echium (Boraginaceae) involved in the biosynthesis of cutin polyesters.

    PubMed

    Mañas-Fernández, Aurora; Li-Beisson, Yonghua; Alonso, Diego López; García-Maroto, Federico

    2010-09-01

    The glycerol-based lipid polyester called cutin is a main component of cuticle, the protective interface of aerial plant organs also controlling compound exchange with the environment. Though recent progress towards understanding of cutin biosynthesis has been made in Arabidopsis thaliana, little is known in other plants. One key step in this process is the acyl transfer reaction to the glycerol backbone. Here we report the cloning and molecular characterization of EpGPAT1, a gene encoding a glycerol-3-phosphate O-acyltransferase (GPAT) from Echium pitardii (Boraginaceae) with high similarity to the AtGPAT4/AtGPAT8 of Arabidopsis. Quantitative analysis by qRT-PCR showed highest expression of EpGPAT1 in seeds, roots, young leaves and flowers. Acyltransferase activity of EpGPAT1 was evidenced by heterologous expression in yeast. Ectopic expression in leaves of tobacco plants lead to an increase of C16 and C18 hydroxyacids and alpha,omega-diacids in the cell wall fraction, indicating a role in the biosynthesis of polyesters. Analysis of the genomic organization in Echium revealed the presence of EpGPAT2, a closely related gene which was found to be mostly expressed in developing leaves and flowers. The presence of a conserved HAD-like domain at the N-terminal moiety of GPATs from Echium, Arabidopsis and other plant species suggests a possible phosphohydrolase activity in addition to the reported acyltransferase activity. Evolutive implications of this finding are discussed.

  7. Functional characterization of the phosphorylating D-glyceraldehyde 3-phosphate dehydrogenase from the archaeon Methanothermus fervidus by comparative molecular modelling and site-directed mutagenesis.

    PubMed

    Talfournier, F; Colloc'h, N; Mornon, J P; Branlant, G

    1999-10-01

    Phosphorylating archaeal D-glyceraldehyde 3-phosphate dehydrogenases (GraP-DHs) share only 15-20% identity with their glycolytic bacterial and eukaryotic counterparts. Unlike the latter which are NAD-specific, archaeal GraP-DHs exhibit a dual-cofactor specificity with a marked preference for NADP. In the present study, we have constructed a three-dimensional model of the Methanothermus fervidus GraP-DH based upon the X-ray structures of the Bacillus stearothermophilus and Escherichia coli GraP-DHs. The overall structure of the archaeal enzyme is globally similar to homology modelling-derived structures, in particular for the cofactor binding domain, which might adopt a classical Rossmann fold. M. fervidus GraP-DH can be considered as a dimer of dimers which exhibits negative and positive cooperativity in binding the coenzymes NAD and NADP, respectively. As expected, the differences between the model and the templates are located mainly within the loops. Based on the predictions derived from molecular modelling, site-directed mutagenesis was performed to characterize better the cofactor binding pocket and the catalytic domain. The Lys32Ala, Lys32Glu and Lys32Asp mutants led to a drastic increase in the Km value for NADP (i.e. 165-, 500- and 1000-fold, respectively), thus demonstrating that the invariant Lys32 residue is one of the most important determinants favouring the adenosine 2'-PO42- binding of NADP. The involvement of the side chain of Asn281, which was postulated to play a role equivalent to that of the Asn313 of bacterial and eukaryotic GraP-DHs in fixing the position of the nicotinamide ring in a syn orientation [Fabry, S. & Hensel, R. (1988) Gene 64, 189-197], was ruled out. Most of the amino acids involved in catalysis and in substrate recognition in bacterial and eukaryotic GraP-DHs are not conserved in the archaeal enzyme except for the essential Cys149. Inspection of our model suggests that side chains of invariant residues Asn150, Arg176, Arg177 and

  8. Two glycerol 3-phosphate dehydrogenase isogenes from Candida versatilis SN-18 play an important role in glycerol biosynthesis under osmotic stress.

    PubMed

    Mizushima, Daiki; Iwata, Hisashi; Ishimaki, Yuki; Ogihara, Jun; Kato, Jun; Kasumi, Takafumi

    2016-05-01

    Two isogenes of glycerol 3-phosphate dehydrogenase (GPD) from Candida versatilis SN-18 were cloned and sequenced. These intronless genes (Cagpd1 and Cagpd2) were both predicted to encode a 378 amino acid polypeptide, and the deduced amino acid sequences mutually showed 76% identity. Interestingly, Cagpd1 and Cagpd2 were located tandemly in a locus of genomic DNA within a 262 bp interval. To our knowledge, this represents a novel instance of isogenic genes relating to glucose metabolism. The stress response element (STRE) was found respectively at -93 to -89 bp upstream of the 5'end of Cagpd1 and -707 to -703 bp upstream of Cagpd2, indicating that these genes are involved in osmotic stress response. In heterologous expression using a gpd1Δgpd2Δ double deletion mutant of Saccharomyces cerevisiae, Cagpd1 and Cagpd2 transformants complemented the function of GPD, with Cagpd2 being much more effective than Cagpd1 in promoting growth and glycerol synthesis. Phylogenetic analysis of the amino acid sequences suggested that Cagpd1p and Cagpd2p are NADP(+)-dependent GPDs (EC 1.1.1.94). However, crude enzyme extract from Cagpd1 and Cagpd2 transformants showed GPD activity with only NAD(+) as cofactor. Hence, both Cagpd1p and Cagpd2p are likely NAD(+)-dependent GPDs (EC 1.1.1.8), similar to GPDs from S. cerevisiae and Candida magnoliae. Copyright © 2015 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  9. Mice deficient in mitochondrial glycerol-3-phosphate acyltransferase-1 have diminished myocardial triacylglycerol accumulation during lipogenic diet and altered phospholipid fatty acid composition

    PubMed Central

    Lewin, Tal M.; de Jong, Hendrik; Schwerbrock, Nicole J. M.; Hammond, Linda E.; Watkins, Steven M.; Combs, Terry P.; Coleman, Rosalind A.

    2008-01-01

    Glycerol-3-phosphate acyltransferase-1 (GPAT1), which is located on the outer mitochondrial membrane comprises up to 30% of total GPAT activity in the heart. It is one of at least four mammalian GPAT isoforms known to catalyze the initial, committed, and rate limiting step of glycerolipid synthesis. Because excess triacylglycerol (TAG) accumulates in cardiomyocytes in obesity and type 2 diabetes, we determined whether lack of GPAT1 would alter the synthesis of heart TAG and phospholipids after a 2-week high sucrose diet or a 3-month high fat diet. Even in the absence of hypertriglyceridemia, TAG increased 2-fold with both diets in hearts from wildtype mice. In contrast, hearts from Gpat1−/− mice contained 20–80% less TAG than the wildtype controls. In addition, hearts from Gpat1−/− mice fed the high-sucrose diet incorporate 60% less [14C]palmitate into heart TAG as compared to wildtype mice. Because GPAT1 prefers 16:0-CoA to other long chain acyl-CoA substrates, we determined the fatty acid composition of heart phospholipids. Compared to wildtype littermate controls, hearts from Gpat1−/− mice contained a lower amount of 16:0 in phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine/phosphatidylinositol and significantly more C20:4n6. Phosphatidylcholine and phosphatidylethanolamine from Gpat1−/− hearts also contained higher amounts of 18:0 and 18:1. Although at least three other GPAT isoforms are expressed in the heart, our data suggest that GPAT1 contributes significantly to cardiomyocyte TAG synthesis during lipogenic or high fat diets and influences the incorporation of 20:4n6 into heart phospholipids. PMID:18522808

  10. Effects of over-expressing a native gene encoding 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) on glyphosate resistance in Arabidopsis thaliana

    PubMed Central

    Beres, Zachery T.; Jin, Lin; Parrish, Jason T.; Zhao, Wanying; Mackey, David; Snow, Allison A.

    2017-01-01

    Widespread overuse of the herbicide glyphosate, the active ingredient in RoundUp®, has led to the evolution of glyphosate-resistant weed biotypes, some of which persist by overproducing the herbicide’s target enzyme, 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS). EPSPS is a key enzyme in the shikimic acid pathway for biosynthesis of aromatic amino acids, lignin, and defensive compounds, but little is known about how overproducing EPSPS affects downstream metabolites, growth, or lifetime fitness in the absence of glyphosate. We are using Arabidopsis as a model system for investigating phenotypic effects of overproducing EPSPS, thereby avoiding confounding effects of genetic background or other mechanisms of herbicide resistance in agricultural weeds. Here, we report results from the first stage of this project. We designed a binary vector expressing a native EPSPS gene from Arabidopsis under control of the CaMV35S promoter (labelled OX, for over-expression). For both OX and the empty vector (labelled EV), we obtained nine independent T3 lines. Subsets of these lines were used to characterize glyphosate resistance in greenhouse experiments. Seven of the nine OX lines exhibited enhanced glyphosate resistance when compared to EV and wild-type control lines, and one of these was discarded due to severe deformities. The remaining six OX lines exhibited enhanced EPSPS gene expression and glyphosate resistance compared to controls. Glyphosate resistance was correlated with the degree of EPSPS over-expression for both vegetative and flowering plants, indicating that glyphosate resistance can be used as a surrogate for EPSPS expression levels in this system. These findings set the stage for examination of the effects of EPSPS over-expression on fitness-related traits in the absence of glyphosate. We invite other investigators to contact us if they wish to study gene expression, downstream metabolic effects, and other questions with these particular lines. PMID

  11. Genetic variability of Yersinia pestis isolates as predicted by PCR-based IS100 genotyping and analysis of structural genes encoding glycerol-3-phosphate dehydrogenase (glpD).

    PubMed

    Motin, Vladimir L; Georgescu, Anca M; Elliott, Jeffrey M; Hu, Ping; Worsham, Patricia L; Ott, Linda L; Slezak, Tomas R; Sokhansanj, Bahrad A; Regala, Warren M; Brubaker, Robert R; Garcia, Emilio

    2002-02-01

    A PCR-based genotyping system that detects divergence of IS100 locations within the Yersinia pestis genome was used to characterize a large collection of isolates of different biovars and geographical origins. Using sequences derived from the glycerol-negative biovar orientalis strain CO92, a set of 27 locus-specific primers was designed to amplify fragments between the end of IS100 and its neighboring gene. Geographically diverse members of the orientalis biovar formed a homogeneous group with identical genotype with the exception of strains isolated in Indochina. In contrast, strains belonging to the glycerol-positive biovar antiqua showed a variety of fingerprinting profiles. Moreover, strains of the biovar medievalis (also glycerol positive) clustered together with the antiqua isolates originated from Southeast Asia, suggesting their close phylogenetic relationships. Interestingly, a Manchurian biovar antiqua strain Nicholisk 51 displayed a genotyping pattern typical of biovar orientalis isolates. Analysis of the glycerol pathway in Y. pestis suggested that a 93-bp deletion within the glpD gene encoding aerobic glycerol-3-phosphate dehydrogenase might account for the glycerol-negative phenotype of the orientalis biovar. The glpD gene of strain Nicholisk 51 did not possess this deletion, although it contained two nucleotide substitutions characteristic of the glpD version found exclusively in biovar orientalis strains. To account for this close relationship between biovar orientalis strains and the antiqua Nicholisk 51 isolate, we postulate that the latter represents a variant of this biovar with restored ability to ferment glycerol. The fact that such a genetic lesion might be repaired as part of the natural evolutionary process suggests the existence of genetic exchange between different Yersinia strains in nature. The relevance of this observation on the emergence of epidemic Y. pestis strains is discussed.

  12. Genetic Variability of Yersinia pestis Isolates as Predicted by PCR-Based IS100 Genotyping and Analysis of Structural Genes Encoding Glycerol-3-Phosphate Dehydrogenase (glpD)

    PubMed Central

    Motin, Vladimir L.; Georgescu, Anca M.; Elliott, Jeffrey M.; Hu, Ping; Worsham, Patricia L.; Ott, Linda L.; Slezak, Tomas R.; Sokhansanj, Bahrad A.; Regala, Warren M.; Brubaker, Robert R.; Garcia, Emilio

    2002-01-01

    A PCR-based genotyping system that detects divergence of IS100 locations within the Yersinia pestis genome was used to characterize a large collection of isolates of different biovars and geographical origins. Using sequences derived from the glycerol-negative biovar orientalis strain CO92, a set of 27 locus-specific primers was designed to amplify fragments between the end of IS100 and its neighboring gene. Geographically diverse members of the orientalis biovar formed a homogeneous group with identical genotype with the exception of strains isolated in Indochina. In contrast, strains belonging to the glycerol-positive biovar antiqua showed a variety of fingerprinting profiles. Moreover, strains of the biovar medievalis (also glycerol positive) clustered together with the antiqua isolates originated from Southeast Asia, suggesting their close phylogenetic relationships. Interestingly, a Manchurian biovar antiqua strain Nicholisk 51 displayed a genotyping pattern typical of biovar orientalis isolates. Analysis of the glycerol pathway in Y. pestis suggested that a 93-bp deletion within the glpD gene encoding aerobic glycerol-3-phosphate dehydrogenase might account for the glycerol-negative phenotype of the orientalis biovar. The glpD gene of strain Nicholisk 51 did not possess this deletion, although it contained two nucleotide substitutions characteristic of the glpD version found exclusively in biovar orientalis strains. To account for this close relationship between biovar orientalis strains and the antiqua Nicholisk 51 isolate, we postulate that the latter represents a variant of this biovar with restored ability to ferment glycerol. The fact that such a genetic lesion might be repaired as part of the natural evolutionary process suggests the existence of genetic exchange between different Yersinia strains in nature. The relevance of this observation on the emergence of epidemic Y. pestis strains is discussed. PMID:11807062

  13. Identification, sequence analysis, and expression of a Corynebacterium glutamicum gene cluster encoding the three glycolytic enzymes glyceraldehyde-3-phosphate dehydrogenase, 3-phosphoglycerate kinase, and triosephosphate isomerase.

    PubMed Central

    Eikmanns, B J

    1992-01-01

    To investigate a possible chromosomal clustering of glycolytic enzyme genes in Corynebacterium glutamicum, a 6.4-kb DNA fragment located 5' adjacent to the structural phosphoenolpyruvate carboxylase (PEPCx) gene ppc was isolated. Sequence analysis of the ppc-proximal part of this fragment identified a cluster of three glycolytic genes, namely, the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene gap, the 3-phosphoglycerate kinase (PGK) gene pgk, and the triosephosphate isomerase (TPI) gene tpi. The four genes are organized in the order gap-pgk-tpi-ppc and are separated by 215 bp (gap and pgk), 78 bp (pgk and tpi), and 185 bp (tpi and ppc). The predicted gene product of gap consists of 336 amino acids (M(r) of 36,204), that of pgk consists of 403 amino acids (M(r) of 42,654), and that of tpi consists of 259 amino acids (M(r) of 27,198). The amino acid sequences of the three enzymes show up to 62% (GAPDH), 48% (PGK), and 44% (TPI) identity in comparison with respective enzymes from other organisms. The gap, pgk, tpi, and ppc genes were cloned into the C. glutamicum-Escherichia coli shuttle vector pEK0 and introduced into C. glutamicum. Relative to the wild type, the recombinant strains showed up to 20-fold-higher specific activities of the respective enzymes. On the basis of codon usage analysis of gap, pgk, tpi, and previously sequenced genes from C. glutamicum, a codon preference profile for this organism which differs significantly from those of E. coli and Bacillus subtilis is presented. Images PMID:1400158

  14. Potential role of D-myo-inositol-3-phosphate synthase and 14-3-3 genes in the crosstalk between Zea mays and Rhizophagus intraradices under drought stress.

    PubMed

    Li, Tao; Sun, Yuqing; Ruan, Yuan; Xu, Lijiiao; Hu, Yajun; Hao, Zhipeng; Zhang, Xin; Li, Hong; Wang, Youshan; Yang, Liguo; Chen, Baodong

    2016-11-01

    Arbuscular mycorrhizal (AM) symbiosis is known to stimulate plant drought tolerance. However, the mechanisms underlying the synergistic responses of the symbiotic partners to drought stress are largely unknown. A split-root experiment was designed to investigate the molecular interactions between a host plant and an AM fungus (AMF) under drought stress. In the two-compartment cultivation system, an entire or only a half root system of a maize plant was inoculated with an AMF, Rhizophagus intraradices, in the presence of localized or systemic drought treatment. Plant physiological parameters including growth, water status, and phosphorus concentration, and the expression of drought tolerance-related genes in both roots and R. intraradices were recorded. Although mycorrhizal inoculation in either one or both compartments systemically decreased abscisic acid (ABA) content in the whole root system subjected to systemic or local drought stress, we observed local and/or systemic AM effects on root physiological traits and the expression of functional genes in both roots and R. intraradices. Interestingly, the simultaneous increase in the expression of plant genes encoding D-myo-inositol-3-phosphate synthase (IPS) and 14-3-3-like protein GF14 (14-3GF), which were responsible for ABA signal transduction, was found to be involved in the activation of 14-3-3 protein and aquaporins (GintAQPF1 and GintAQPF2) in R. intraradices. These findings suggest that coexpression of IPS and 14-3GF is responsible for the crosstalk between maize and R. intraradices under drought stress, and potentially induces the synergistic actions of the symbiotic partners in enhancing plant drought tolerance.

  15. Glycerol-3-phosphate Acyltransferase (GPAT)-1, but Not GPAT4, Incorporates Newly Synthesized Fatty Acids into Triacylglycerol and Diminishes Fatty Acid Oxidation*

    PubMed Central

    Wendel, Angela A.; Cooper, Daniel E.; Ilkayeva, Olga R.; Muoio, Deborah M.; Coleman, Rosalind A.

    2013-01-01

    Four glycerol-3-phosphate acyltransferase (GPAT) isoforms, each encoded by a separate gene, catalyze the initial step in glycerolipid synthesis; in liver, the major isoforms are GPAT1 and GPAT4. To determine whether each of these hepatic isoforms performs a unique function in the metabolism of fatty acid, we measured the incorporation of de novo synthesized fatty acid or exogenous fatty acid into complex lipids in primary mouse hepatocytes from control, Gpat1−/−, and Gpat4−/− mice. Although hepatocytes from each genotype incorporated a similar amount of exogenous fatty acid into triacylglycerol (TAG), only control and Gpat4−/− hepatocytes were able to incorporate de novo synthesized fatty acid into TAG. When compared with controls, Gpat1−/− hepatocytes oxidized twice as much exogenous fatty acid. To confirm these findings and to assess hepatic β-oxidation metabolites, we measured acylcarnitines in liver from mice after a 24-h fast and after a 24-h fast followed by 48 h of refeeding with a high sucrose diet to promote lipogenesis. Confirming the in vitro findings, the hepatic content of long-chain acylcarnitine in fasted Gpat1−/− mice was 3-fold higher than in controls. When compared with control and Gpat4−/− mice, after the fasting-refeeding protocol, Gpat1−/− hepatic TAG was depleted, and long-chain acylcarnitine content was 3.5-fold higher. Taken together, these data demonstrate that GPAT1, but not GPAT4, is required to incorporate de novo synthesized fatty acids into TAG and to divert them away from oxidation. PMID:23908354

  16. Evolution of a Double Amino Acid Substitution in the 5-Enolpyruvylshikimate-3-Phosphate Synthase in Eleusine indica Conferring High-Level Glyphosate Resistance1

    PubMed Central

    Yu, Qin; Jalaludin, Adam; Han, Heping; Chen, Ming; Sammons, R. Douglas; Powles, Stephen B.

    2015-01-01

    Glyphosate is the most important and widely used herbicide in world agriculture. Intensive glyphosate selection has resulted in the widespread evolution of glyphosate-resistant weed populations, threatening the sustainability of this valuable once-in-a-century agrochemical. Field-evolved glyphosate resistance due to known resistance mechanisms is generally low to modest. Here, working with a highly glyphosate-resistant Eleusine indica population, we identified a double amino acid substitution (T102I + P106S [TIPS]) in the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene in glyphosate-resistant individuals. This TIPS mutation recreates the biotechnology-engineered commercial first generation glyphosate-tolerant EPSPS in corn (Zea mays) and now in other crops. In E. indica, the naturally evolved TIPS mutants are highly (more than 180-fold) resistant to glyphosate compared with the wild type and more resistant (more than 32-fold) than the previously known P106S mutants. The E. indica TIPS EPSPS showed very high-level (2,647-fold) in vitro resistance to glyphosate relative to the wild type and is more resistant (600-fold) than the P106S variant. The evolution of the TIPS mutation in crop fields under glyphosate selection is likely a sequential event, with the P106S mutation being selected first and fixed, followed by the T102I mutation to create the highly resistant TIPS EPSPS. The sequential evolution of the TIPS mutation endowing high-level glyphosate resistance is an important mechanism by which plants adapt to intense herbicide selection and a dramatic example of evolution in action. PMID:25717039

  17. Inter-species variation in the oligomeric states of the higher plant Calvin cycle enzymes glyceraldehyde-3-phosphate dehydrogenase and phosphoribulokinase

    PubMed Central

    Lloyd, Julie C.; Raines, Christine A.

    2011-01-01

    In darkened leaves the Calvin cycle enzymes glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and phosphoribulokinase (PRK) form a regulatory multi-enzyme complex with the small chloroplast protein CP12. GAPDH also forms a high molecular weight regulatory mono-enzyme complex. Given that there are different reports as to the number and subunit composition of these complexes and that enzyme regulatory mechanisms are known to vary between species, it was reasoned that protein–protein interactions may also vary between species. Here, this variation is investigated. This study shows that two different tetramers of GAPDH (an A2B2 heterotetramer and an A4 homotetramer) have the capacity to form part of the PRK/GAPDH/CP12 complex. The role of the PRK/GAPDH/CP12 complex is not simply to regulate the ‘non-regulatory’ A4 GAPDH tetramer. This study also demonstrates that the abundance and nature of PRK/GAPDH/CP12 interactions are not equal in all species and that whilst NAD enhances complex formation in some species, this is not sufficient for complex formation in others. Furthermore, it is shown that the GAPDH mono-enzyme complex is more abundant as a 2(A2B2) complex, rather than the larger 4(A2B2) complex. This smaller complex is sensitive to cellular metabolites indicating that it is an important regulatory isoform of GAPDH. This comparative study has highlighted considerable heterogeneity in PRK and GAPDH protein interactions between closely related species and the possible underlying physiological basis for this is discussed. PMID:21498632

  18. Oxygen transfer as a tool for fine-tuning recombinant protein production by Pichia pastoris under glyceraldehyde-3-phosphate dehydrogenase promoter.

    PubMed

    Güneş, Hande; Çalık, Pınar

    2016-07-01

    Effects of oxygen transfer on recombinant protein production by Pichia pastoris under glyceraldehyde-3-phosphate dehydrogenase promoter were investigated. Recombinant glucose isomerase was chosen as the model protein. Two groups of oxygen transfer strategies were applied, one of which was based on constant oxygen transfer rate where aeration rate was Q O/V = 3 and 10 vvm, and agitation rate was N = 900 min(-1); while the other one was based on constant dissolved oxygen concentrations, C DO = 5, 10, 15, 20 and 40 % in the fermentation broth, by using predetermined exponential glucose feeding with μ o = 0.15 h(-1). The highest cell concentration was obtained as 44 g L(-1) at t = 9 h of the glucose fed-batch phase at C DO = 20 % operation while the highest volumetric and specific enzyme activities were obtained as 4440 U L(-1) and 126 U g(-1) cell, respectively at C DO = 15 % operation. Investigation of specific enzyme activities revealed that keeping C DO at 15 % was more advantageous with an expense of relatively higher by-product formation and lower specific cell growth rate. For this strategy, the highest oxygen transfer coefficient and oxygen uptake rate were K L a = 0.045 s(-1) and OUR = 8.91 mmol m(-3) s(-1), respectively.

  19. Calcium- and Nitric Oxide-Dependent Nuclear Accumulation of Cytosolic Glyceraldehyde-3-Phosphate Dehydrogenase in Response to Long Chain Bases in Tobacco BY-2 Cells.

    PubMed

    Testard, Ambroise; Da Silva, Daniel; Ormancey, Mélanie; Pichereaux, Carole; Pouzet, Cécile; Jauneau, Alain; Grat, Sabine; Robe, Eugénie; Brière, Christian; Cotelle, Valérie; Mazars, Christian; Thuleau, Patrice

    2016-10-01

    Sphinganine or dihydrosphingosine (d18:0, DHS), one of the most abundant free sphingoid long chain bases (LCBs) in plants, is known to induce a calcium-dependent programmed cell death (PCD) in plants. In addition, in tobacco BY-2 cells, it has been shown that DHS triggers a rapid production of H2O2 and nitric oxide (NO). Recently, in analogy to what is known in the animal field, plant cytosolic glyceraldehyde-3-phosphate dehydrogenase (GAPC), a ubiquitous enzyme involved in glycolysis, has been suggested to fulfill other functions associated with its oxidative post-translational modifications such as S-nitrosylation on cysteine residues. In particular, in mammals, stress signals inducing NO production promote S-nitrosylation of GAPC and its subsequent translocation into the nucleus where the protein participates in the establishment of apoptosis. In the present study, we investigated the behavior of GAPC in tobacco BY-2 cells treated with DHS. We found that upon DHS treatment, an S-nitrosylated form of GAPC accumulated in the nucleus. This accumulation was dependent on NO production. Two genes encoding GAPCs, namely Nt(BY-2)GAPC1 and Nt(BY-2)GAPC2, were cloned. Transient overexpression of Nt(BY-2)GAPC-green fluorescent protein (GFP) chimeric constructs indicated that both proteins localized in the cytoplasm as well as in the nucleus. Mutating into serine the two cysteine residues thought to be S-nitrosylated in response to DHS did not modify the localization of the proteins, suggesting that S-nitrosylation of GAPCs was probably not necessary for their nuclear relocalization. Interestingly, using Förster resonance energy transfer experiments, we showed that Nt(BY-2)GAPCs interact with nucleic acids in the nucleus. When GAPCs were mutated on their cysteine residues, their interaction with nucleic acids was abolished, suggesting a role for GAPCs in the protection of nucleic acids against oxidative stress.

  20. S100B impairs glycolysis via enhanced poly(ADP-ribosyl)ation of glyceraldehyde 3-phosphate dehydrogenase in rodent muscle cells.

    PubMed

    Hosokawa, Kaori; Hamada, Yoji; Fujiya, Atsushi; Murase, Masatoshi; Maekawa, Ryuya; Niwa, Yasuhiro; Izumoto, Takako; Seino, Yusuke; Tsunekawa, Shin; Arima, Hiroshi

    2017-02-07

    S100 calcium-binding protein B (S100B), a multifunctional macromolecule mainly expressed in nerve tissues and adipocytes, has been suggested to contribute to the pathogenesis of obesity. To clarify the role of S100B in insulin action and glucose metabolism in peripheral tissues, we investigated the effect of S100B on glycolysis in myoblast and myotube cells. Rat myoblast L6 cells were treated with recombinant mouse S100B to examine glucose consumption, lactate production, glycogen accumulation, glycolytic metabolites and enzyme activity, insulin signaling, and poly(ADP-ribosyl)ation of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Glycolytic metabolites were investigated by enzyme assays or metabolome analysis, and insulin signaling was assessed by western blot analysis. Enzyme activity and poly(ADP-ribosyl)ation of GAPDH was evaluated by an enzyme assay and immunoprecipitation followed by dot blot with an anti-poly(ADP-ribose) antibody, respectively. S100B significantly decreased glucose consumption, glucose analog uptake, and lactate production in L6 cells, in either the presence or absence of insulin. In contrast, S100B had no effect on glycogen accumulation and insulin signaling. Metabolome analysis revealed that S100B increased the concentration of glycolytic intermediates upstream of GAPDH. S100B impaired GAPDH activity and increased poly(ADP-ribosyl)ated GAPDH proteins. The effects of S100B on glucose metabolism were mostly canceled by a poly(ADP-ribose) polymerase (PARP) inhibitor. Similar results were obtained in C2C12 myotube cells. We conclude that S100B as a humoral factor may impair glycolysis in muscle cells independently of insulin action, and the effect may be attributed to the inhibition of GAPDH activity from enhanced poly(ADP-ribosyl)ation of the enzyme.

  1. S-glutathionylation of glyceraldehyde-3-phosphate dehydrogenase induces formation of C150-C154 intrasubunit disulfide bond in the active site of the enzyme.

    PubMed

    Barinova, K V; Serebryakova, M V; Muronetz, V I; Schmalhausen, E V

    2017-09-19

    Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a glycolytic protein involved in numerous non-glycolytic functions. S-glutathionylated GAPDH was revealed in plant and animal tissues. The role of GAPDH S-glutathionylation is not fully understood. Rabbit muscle GAPDH was S-glutathionylated in the presence of H2O2 and reduced glutathione (GSH). The modified protein was assayed by MALDI-MS analysis, differential scanning calorimetry, dynamic light scattering, and ultracentrifugation. Incubation of GAPDH in the presence of H2O2 together with GSH resulted in the complete inactivation of the enzyme. In contrast to irreversible oxidation of GAPDH by H2O2, this modification could be reversed in the excess of GSH or dithiothreitol. By data of MALDI-MS analysis, the modified protein contained both mixed disulfide between Cys150 and GSH and the intrasubunit disulfide bond between Cys150 and Cys154 (different subunits of tetrameric GAPDH may contain different products). S-glutathionylation results in loosening of the tertiary structure of GAPDH, decreases its affinity to NAD(+) and thermal stability. The mixed disulfide between Cys150 and GSH is an intermediate product of S-glutathionylation: its subsequent reaction with Cys154 results in the intrasubunit disulfide bond in the active site of GAPDH. The mixed disulfide and the C150-C154 disulfide bond protect GAPDH from irreversible oxidation and can be reduced in the excess of thiols. Conformational changes that were observed in S-glutathionylated GAPDH may affect interactions between GAPDH and other proteins (ligands), suggesting the role of S-glutathionylation in the redox signaling. The manuscript considers one of the possible mechanisms of redox regulation of cell functions. Copyright © 2017 Elsevier B.V. All rights reserved.

  2. Glycerol-3-phosphate acyltransferase-4-deficient mice are protected from diet-induced insulin resistance by the enhanced association of mTOR and rictor.

    PubMed

    Zhang, Chongben; Cooper, Daniel E; Grevengoed, Trisha J; Li, Lei O; Klett, Eric L; Eaton, James M; Harris, Thurl E; Coleman, Rosalind A

    2014-08-01

    Glycerol-3-phosphate acyltransferase (GPAT) activity is highly induced in obese individuals with insulin resistance, suggesting a correlation between GPAT function, triacylglycerol accumulation, and insulin resistance. We asked whether microsomal GPAT4, an isoform regulated by insulin, might contribute to the development of hepatic insulin resistance. Compared with control mice fed a high fat diet, Gpat4(-/-) mice were more glucose tolerant and were protected from insulin resistance. Overexpression of GPAT4 in mouse hepatocytes impaired insulin-suppressed gluconeogenesis and insulin-stimulated glycogen synthesis. Impaired glucose homeostasis was coupled to inhibited insulin-stimulated phosphorylation of Akt(Ser⁴⁷³) and Akt(Thr³⁰⁸). GPAT4 overexpression inhibited rictor's association with the mammalian target of rapamycin (mTOR), and mTOR complex 2 (mTORC2) activity. Compared with overexpressed GPAT3 in mouse hepatocytes, GPAT4 overexpression increased phosphatidic acid (PA), especially di16:0-PA. Conversely, in Gpat4(-/-) hepatocytes, both mTOR/rictor association and mTORC2 activity increased, and the content of PA in Gpat4(-/-) hepatocytes was lower than in controls, with the greatest decrease in 16:0-PA species. Compared with controls, liver and skeletal muscle from Gpat4(-/-)-deficient mice fed a high-fat diet were more insulin sensitive and had a lower hepatic content of di16:0-PA. Taken together, these data demonstrate that a GPAT4-derived lipid signal, likely di16:0-PA, impairs insulin signaling in mouse liver and contributes to hepatic insulin resistance. Copyright © 2014 the American Physiological Society.

  3. Engineering Camelina sativa (L.) Crantz for Enhanced Oil and Seed Yields by Combining Diacylglycerol Acyltransferase1 and Glycerol-3-Phosphate Dehydrogenase Expression.

    PubMed

    Chhikara, Sudesh; Abdullah, Hesham M; Akbari, Parisa; Schnell, Danny; Dhankher, Om Parkash

    2017-10-04

    Plant seed oils based liquid transportation fuels (i.e., biodiesel and green diesel) have tremendous potential as environmentally, economically and technologically feasible alternatives to petroleum-derived fuels. Due to their nutritional and industrial importance, one of the major objectives is to increase the seed yield and oil production of oilseed crops via biotechnological approaches. Camelina sativa, an emerging oilseed crop, has been proposed as an ideal crop for biodiesel and bioproducts applications. Further increase in seed oil yield by increasing the flux of carbon from increased photosynthesis into triacylglycerol (TAG) synthesis will make this crop more profitable. To increase the oil yield, we engineered Camelina by co-expressing the Arabidopsis thaliana (L.) Heynh. diacylglycerol acyltransferase1 (DGAT1) and a yeast cytosolic glycerol-3-phosphate dehydrogenase (GPD1) genes under the control of seed-specific promoters. Plants co-expressing DGAT1 and GPD1 exhibited up to 13% higher seed oil content and up to 52% increase in seed mass compared to wild type plants. Further, DGAT1 and GDP1 co-expressing lines showed significantly higher seed and oil yields on a dry-weight basis than the wild type controls or plants expressing DGAT1 and GPD1 alone. The oil harvest index (g oil per g total dry matter) for DGTA1 and GPD1 co-expressing lines was almost two-folds higher as compared to wild type and the lines expressing DGAT1 and GPD1 alone. Therefore, combining the overexpression of TAG biosynthetic genes, DGAT1 and GPD1, appears to be a positive strategy to achieve a synergistic effect on the flux through the TAG synthesis pathway, and thereby further increase the oil yield. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

  4. A BAR-Domain Protein SH3P2, Which Binds to Phosphatidylinositol 3-Phosphate and ATG8, Regulates Autophagosome Formation in Arabidopsis[C][W

    PubMed Central

    Zhuang, Xiaohong; Wang, Hao; Lam, Sheung Kwan; Gao, Caiji; Wang, Xiangfeng; Cai, Yi; Jiang, Liwen

    2013-01-01

    Autophagy is a well-defined catabolic mechanism whereby cytoplasmic materials are engulfed into a structure termed the autophagosome. In plants, little is known about the underlying mechanism of autophagosome formation. In this study, we report that SH3 DOMAIN-CONTAINING PROTEIN2 (SH3P2), a Bin-Amphiphysin-Rvs domain–containing protein, translocates to the phagophore assembly site/preautophagosome structure (PAS) upon autophagy induction and actively participates in the membrane deformation process. Using the SH3P2–green fluorescent protein fusion as a reporter, we found that the PAS develops from a cup-shaped isolation membranes or endoplasmic reticulum–derived omegasome-like structures. Using an inducible RNA interference (RNAi) approach, we show that RNAi knockdown of SH3P2 is developmentally lethal and significantly suppresses autophagosome formation. An in vitro membrane/lipid binding assay demonstrates that SH3P2 is a membrane-associated protein that binds to phosphatidylinositol 3-phosphate. SH3P2 may facilitate membrane expansion or maturation in coordination with the phosphatidylinositol 3-kinase (PI3K) complex during autophagy, as SH3P2 promotes PI3K foci formation, while PI3K inhibitor treatment inhibits SH3P2 from translocating to autophagosomes. Further interaction analysis shows that SH3P2 associates with the PI3K complex and interacts with ATG8s in Arabidopsis thaliana, whereby SH3P2 may mediate autophagy. Thus, our study has identified SH3P2 as a novel regulator of autophagy and provided a conserved model for autophagosome biogenesis in Arabidopsis. PMID:24249832

  5. A Land-Plant-Specific Glycerol-3-Phosphate Acyltransferase Family in Arabidopsis: Substrate Specificity, sn-2 Preference, and Evolution1[W][OA

    PubMed Central

    Yang, Weili; Simpson, Jeffrey P.; Li-Beisson, Yonghua; Beisson, Fred; Pollard, Mike; Ohlrogge, John B.

    2012-01-01

    Arabidopsis (Arabidopsis thaliana) has eight glycerol-3-phosphate acyltransferase (GPAT) genes that are members of a plant-specific family with three distinct clades. Several of these GPATs are required for the synthesis of cutin or suberin. Unlike GPATs with sn-1 regiospecificity involved in membrane or storage lipid synthesis, GPAT4 and -6 are unique bifunctional enzymes with both sn-2 acyltransferase and phosphatase activity resulting in 2-monoacylglycerol products. We present enzymology, pathway organization, and evolutionary analysis of this GPAT family. Within the cutin-associated clade, GPAT8 is demonstrated as a bifunctional sn-2 acyltransferase/phosphatase. GPAT4, -6, and -8 strongly prefer C16:0 and C18:1 ω-oxidized acyl-coenzyme As (CoAs) over unmodified or longer acyl chain substrates. In contrast, suberin-associated GPAT5 can accommodate a broad chain length range of ω-oxidized and unsubstituted acyl-CoAs. These substrate specificities (1) strongly support polyester biosynthetic pathways in which acyl transfer to glycerol occurs after oxidation of the acyl group, (2) implicate GPAT specificities as one major determinant of cutin and suberin composition, and (3) argue against a role of sn-2-GPATs (Enzyme Commission 2.3.1.198) in membrane/storage lipid synthesis. Evidence is presented that GPAT7 is induced by wounding, produces suberin-like monomers when overexpressed, and likely functions in suberin biosynthesis. Within the third clade, we demonstrate that GPAT1 possesses sn-2 acyltransferase but not phosphatase activity and can utilize dicarboxylic acyl-CoA substrates. Thus, sn-2 acyltransferase activity extends to all subbranches of the Arabidopsis GPAT family. Phylogenetic analyses of this family indicate that GPAT4/6/8 arose early in land-plant evolution (bryophytes), whereas the phosphatase-minus GPAT1 to -3 and GPAT5/7 clades diverged later with the appearance of tracheophytes. PMID:22864585

  6. Identification of a Glyphosate-Resistant Mutant of Rice 5-Enolpyruvylshikimate 3-Phosphate Synthase Using a Directed Evolution Strategy1[W][OA

    PubMed Central

    Zhou, Min; Xu, Honglin; Wei, Xiaoli; Ye, Zhiqiang; Wei, Liping; Gong, Weimin; Wang, Yongqin; Zhu, Zhen

    2006-01-01

    5-Enolpyruvylshikimate 3-phosphate synthase (EPSPS) is a key enzyme in the shikimate pathway and is targeted by the wide-spectrum herbicide glyphosate. Here, we describe the use of a selection system based on directed evolution to select glyphosate-resistant mutants of EPSPS. Using this system, the rice (Oryza sativa) EPSPS gene, mutagenized by Error-Prone polymerase chain reaction, was introduced into an EPSPS-deficient Escherichia coli strain, AB2829, and transformants were selected on minimal medium by functional complementation. Three mutants with high glyphosate resistance were identified in three independent glyphosate selection experiments. Each mutant contained a C317→T transition within the EPSPS coding sequence, causing a change of proline-106 to leucine (P106L) in the protein sequence. Glyphosate resistance assays indicated a 3-fold increase in glyphosate resistance of E. coli expressing the P106L mutant. Affinity of the P106L mutant for glyphosate and phosphoenolpyruvate was decreased about 70-fold and 4.6-fold, respectively, compared to wild-type EPSPS. Analysis based on a kinetic model demonstrates that the P106L mutant has a high glyphosate resistance while retaining relatively high catalytic efficiency at low phosphoenolpyruvate concentrations. A mathematical model derived from the Michaelis-Menten equation was used to characterize the effect of expression level and selection conditions on kinetic (Ki and Km) variation of the mutants. This prediction suggests that the expression level is an important aspect of the selection system. Furthermore, glyphosate resistance of the P106L mutant was confirmed in transgenic tobacco (Nicotiana tabacum), demonstrating the potential for using the P106L mutant in transgenic crops. PMID:16361526

  7. An improved amperometric triglyceride biosensor based on co-immobilization of nanoparticles of lipase, glycerol kinase and glycerol 3-phosphate oxidase onto pencil graphite electrode.

    PubMed

    Narwal, Vinay; Pundir, C S

    2017-05-01

    Nanoparticles (NPs) of commercial lipase from Candida rugosa, of glycerol kinase (GK) from Cellulomonas species, of glycerol-3- phosphate oxidase (GPO) from Aerococcus viridans were prepared, characterized and co-immobilized onto a pencil graphite (PG) electrode. The morphological and electrochemical characterization of PG electrode was performed by scanning electron microscopy (SEM), cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) before and after co-immobilization of enzyme nanoparticles (ENPs). An improved amperometric triglyceride (TG) biosensor was fabricated using Lipase NPs/GKNPs/GPONPs/PG electrode as the working electrode, Ag/AgCl as the standard electrode and Pt wire as auxiliary electrode. The biosensor showed optimum response within 2.5s at a pH 7.0 and temperature of 35°C. The biosensor measured current due to electrons generated at 0.1V against Ag/AgCl, from H2O2, which is produced from triolein by co-immobilized ENPs. A linear relationship was obtained over between a wide triolein concentration range (0.1mM-45mM) and current (mA) under optimal conditions. The Lipase NPs/GKNPs/GPONPs/PG electrode showed high sensitivity (1241±20mAcm(-2)mM(-1)); a lower detection limit (0.1nM) and good correlation coeficient (R(2)=0.99) with a standard enzymic colorimetric method. Analytical recovery of added triolein in serum was 98.01%, within and between batch coefficients of variation (CV) were 0.05% and 0.06% respectively. The biosensor was evaluated and employed for determination of TG in the serum of apparently healthy subject and persons suffering from hypertriglyceridemia. The biosensor lost 20% of its initial activity after its continued uses over a period of 240days, while being stored at 4°C. Copyright © 2017 Elsevier Inc. All rights reserved.

  8. Effects of over-expressing a native gene encoding 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) on glyphosate resistance in Arabidopsis thaliana.

    PubMed

    Yang, Xiao; Beres, Zachery T; Jin, Lin; Parrish, Jason T; Zhao, Wanying; Mackey, David; Snow, Allison A

    2017-01-01

    Widespread overuse of the herbicide glyphosate, the active ingredient in RoundUp®, has led to the evolution of glyphosate-resistant weed biotypes, some of which persist by overproducing the herbicide's target enzyme, 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS). EPSPS is a key enzyme in the shikimic acid pathway for biosynthesis of aromatic amino acids, lignin, and defensive compounds, but little is known about how overproducing EPSPS affects downstream metabolites, growth, or lifetime fitness in the absence of glyphosate. We are using Arabidopsis as a model system for investigating phenotypic effects of overproducing EPSPS, thereby avoiding confounding effects of genetic background or other mechanisms of herbicide resistance in agricultural weeds. Here, we report results from the first stage of this project. We designed a binary vector expressing a native EPSPS gene from Arabidopsis under control of the CaMV35S promoter (labelled OX, for over-expression). For both OX and the empty vector (labelled EV), we obtained nine independent T3 lines. Subsets of these lines were used to characterize glyphosate resistance in greenhouse experiments. Seven of the nine OX lines exhibited enhanced glyphosate resistance when compared to EV and wild-type control lines, and one of these was discarded due to severe deformities. The remaining six OX lines exhibited enhanced EPSPS gene expression and glyphosate resistance compared to controls. Glyphosate resistance was correlated with the degree of EPSPS over-expression for both vegetative and flowering plants, indicating that glyphosate resistance can be used as a surrogate for EPSPS expression levels in this system. These findings set the stage for examination of the effects of EPSPS over-expression on fitness-related traits in the absence of glyphosate. We invite other investigators to contact us if they wish to study gene expression, downstream metabolic effects, and other questions with these particular lines.

  9. Role of secreted glyceraldehyde-3-phosphate dehydrogenase in the infection mechanism of enterohemorrhagic and enteropathogenic Escherichia coli: interaction of the extracellular enzyme with human plasminogen and fibrinogen.

    PubMed

    Egea, L; Aguilera, L; Giménez, R; Sorolla, M A; Aguilar, J; Badía, J; Baldoma, L

    2007-01-01

    Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (EC 1.2.1.12) is an anchorless, multifunctional protein displayed on the surface of several fungi and Gram-positive pathogens, which contributes to their adhesion and virulence. To date a role for extracellular GAPDH in the pathogenesis of Gram-negative bacteria has not been described. The aim of this study was to analyze the extracellular localization of GAPDH in enterohemorrhagic (EHEC) and enteropathogenic (EPEC) Escherichia coli strains and to examine its interaction with host components that could be related to the infection mechanism. Recombinant E. coli GAPDH was purified and polyclonal antibodies were obtained. Western blotting and immunoelectron microscopy showed that GAPDH is located on the bacterial surface and released to the culture medium of EHEC and EPEC strains. GAPDH export in these Gram-negative pathogens depends on the external medium, is not mediated by vesicles and leads to an extracellular active enzyme. Non-pathogenic E. coli strains do not secrete GAPDH. Two-dimensional electrophoresis analysis showed that in E. coli GAPDH is present at least in two major forms with different isoelectric points. Of these forms, the more basic is secreted. Purified GAPDH was found to bind human plasminogen and fibrinogen in Far-Western blot and ELISA-based assays. In addition, GAPDH remained associated with colonic Caco-2 epithelial cells after adhesion of EHEC or EPEC. These observations indicate that exported GAPDH may act as a virulence factor which could contribute to EHEC and EPEC pathogenesis. This is the first description of an extracellular localization for this enzyme, with a function other than its glycolytic role in Gram-negative pathogens.

  10. Inter-species variation in the oligomeric states of the higher plant Calvin cycle enzymes glyceraldehyde-3-phosphate dehydrogenase and phosphoribulokinase.

    PubMed

    Howard, Thomas P; Lloyd, Julie C; Raines, Christine A

    2011-07-01

    In darkened leaves the Calvin cycle enzymes glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and phosphoribulokinase (PRK) form a regulatory multi-enzyme complex with the small chloroplast protein CP12. GAPDH also forms a high molecular weight regulatory mono-enzyme complex. Given that there are different reports as to the number and subunit composition of these complexes and that enzyme regulatory mechanisms are known to vary between species, it was reasoned that protein-protein interactions may also vary between species. Here, this variation is investigated. This study shows that two different tetramers of GAPDH (an A2B2 heterotetramer and an A4 homotetramer) have the capacity to form part of the PRK/GAPDH/CP12 complex. The role of the PRK/GAPDH/CP12 complex is not simply to regulate the 'non-regulatory' A4 GAPDH tetramer. This study also demonstrates that the abundance and nature of PRK/GAPDH/CP12 interactions are not equal in all species and that whilst NAD enhances complex formation in some species, this is not sufficient for complex formation in others. Furthermore, it is shown that the GAPDH mono-enzyme complex is more abundant as a 2(A2B2) complex, rather than the larger 4(A2B2) complex. This smaller complex is sensitive to cellular metabolites indicating that it is an important regulatory isoform of GAPDH. This comparative study has highlighted considerable heterogeneity in PRK and GAPDH protein interactions between closely related species and the possible underlying physiological basis for this is discussed.

  11. DNA vaccine encoding the moonlighting protein Onchocerca volvulus glyceraldehyde-3-phosphate dehydrogenase (Ov-GAPDH) leads to partial protection in a mouse model of human filariasis.

    PubMed

    Steisslinger, Vera; Korten, Simone; Brattig, Norbert W; Erttmann, Klaus D

    2015-10-26

    River blindness, caused by the filarial parasite Onchocerca volvulus, is a major socio-economic and public health problem in Sub-Saharan Africa. In January 2015, The Onchocerciasis Vaccine for Africa (TOVA) Initiative has been launched with the aim of providing new tools to complement mass drug administration (MDA) of ivermectin, thereby promoting elimination of onchocerciasis in Africa. In this context we here present Onchocerca volvulus glyceraldehyde-3-phosphate dehydrogenase (Ov-GAPDH) as a possible DNA vaccine candidate. We report that in a laboratory model for filariasis, immunization with Ov-GAPDH led to a significant reduction of adult worm load and microfilaraemia in BALB/c mice after challenge infection with the filarial parasite Litomosoides sigmodontis. Mice were either vaccinated with Ov-GAPDH.DNA plasmid (Ov-pGAPDH.DNA) alone or in combination with recombinantly expressed Ov-GAPDH protein (Ov-rGAPDH). During the following challenge infection of immunized and control mice with L. sigmodontis, those formulations which included the DNA plasmid, led to a significant reduction of adult worm loads (up to 57% median reduction) and microfilaraemia (up to 94% reduction) in immunized animals. In a further experiment, immunization with a mixture of four overlapping, synthetic Ov-GAPDH peptides (Ov-GAPDHpept), with alum as adjuvant, did not significantly reduce worm loads. Our results indicate that DNA vaccination with Ov-GAPDH has protective potential against filarial challenge infection in the mouse model. This suggests a transfer of the approach into the cattle Onchocerca ochengi model, where it is possible to investigate the effects of this vaccination in the context of a natural host-parasite relationship.

  12. Direct evidence that genetic variation in glycerol-3-phosphate and malate dehydrogenase genes (Gpdh and Mdh1) affects adult ethanol tolerance in Drosophila melanogaster.

    PubMed

    Eanes, Walter F; Merritt, Thomas J S; Flowers, Jonathan M; Kumagai, Seiji; Zhu, Chen-Tseh

    2009-02-01

    Many studies of alcohol adaptation in Drosophila melanogaster have focused on the Adh polymorphism, yet the metabolic elimination of alcohol should involve many enzymes and pathways. Here we evaluate the effects of glycerol-3-phosphate dehydrogenase (Gpdh) and cytosolic malate dehydrogenase (Mdh1) genotype activity on adult tolerance to ethanol. We have created a set of P-element-excision-derived Gpdh, Mdh1, and Adh alleles that generate a range of activity phenotypes from full to zero activity. Comparisons of paired Gpdh genotypes possessing 10 and 60% normal activity and 66 and 100% normal activity show significant effects where higher activity increases tolerance. Mdh1 null allele homozygotes show reductions in tolerance. We use piggyBac FLP-FRT site-specific recombination to create deletions and duplications of Gpdh. Duplications show an increase of 50% in activity and an increase of adult tolerance to ethanol exposure. These studies show that the molecular polymorphism associated with GPDH activity could be maintained in natural populations by selection related to adaptation to alcohols. Finally, we examine the interactions between activity genotypes for Gpdh, Mdh1, and Adh. We find no significant interlocus interactions. Observations on Mdh1 in both Gpdh and Adh backgrounds demonstrate significant increases in ethanol tolerance with partial reductions (50%) in cytosolic MDH activity. This observation strongly suggests the operation of pyruvate-malate and, in particular, pyruvate-citrate cycling in adaptation to alcohol exposure. We propose that an understanding of the evolution of tolerance to alcohols will require a system-level approach, rather than a focus on single enzymes.

  13. An asynchronous unfolding among molecular different regions of lobster D-glyceraldehyde-3-phosphate dehydrogenase and maltotetraose-forming amylase from an Alcaligenes sp. during guanidine denaturation.

    PubMed

    He, R Q; Zhao, K Y; Yan, Z Z; Li, M

    1993-06-04

    Changes in ultraviolet absorbance and intrinsic protein fluorescence of 1,4-alpha-D-glucan maltotetrahydrolase (EC 3.2.1.60) from an Alcaligenes sp. (Gram-negative bacteria 537.1) and D-glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12) have been compared with their inactivation during denaturation in guanidinium-Cl solutions. The two enzymes were completely inactivated at GuHCl concentrations less than 0.6 M and this was accompanied by marked absorbance and intrinsic fluorescence changes suggesting exposure of aromatic residues. The changes of the intrinsic fluorescence of the amylase have a relatively constant plateau in emission intensities and maxima at GuHCl concentrations from 0.8-2.0 M, similar to that of muscle GAPDH. The relative activity of the enzyme increased markedly in dilute GuHCl solutions accompanied by very little change of its intrinsic fluorescence at 8 degrees C. The kinetic decrease in emission intensities, excited respectively by 230 nm and 292 nm, was different for the two enzymes. The inactivation was a biphasic process with a fast phase faster than the unfolding rate as measured by fluorescence changes in 0.5 M GuHCl solution. Similar to the inactivation process, changes in intensity of 410 nm NAD fluorescent derivative of GAPDH which is in situ at the active site is also a biphasic process under the same condition. It appears that there may be an unfolding intermediate state of the enzymes and an asynchronous unfolding process among the different regions in the molecules during GuHCl denaturation, this may be due to differences in their flexibility.

  14. Evolution of a double amino acid substitution in the 5-enolpyruvylshikimate-3-phosphate synthase in Eleusine indica conferring high-level glyphosate resistance.

    PubMed

    Yu, Qin; Jalaludin, Adam; Han, Heping; Chen, Ming; Sammons, R Douglas; Powles, Stephen B

    2015-04-01

    Glyphosate is the most important and widely used herbicide in world agriculture. Intensive glyphosate selection has resulted in the widespread evolution of glyphosate-resistant weed populations, threatening the sustainability of this valuable once-in-a-century agrochemical. Field-evolved glyphosate resistance due to known resistance mechanisms is generally low to modest. Here, working with a highly glyphosate-resistant Eleusine indica population, we identified a double amino acid substitution (T102I+P106S [TIPS]) in the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene in glyphosate-resistant individuals. This TIPS mutation recreates the biotechnology-engineered commercial first generation glyphosate-tolerant EPSPS in corn (Zea mays) and now in other crops. In E. indica, the naturally evolved TIPS mutants are highly (more than 180-fold) resistant to glyphosate compared with the wild type and more resistant (more than 32-fold) than the previously known P106S mutants. The E. indica TIPS EPSPS showed very high-level (2,647-fold) in vitro resistance to glyphosate relative to the wild type and is more resistant (600-fold) than the P106S variant. The evolution of the TIPS mutation in crop fields under glyphosate selection is likely a sequential event, with the P106S mutation being selected first and fixed, followed by the T102I mutation to create the highly resistant TIPS EPSPS. The sequential evolution of the TIPS mutation endowing high-level glyphosate resistance is an important mechanism by which plants adapt to intense herbicide selection and a dramatic example of evolution in action. © 2015 American Society of Plant Biologists. All Rights Reserved.

  15. Insulin activation of vacuolar protein sorting 34 mediates localized phosphatidylinositol 3-phosphate production at lamellipodia and activation of mTOR/S6K1.

    PubMed

    Hirsch, Dianne S; Shen, Yi; Dokmanovic, Milos; Yu, Joyce; Mohan, Nishant; Elzarrad, Mohammed Khair; Wu, Wen Jin

    2014-06-01

    The class III phosphatidylinositol 3-kinase, VPS34, phosphorylates the D3 hydroxyl of inositol generating phosphatidylinositol 3-phosphate (ptdins(3)p). Initial studies suggested that ptdins(3)p solely functioned as a component of vesicular and endosomal membranes and that VPS34 did not function in signal transduction. However, VPS34 has recently been shown to be required for insulin-mediated activation of S6 kinase 1 (S6K1). Whether VPS34 activity is directly regulated by insulin is unclear. It is also not known whether VPS34 activity can be spatially restricted in response to extracellular stimuli. Data presented here demonstrate that in response to insulin, VPS34 is activated and translocated to lamellipodia where it produces ptdins(3)p. The localized production of ptdins(3)p is dependent on Src phosphorylation of VPS34. In cells expressing VPS34 with mutations at Y231 or Y310, which are Src-phosphorylation sites, insulin-stimulated VPS34 translocation to the plasma membrane and lamellipodia formation are blocked. mTOR also colocalizes with VPS34 and ptdins(3)p at lamellipodia following insulin-stimulation. In cells expressing the VPS34-Y231F mutant, which blocks lamellipodia formation, mTOR localization at the plasma membrane and insulin-mediated S6K1 activation are reduced. This suggests that mTOR localization at lamellipodia is important for full activation of S6K1 induced by insulin. These data demonstrate that insulin can spatially regulate VPS34 activity through Src-mediated tyrosine phosphorylation and that this membrane localized activity contributes to lamellipodia formation and activation of mTOR/S6K1signaling.

  16. Enzyme Architecture: Self-Assembly of Enzyme and Substrate Pieces of Glycerol-3-Phosphate Dehydrogenase into a Robust Catalyst of Hydride Transfer.

    PubMed

    Reyes, Archie C; Amyes, Tina L; Richard, John P

    2016-11-23

    The stabilization of the transition state for hlGPDH-catalyzed reduction of DHAP due to the action of the phosphodianion of DHAP and the cationic side chain of R269 is between 12.4 and 17 kcal/mol. The R269A mutation of glycerol-3-phosphate dehydrogenase (hlGPDH) results in a 9.1 kcal/mol destabilization of the transition state for enzyme-catalyzed reduction of dihydroxyacetone phosphate (DHAP) by NADH, and there is a 6.7 kcal/mol stabilization of this transition state by 1.0 M guanidine cation (Gua(+)) [J. Am. Chem. Soc. 2015, 137, 5312-5315]. The R269A mutant shows no detectable activity toward reduction of glycolaldehyde (GA), or activation of this reaction by 30 mM HPO3(2-). We report the unprecedented self-assembly of R269A hlGPDH, dianions (X(2-) = FPO3(2-), HPO3(2-), or SO4(2-)), Gua(+) and GA into a functioning catalyst of the reduction of GA, and fourth-order reaction rate constants kcat/KGAKXKGua. The linear logarithmic correlation (slope = 1.0) between values of kcat/KGAKX for dianion activation of wildtype hlGPDH-catalyzed reduction of GA and kcat/KGAKXKGua shows that the electrostatic interaction between exogenous dianions and the side chain of R269 is not significantly perturbed by cutting hlGPDH into R269A and Gua(+) pieces. The advantage for connection of hlGPDH (R269A mutant + Gua(+)) and substrate pieces (GA + HPi) pieces, (ΔGS(‡))HPi+E+Gua = 5.6 kcal/mol, is nearly equal to the sum of the advantage to connection of the substrate pieces, (ΔGS(‡))GA+HPi = 3.3 kcal/mol, for wildtype hlGPDH-catalyzed reaction of GA + HPi, and for connection of the enzyme pieces, (ΔGS(‡))E+Gua = 2.4 kcal/mol, for Gua(+) activation of the R269A hlGPDH-catalyzed reaction of DHAP.

  17. Biosynthesis of isoprenoids (carotenoids, sterols, prenyl side-chains of chlorophylls and plastoquinone) via a novel pyruvate/glyceraldehyde 3-phosphate non-mevalonate pathway in the green alga Scenedesmus obliquus.

    PubMed

    Schwender, J; Seemann, M; Lichtenthaler, H K; Rohmer, M

    1996-05-15

    Isoprenoid biosynthesis was investigated in the green alga Scenedesmus obliquus grown heterotrophically on 13C-labelled glucose and acetate. Several isoprenoid compounds were isolated and investigated by 13C-NMR spectroscopy. According to the 13C-labelling pattern indicated by the 13C-NMR spectra, the biosynthesis of all plastidic isoprenoids investigated (prenyl side-chains of chlorophylls and plastoquinone-9, and the carotenoids beta-carotene and lutein), as well as of the non-plastidic cytoplasmic sterols, does not proceed via the classical acetate/mevalonate pathway (which leads from acetyl-CoA via mevalonate to isopentenyl diphosphate), but via the novel glyceraldehyde 3-phosphate/pyruvate route recently detected in eubacteria. Formation of isopentenyl diphosphate involves the condensation of a C2 unit derived from pyruvate decarboxylation with glyceraldehyde 3-phosphate and a transposition yielding the branched C5 skeleton of isoprenic units.

  18. Effect of diet and triiodothyronine on the activity of sn-glycerol-3-phosphate dehydrogenase and on the metabolism of glucose and pyruvate by adipose tissue of obese patients

    PubMed Central

    Bray, G. A.; Mothon, Stella; Cohen, Andrea

    1969-01-01

    Lipogenesis and the metabolism of sn-glycerol-3-phosphate were studied in 23 fat biopsies from eight grossly obese patients. The first biopsy was obtained after a minimum of 12 days on a 3500 cal diet, the second biopsy after 2 wk on a 900 cal diet, and the third biopsy after an additional 2 wk on 900 cal supplemented with thiiodothyronine, 250 μg/day. Oxygen consumption and respiratory quotient declined during caloric restriction. Oxygen consumption was restored to the initial level during treatment with triiodothyronine, and the respiratory quotient rose somewhat. Lipogenesis from glucose and pyruvate was demonstrated in fat obtained from the first biopsy but could not be detected in the other biopsies. The incorporation of radioactivity from pyruvate into fatty acids was stimulated by the addition of glucose. Insulin stimulated lipogenesis in pieces of fat from the first biopsy, but isolated fat cells were unaffected by insulin. After caloric restriction no effects of insulin could be detected. The activity of both the cytoplasmic and mitochondrial sn-glycerol-3-phosphate dehydrogenase in homogenates of adipose tissue declined with caloric restriction. Treatment with triiodothyronine enhanced the activity of the mitochondrial sn-glycerol-3-phosphate dehydrogenase but did not affect the cytoplasmic enzyme. PMID:5796353

  19. Antitrypanosomal compounds from the essential oil and extracts of Keetia leucantha leaves with inhibitor activity on Trypanosoma brucei glyceraldehyde-3-phosphate dehydrogenase.

    PubMed

    Bero, J; Beaufay, C; Hannaert, V; Hérent, M-F; Michels, P A; Quetin-Leclercq, J

    2013-02-15

    Keetia leucantha is a West African tree used in traditional medicine to treat several diseases among which parasitic infections. The dichloromethane extract of leaves was previously shown to possess growth-inhibitory activities on Plasmodium falciparum, Trypanosoma brucei brucei and Leishmania mexicana mexicana with low or no cytotoxicity (>100 μg/ml on human normal fibroblasts) (Bero et al. 2009, 2011). In continuation of our investigations on the antitrypanosomal compounds from this dichloromethane extract, we analyzed by GC-FID and GC-MS the essential oil of its leaves obtained by hydrodistillation and the major triterpenic acids in this extract by LC-MS. Twenty-seven compounds were identified in the oil whose percentages were calculated using the normalization method. The essential oil, seven of its constituents and the three triterpenic acids were evaluated for their antitrypanosomal activity on Trypanosoma brucei brucei bloodstream forms (Tbb BSF) and procyclic forms (Tbb PF) to identify an activity on the glycolytic process of trypanosomes. The oil showed an IC(50) of 20.9 μg/ml on Tbb BSF and no activity was observed on Tbb PF. The best antitrypanosomal activity was observed for ursolic acid with IC(50) of 2.5 and 6.5 μg/ml respectively on Tbb BSF and Tbb PF. The inhibitory activity on a glycolytic enzyme of T. brucei, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), was also evaluated for betulinic acid, olenaolic acid, ursolic acid, phytol, α-ionone and β-ionone. The three triterpenic acids and β-ionone showed inhibitory activities on GAPDH with oleanolic acid being the most active with an inhibition of 72.63% at 20 μg/ml. This paper reports for the first time the composition and antitrypanosomal activity of the essential oil of Keetia leucantha. Several of its constituents and three triterpenic acids present in the dichloromethane leaves extract showed a higher antitrypanosomal activity on bloodstream forms of Tbb as compared to procyclic forms

  20. Functional complementation of an Escherichia coli gap mutant supports an amphibolic role for NAD(P)-dependent glyceraldehyde-3-phosphate dehydrogenase of Synechocystis sp. strain PCC 6803.

    PubMed Central

    Valverde, F; Losada, M; Serrano, A

    1997-01-01

    The gap-2 gene, encoding the NAD(P)-dependent D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH2) of the cyanobacterium Synechocystis sp. strain PCC 6803, was cloned by functional complementation of an Escherichia coli gap mutant with a genomic DNA library; this is the first time that this cloning strategy has been used for a GAPDH involved in photosynthetic carbon assimilation. The Synechocystis DNA region able to complement the E. coli gap mutant was narrowed down to 3 kb and fully sequenced. A single complete open reading frame of 1,011 bp encoding a protein of 337 amino acids was found and identified as the putative gap-2 gene identified in the complete genome sequence of this organism. Determination of the transcriptional start point, identification of putative promoter and terminator sites, and orientation of the truncated flanking genes suggested the gap-2 transcript should be monocystronic, a possibility further confirmed by Northern blot studies. Both natural and recombinant homotetrameric GAPDH2s were purified and found to exhibit virtually identical physicochemical and kinetic properties. The recombinant GAPDH2 showed the dual pyridine nucleotide specificity characteristic of the native cyanobacterial enzyme, and similar ratios of NAD- to NADP-dependent activities were found in cell extracts from Synechocystis as well as in those from the complemented E. coli clones. The deduced amino acid sequence of Synechocystis GAPDH2 presented a high degree of identity with sequences of the chloroplastic NADP-dependent enzymes. In agreement with this result, immunoblot analysis using monospecific antibodies raised against GAPDH2 showed the presence of the 38-kDa GAPDH subunit not only in crude extracts from the gap-2-expressing E. coli clones and all cyanobacteria that were tested but also in those from eukaryotic microalgae and plants. Western and Northern blot experiments showed that gap-2 is conspicuously expressed, although at different levels, in Synechocystis

  1. Protective immune responses against Schistosoma mansoni infection by immunization with functionally active gut-derived cysteine peptidases alone and in combination with glyceraldehyde 3-phosphate dehydrogenase

    PubMed Central

    Tallima, Hatem; Dvořák, Jan; Kareem, Sahira; Abou El Dahab, Marwa; Abdel Aziz, Nada; El Ridi, Rashika

    2017-01-01

    Background Schistosomiasis, a severe disease caused by parasites of the genus Schistosoma, is prevalent in 74 countries, affecting more than 250 million people, particularly children. We have previously shown that the Schistosoma mansoni gut-derived cysteine peptidase, cathepsin B1 (SmCB1), administered without adjuvant, elicits protection (>60%) against challenge infection of S. mansoni or S. haematobium in outbred, CD-1 mice. Here we compare the immunogenicity and protective potential of another gut-derived cysteine peptidase, S. mansoni cathepsin L3 (SmCL3), alone, and in combination with SmCB1. We also examined whether protective responses could be boosted by including a third non-peptidase schistosome secreted molecule, glyceraldehyde 3-phosphate dehydrogenase (SG3PDH), with the two peptidases. Methodology/Principal findings While adjuvant-free SmCB1 and SmCL3 induced type 2 polarized responses in CD-1 outbred mice those elicited by SmCL3 were far weaker than those induced by SmCB1. Nevertheless, both cysteine peptidases evoked highly significant (P < 0.005) reduction in challenge worm burden (54–65%) as well as worm egg counts and viability. A combination of SmCL3 and SmCB1 did not induce significantly stronger immune responses or higher protection than that achieved using each peptidase alone. However, when the two peptidases were combined with SG3PDH the levels of protection against challenge S. mansoni infection reached 70–76% and were accompanied by highly significant (P < 0.005) decreases in worm egg counts and viability. Similarly, high levels of protection were achieved in hamsters immunized with the cysteine peptidase/SG3PDH-based vaccine. Conclusions/Significance Gut-derived cysteine peptidases are highly protective against schistosome challenge infection when administered subcutaneously without adjuvant to outbred CD-1 mice and hamsters, and can also act to enhance the efficacy of other schistosome antigens, such as SG3PDH. This cysteine

  2. Observation of thiamin-bound intermediates and microscopic rate constants for their interconversion on 1-deoxy-D-xylulose 5-phosphate synthase: 600-fold rate acceleration of pyruvate decarboxylation by D-glyceraldehyde-3-phosphate.

    PubMed

    Patel, Hetalben; Nemeria, Natalia S; Brammer, Leighanne A; Freel Meyers, Caren L; Jordan, Frank

    2012-11-07

    The thiamin diphosphate (ThDP)-dependent enzyme 1-deoxy-D-xylulose 5-phosphate (DXP) synthase carries out the condensation of pyruvate as a 2-hydroxyethyl donor with d-glyceraldehyde-3-phosphate (d-GAP) as acceptor forming DXP. Toward understanding catalysis of this potential anti-infective drug target, we examined the pathway of the enzyme using steady state and presteady state kinetic methods. It was found that DXP synthase stabilizes the ThDP-bound predecarboxylation intermediate formed between ThDP and pyruvate (C2α-lactylThDP or LThDP) in the absence of D-GAP, while addition of D-GAP enhanced the rate of decarboxylation by at least 600-fold. We postulate that decarboxylation requires formation of a ternary complex with both LThDP and D-GAP bound, and the central enzyme-bound enamine reacts with D-GAP to form DXP. This appears to be the first study of a ThDP enzyme where the individual rate constants could be evaluated by time-resolved circular dichroism spectroscopy, and the results could have relevance to other ThDP enzymes in which decarboxylation is coupled to a ligation reaction. The acceleration of the rate of decarboxylation of enzyme-bound LThDP in the presence of D-GAP suggests a new approach to inhibitor design.

  3. Rational Design of Nanoparticle Platforms for "Cutting-the-Fat": Covalent Immobilization of Lipase, Glycerol Kinase, and Glycerol-3-Phosphate Oxidase on Metal Nanoparticles.

    PubMed

    Aggarwal, V; Pundir, C S

    2016-01-01

    The aggregates of nanoparticles (NPs) are considered better supports for the immobilization of enzymes, as these promote enzyme kinetics, due to their unusual but favorable properties such as larger surface area to volume ratio, high catalytic efficiency of certain immobilized enzymes, non-toxicity of some of the nanoparticle matrices, high stability, strong adsorption of the enzyme of interest by a number of different approaches, and faster electron transportability. Co-immobilization of multiple enzymes required for a multistep reaction cascade on a single support is more efficient than separately immobilizing the corresponding enzymes and mixing them physically, since products of one enzyme could serve as reactants for another. These products can diffuse much more easily between enzymes on the same particle than diffusion from one particle to the next, in the reaction medium. Thus, co-immobilization of enzymes onto NP aggregates is expected to produce faster kinetics than their individual immobilizations on separate matrices. Lipase, glycerol kinase, and glycerol-3-phosphate oxidase are required for lipid analysis in a cascade reaction, and we describe the co-immobilization of these three enzymes on nanocomposites of zinc oxide nanoparticles (ZnONPs)-chitosan (CHIT) and gold nanoparticles-polypyrrole-polyindole carboxylic acid (AuPPy-Pin5COOH) which are electrodeposited on Pt and Au electrodes, respectively. The kinetic properties and analytes used for amperometric determination of TG are fully described for others to practice in a trained laboratory. Cyclic voltammetry, scanning electron microscopy, Fourier transform infra-red spectra, and electrochemical impedance spectra confirmed their covalent co-immobilization onto electrode surfaces through glutaraldehyde coupling on CHIT-ZnONPs and amide bonding on AuPPy/Pin5COOH. The combined activities of co-immobilized enzymes was tested amperometrically, and these composite nanobiocatalysts showed optimum activity

  4. Synthesis of 4-nitrophenyl acetate using molecular sieve-immobilized lipase from Bacillus coagulans.

    PubMed

    Raghuvanshi, Shilpa; Gupta, Reena

    2009-03-01

    Extracellular lipase from Bacillus coagulans BTS-3 was immobilized on (3 A x 1.5 mm) molecular sieve. The molecular sieve showed approximately 68.48% binding efficiency for lipase (specific activity 55 IU mg(-1)). The immobilized enzyme achieved approx 90% conversion of acetic acid and 4-nitrophenol (100 mM each) into 4-nitrophenyl acetate in n-heptane at 65 degrees C in 3 h. When alkane of C-chain length other than n-heptane was used as the organic solvent, the conversion of 4-nitrophenol and acetic acid was found to decrease. About 88.6% conversion of the reactants into ester was achieved when reactants were used at molar ratio of 1:1. The immobilized lipase brought about conversion of approximately 58% for esterification of 4-nitrophenol and acetic acid into 4-nitrophenyl acetate at a temperature of 65 degrees C after reuse for 5 cycles.

  5. Octa, deca, and dodeca(4-nitrophenyl) cage silsesquioxanes via 4-trimethylsilylphenyl derivatives.

    PubMed

    Miyazato, Akio; Pakjamsai, Chitsakon; Kawakami, Yusuke

    2010-04-07

    Pure octa, deca, and dodeca(4-nitrophenyl) cage silsesquioxanes were obtained by regio-selective 4-nitration of octa, deca, and dodeca(4-trimethylsilylphenyl) cage silsesquioxanes via ipso-substitution of trimethylsilyl-phenyl bonds by fuming nitric acid. 3-Nitration of octa(4-methylphenyl)octasilesquioxane was also described. The starting octa(4-methyl-, 4-isopropyl- and 4-trimethylsilylphenyl)octasilsesquioxanes were selectively formed in 9-21% isolated yield in the presence of hydrochloric acid. Mixtures of octa, deca and dodecasilsesquioxanes, with decasilsesquioxane as the main component, were formed in the presence of tetrabutylammmonium fluoride as a catalyst. All the cage compounds could be separated mainly by crystallization.

  6. Acute Oral Toxicity (LD50) of 4-Nitrophenyl Monochloromethyl (Phenyl) Phosphinate (TA009) in Female Rats

    DTIC Science & Technology

    1984-10-01

    chosen was a mixture of Tween 80 (Fisher Scientific Company, Fairlawn, NJ) ethanol and citrate buffer (pH 2.9). Additional information on the vehicle...phosphinate formulated with Tween 80 , EtOH, and citrate buffer (LAIR SOP-OP-STX-45, Preparation of Compounds Unstable in Water for SLRL Assay). A 2.0...percent phosphinate solution was prepared with 1.5 g 4- nitrophenyl monochloromethyl (phenyl) phosphinate. 15.0 ml Tween 80 7.5 ml (100 %) ethanol

  7. The FAD-dependent glycerol-3-phosphate dehydrogenase of Giardia duodenalis: an unconventional enzyme that interacts with the g14-3-3 and it is a target of the antitumoral compound NBDHEX

    PubMed Central

    Lalle, Marco; Camerini, Serena; Cecchetti, Serena; Finelli, Renata; Sferra, Gabriella; Müller, Joachim; Ricci, Giorgio; Pozio, Edoardo

    2015-01-01

    The flagellated protozoan Giardia duodenalis is a worldwide parasite causing giardiasis, an acute and chronic diarrheal disease. Metabolism in G. duodenalis has a limited complexity thus making metabolic enzymes ideal targets for drug development. However, only few metabolic pathways (i.e., carbohydrates) have been described so far. Recently, the parasite homolog of the mitochondrial-like glycerol-3-phosphate dehydrogenase (gG3PD) has been identified among the interactors of the g14-3-3 protein. G3PD is involved in glycolysis, electron transport, glycerophospholipids metabolism, and hyperosmotic stress response, and is emerging as promising target in tumor treatment. In this work, we demonstrate that gG3PD is a functional flavoenzyme able to convert glycerol-3-phosphate into dihydroxyacetone phosphate and that its activity and the intracellular glycerol level increase during encystation. Taking advantage of co-immunoprecipitation assays and deletion mutants, we provide evidence that gG3PD and g14-3-3 interact at the trophozoite stage, the intracellular localization of gG3PD is stage dependent and it partially co-localizes with mitosomes during cyst development. Finally, we demonstrate that the gG3PD activity is affected by the antitumoral compound 6-(7-nitro-2,1,3-benzoxadiazol-4-ylthio)hexanol, that results more effective in vitro at killing G. duodenalis trophozoites than the reference drug metronidazole. Overall, our results highlight the involvement of gG3PD in processes crucial for the parasite survival thus proposing this enzyme as target for novel antigiardial interventions. PMID:26082764

  8. The FAD-dependent glycerol-3-phosphate dehydrogenase of Giardia duodenalis: an unconventional enzyme that interacts with the g14-3-3 and it is a target of the antitumoral compound NBDHEX.

    PubMed

    Lalle, Marco; Camerini, Serena; Cecchetti, Serena; Finelli, Renata; Sferra, Gabriella; Müller, Joachim; Ricci, Giorgio; Pozio, Edoardo

    2015-01-01

    The flagellated protozoan Giardia duodenalis is a worldwide parasite causing giardiasis, an acute and chronic diarrheal disease. Metabolism in G. duodenalis has a limited complexity thus making metabolic enzymes ideal targets for drug development. However, only few metabolic pathways (i.e., carbohydrates) have been described so far. Recently, the parasite homolog of the mitochondrial-like glycerol-3-phosphate dehydrogenase (gG3PD) has been identified among the interactors of the g14-3-3 protein. G3PD is involved in glycolysis, electron transport, glycerophospholipids metabolism, and hyperosmotic stress response, and is emerging as promising target in tumor treatment. In this work, we demonstrate that gG3PD is a functional flavoenzyme able to convert glycerol-3-phosphate into dihydroxyacetone phosphate and that its activity and the intracellular glycerol level increase during encystation. Taking advantage of co-immunoprecipitation assays and deletion mutants, we provide evidence that gG3PD and g14-3-3 interact at the trophozoite stage, the intracellular localization of gG3PD is stage dependent and it partially co-localizes with mitosomes during cyst development. Finally, we demonstrate that the gG3PD activity is affected by the antitumoral compound 6-(7-nitro-2,1,3-benzoxadiazol-4-ylthio)hexanol, that results more effective in vitro at killing G. duodenalis trophozoites than the reference drug metronidazole. Overall, our results highlight the involvement of gG3PD in processes crucial for the parasite survival thus proposing this enzyme as target for novel antigiardial interventions.

  9. Cloning of glycerol-3-phosphate dehydrogenase genes (ZrGPD1 and ZrGPD2) and glycerol dehydrogenase genes (ZrGCY1 and ZrGCY2) from the salt-tolerant yeast Zygosaccharomyces rouxii.

    PubMed

    Iwaki, T; Kurono, S; Yokose, Y; Kubota, K; Tamai, Y; Watanabe, Y

    2001-06-01

    The ZrGPD1 and ZrGPD2 genes encoding putative glycerol-3-phosphate dehydrogenases were isolated from the salt-tolerant yeast, Zygosaccharomyces rouxii. Both genes are homologous to GPD1 of Saccharomyces cerevisiae and are constitutively expressed in Z. rouxii cells. Putative glycerol dehydrogenase genes, ZrGCY1 and ZrGCY2, which are highly homologous to GCY1 of S. cerevisiae, were also isolated. Since the level of transcripts of ZrGCY1 and ZrGCY2 increased in Z. rouxii cells subjected to salt stress, it is suggested that the pathway of the signal transduction of salt stress controls the expression of these genes. The Accession Nos of these sequences in GenBank are as follows: ZrGPD1, AB047394; ZrGPD2, AB047395; ZrGCY1, AB047396; ZrGCY2, AB047397. Copyright 2001 John Wiley & Sons, Ltd.

  10. Generation of stable 'low phytic acid' transgenic rice through antisense repression of the 1D-myo-inositol 3-phosphate synthase gene (RINO1) using the 18-kDa oleosin promoter.

    PubMed

    Kuwano, Mio; Mimura, Tetsuro; Takaiwa, Fumio; Yoshida, Kaoru T

    2009-01-01

    Phytic acid acts as the major storage form of phosphorus in plant seeds and is poorly digested by monogastric animals. The degradation of phytic acid in animal diets is necessary to overcome both environmental and nutritional issues. The enzyme 1D-myo-inositol 3-phosphate [Ins(3)P(1)] synthase (EC 5.5.1.4) catalyses the first step of myo-inositol biosynthesis and directs phytic acid biosynthesis in seeds. The rice Ins(3)P(1) synthase gene (RINO1) is highly expressed in developing seed embryos and in the aleurone layer, where phytic acid is synthesized and stored. In rice seeds, 18-kDa oleosin (Ole18) is expressed in a seed-specific manner, and its transcripts are restricted to the embryo and the aleurone layer. Therefore, to effectively suppress phytic acid biosynthesis, antisense RINO1 cDNA was expressed under the control of the Ole18 promoter, directing the same spatial pattern in seeds as RINO1 in transgenic rice plants. The generated transgenic rice plants showed strong 'low phytic acid' (lpa) phenotypes, in which seed phytic acid was reduced by 68% and free available phosphate was concomitantly increased. No negative effects on seed weight, germination or plant growth were observed. The available phosphate levels of the stable transgenic plants surpassed those of currently available rice lpa mutants.

  11. Temperature induced phase transition of CaMn{sub 0.5}Zr{sub 1.5}(PO{sub 4}){sub 3} phosphate

    SciTech Connect

    Orlova, Maria; Perfler, Lukas; Tribus, Martina; Salnikov, Petr; Glorieux, Benoit; Orlova, Albina

    2016-03-15

    In this work we investigated the structural behaviour of a CaMn{sub 0.5}Zr{sub 1.5}(PO{sub 4}){sub 3}. Due to the presence of divalent Mn{sup 2+} cations this compound can possess interesting luminescence properties. It was recently understood that this phosphate undergoes a temperature induced irreversible phase transition in the range of 800–875 °C. It has also been shown that the 3d–3d luminescence of Mn{sup 2+} increases 10 fold for the high temperature polymorph. To determine the Mn environment structural investigations of both phases have been performed by the X-ray powder diffraction and Raman spectroscopy methods. The low temperature modification adopts the trigonal NZP structure type with a slightly lower symmetry (space group R32, a=8.7850(2) Å, c=22.6496(7) Å, V=1514.8(1) Å{sup 3}). The high temperature form in turn has orthorhombic symmetry (space group Pnma, a=6.2350(3) Å, b=6.6281(3) Å, c=14.4731(6) Å, V=598.13(5) Å{sup 3}). Both structures were solved ab-initio from powder data and structural analysis was performed. In-situ and RT Raman spectra are consistent with the XRD derived structural model. Mn{sup 2+} cations occupy different types of positions in these structures and a change in Mn coordination number (6 for LT phase, 7 for HT phase) results in different Mn–O bond lengths. These differences may explain the change in the optical properties between the polymorphs. - Graphical abstract: The compound CaMn{sub 0.5}Zr{sub 1.5}(PO{sub 4}){sub 3} was synthesized in order to create a material with enhanced luminescent properties. The goal of present studies is to define Mn{sup 2+} environment and its changes due to the structural transformations of the phosphate along phase transition at the T range of 800–875 °C. It was found that LT modification adopts the trigonal NZP structure type, sp.gr. R32, the HT form in turn exhibits orthorhombic symmetry sp.gr. Pnma. Mn2+ cations occupy different types of positions in those structures and a

  12. Effects of supplementation on food intake, body weight and hepatic metabolites in the citrin/mitochondrial glycerol-3-phosphate dehydrogenase double-knockout mouse model of human citrin deficiency.

    PubMed

    Saheki, Takeyori; Inoue, Kanako; Ono, Hiromi; Katsura, Natsumi; Yokogawa, Mana; Yoshidumi, Yukari; Furuie, Sumie; Kuroda, Eishi; Ushikai, Miharu; Asakawa, Akihiro; Inui, Akio; Eto, Kazuhiro; Kadowaki, Takashi; Sinasac, David S; Yamamura, Ken-Ichi; Kobayashi, Keiko

    2012-11-01

    The C57BL/6:Slc23a13(-/-);Gpd2(-/-) double-knockout (a.k.a., citrin/mitochondrial glycerol 3-phosphate dehydrogenase double knockout or Ctrn/mGPD-KO) mouse displays phenotypic attributes of both neonatal intrahepatic cholestasis (NICCD) and adult-onset type II citrullinemia (CTLN2), making it a suitable model of human citrin deficiency. In the present study, we show that when mature Ctrn/mGPD-KO mice are switched from a standard chow diet (CE-2) to a purified maintenance diet (AIN-93M), this resulted in a significant loss of body weight as a result of reduced food intake compared to littermate mGPD-KO mice. However, supplementation of the purified maintenance diet with additional protein (from 14% to 22%; and concomitant reduction or corn starch), or with specific supplementation with alanine, sodium glutamate, sodium pyruvate or medium-chain triglycerides (MCT), led to increased food intake and body weight gain near or back to that on chow diet. No such effect was observed when supplementing the diet with other sources of fat that contain long-chain fatty acids. Furthermore, when these supplements were added to a sucrose solution administered enterally to the mice, which has been shown previously to lead to elevated blood ammonia as well as altered hepatic metabolite levels in Ctrn/mGPP-KO mice, this led to metabolic correction. The elevated hepatic glycerol 3-phosphate and citrulline levels after sucrose administration were suppressed by the administration of sodium pyruvate, alanine, sodium glutamate and MCT, although the effect of MCT was relatively small. Low hepatic citrate and increased lysine levels were only found to be corrected by sodium pyruvate, while alanine and sodium glutamate both corrected hepatic glutamate and aspartate levels. Overall, these results suggest that dietary factors including increased protein content, supplementation of specific amino acids like alanine and sodium glutamate, as well as sodium pyruvate and MCT all show beneficial

  13. Synthesis, growth and characterization of a new promising organic nonlinear optical crystal: 4-Nitrophenyl hydrazone.

    PubMed

    Hemaraju, B C; Ahlam, M A; Pushpa, N; Mahadevan, K M; Gnana Prakash, A P

    2015-12-05

    4-Nitrophenyl hydrazone single crystals were grown by slow evaporation of solvent method using acetone as a solvent. The grown crystals were subjected to various characterizations such as X-ray diffraction studies, UV-visible studies, thermogravimetric analysis and differential thermal analysis (TGA/DTA) and second harmonic generation (SHG). The cell parameters were calculated by using single crystal XRD measurement and the crystal system was found as orthorhombic with non-centro-symmetric space group Pca21. The crystallinity of the grown crystal was confirmed by powder X-ray diffraction analysis. The Fourier transform infrared (FTIR) and nuclear magnetic resonance (NMR) studies confirmed the presence of functional groups in the sample. The optical transmittance spectrum shows that the crystal is transparent in the visible wavelength range. The scanning electron microscopy (SEM) was used to study the surface morphology of the grown crystal. The chemical composition of the grown crystal was confirmed by energy dispersive X-ray (EDX) analysis. The TGA/DTA results showed that the material is stable up to 146°C. The NLO property of the crystal was confirmed by Kurtz and Perry method and SHG conversion efficiency is 15.39 times when compared to KDP crystal. Copyright © 2015 Elsevier B.V. All rights reserved.

  14. The expressed protein in glyphosate-tolerant soybean, 5-enolpyruvylshikimate-3-phosphate synthase from Agrobacterium sp. strain CP4, is rapidly digested in vitro and is not toxic to acutely gavaged mice.

    PubMed

    Harrison, L A; Bailey, M R; Naylor, M W; Ream, J E; Hammond, B G; Nida, D L; Burnette, B L; Nickson, T E; Mitsky, T A; Taylor, M L; Fuchs, R L; Padgette, S R

    1996-03-01

    The safety of 5-enolpyruvylshikimate-3-phosphate synthase enzyme derived from Agrobacterium sp. strain CP4 (CP4 EPSPS) was assessed. CP4 EPSPS is the only protein introduced by genetic manipulation that is expressed in glyphosate-tolerant soybeans, which are being developed to provide new weed-control options for farmers. Expression of this protein in plants imparts high levels of glyphosate tolerance. The safety of CP4 EPSPS was ascertained by evaluating both physical and functional characteristics. CP4 EPSPS degrades readily in simulated gastric and intestinal fluids, suggesting that this protein will be degraded in the mammalian digestive tract upon ingestion as a component of food or feed, There were no deleterious effects due to the acute administration of CP4 EPSPS to mice by gavage at a high dosage of 572 mg/kg body wt, which exceeds 1000-fold tha anticipated consumption level of food products potentially containing CP4 EPSPS protein. CP4 EPSPS does not pose any important allergen concerns because this protein does not possess characteristics typical of allergenic proteins. These data, in combination with seed compositional analysis and animal feeding studies, support the conclusion that glyphosate-tolerant soybean are as safe and nutritious as traditional soybeans currently being marketed.

  15. The proline-rich region of glyceraldehyde-3-phosphate dehydrogenase from human sperm may bind SH3 domains, as revealed by a bioinformatic study of low-complexity protein segments.

    PubMed

    Tatjewski, Marcin; Gruca, Aleksandra; Plewczynski, Dariusz; Grynberg, Marcin

    2016-02-01

    Glyceraldehyde-3-phosphate dehydrogenase from human sperm (GAPDHS) provides energy to the sperm flagellum, and is therefore essential for sperm motility and male fertility. This isoform is distinct from somatic GAPDH, not only in being specific for the testis but also because it contains an additional amino-terminal region that encodes a proline-rich motif that is known to bind to the fibrous sheath of the sperm tail. By conducting a large-scale sequence comparison on low-complexity sequences available in databases, we identified a strong similarity between the proline-rich motif from GAPDHS and the proline-rich sequence from Ena/vasodilator-stimulated phosphoprotein-like (EVL), which is known to bind an SH3 domain of dynamin-binding protein (DNMBP). The putative binding partners of the proline-rich GAPDHS motif include SH3 domain-binding protein 4 (SH3BP4) and the IL2-inducible T-cell kinase/tyrosine-protein kinase ITK/TSK (ITK). This result implies that GAPDHS participates in specific signal-transduction pathways. Gene Ontology category-enrichment analysis showed several functional classes shared by both proteins, of which the most interesting ones are related to signal transduction and regulation of hydrolysis. Furthermore, a mutation of one EVL proline to leucine is known to cause colorectal cancer, suggesting that mutation of homologous amino acid residue in the GAPDHS motif may be functionally deleterious.

  16. Efficient promotion of phosphate diester cleavage by a face-to-face cyclodextrin dimer without metal.

    PubMed

    Hu, Ping; Liu, Gao-Feng; Ji, Liang-Nian; Mao, Zong-Wan

    2012-06-04

    An organic face-to-face cyclodextrin dimer promotes the cleavage of bis(4-nitrophenyl) phosphate efficiently in neutral pH without the addition of metal. Both of the phosphate diester bonds can be cleaved.

  17. Inactivation of Glyceraldehyde-3-Phosphate Dehydrogenase by Fumarate in Diabetes: Formation of S-(2-Succinyl)Cysteine, a Novel Chemical Modification of Protein and Possible Biomarker of Mitochondrial Stress

    PubMed Central

    Blatnik, Matthew; Frizzell, Norma; Thorpe, Suzanne R.; Baynes, John W.

    2008-01-01

    OBJECTIVE S-(2-succinyl)cysteine (2SC) is formed by a Michael addition reaction of the Krebs cycle intermediate, fumarate, with cysteine residues in protein. We investigated the role of fumarate in chemical modification and inhibition of the sulfhydryl enzyme, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), in vitro and in tissues of diabetic rats. RESEARCH DESIGN AND METHODS GAPDH was incubated with fumarate in PBS to assess effects of fumarate on enzyme activity in vitro. Sites of 2SC formation were determined by analysis of tryptic peptides by high-performance liquid chromatography–quadrupole/time-of-flight mass spectrometry. 2SC and fumarate in gastrocnemius muscle of control and streptozotocin-induced diabetic rats were measured by liquid chromatography/tandem mass spectrometry and by gas chromatography/mass spectrometry, respectively. GAPDH was isolated from muscle by immunoprecipitation, and sites of modification of GAPDH were determined by mass spectrometry analysis. RESULTS 2SC was found, both in vitro and in vivo, about equally at active-site Cys-149 and nucleophilic Cys-244. Inactivation of GAPDH by fumarate in vitro correlated with formation of 2SC. In diabetic compared with control rats, fumarate and 2SC concentration increased approximately fivefold, accompanied by an ~25% decrease in GAPDH specific activity. The fractional modification of GAPDH by 2SC was significantly increased in diabetic versus control animals, consistent with the decreased specific activity of GAPDH in muscle of diabetic animals. CONCLUSIONS Fumarate contributes to inactivation of GAPDH in diabetes. 2SC may be a useful biomarker of mitochondrial stress in diabetes. Modification of GAPDH and other enzymes and proteins by fumarate may contribute to the metabolic changes underlying the development of diabetes complications. PMID:17934141

  18. Inactivation of glyceraldehyde-3-phosphate dehydrogenase by fumarate in diabetes: formation of S-(2-succinyl)cysteine, a novel chemical modification of protein and possible biomarker of mitochondrial stress.

    PubMed

    Blatnik, Matthew; Frizzell, Norma; Thorpe, Suzanne R; Baynes, John W

    2008-01-01

    (2-succinyl)cysteine (2SC) is formed by a Michael addition reaction of the Krebs cycle intermediate, fumarate, with cysteine residues in protein. We investigated the role of fumarate in chemical modification and inhibition of the sulfhydryl enzyme, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), in vitro and in tissues of diabetic rats. GAPDH was incubated with fumarate in PBS to assess effects of fumarate on enzyme activity in vitro. Sites of 2SC formation were determined by analysis of tryptic peptides by high-performance liquid chromatography-quadrupole/time-of-flight mass spectrometry. 2SC and fumarate in gastrocnemius muscle of control and streptozotocin-induced diabetic rats were measured by liquid chromatography/tandem mass spectrometry and by gas chromatography/mass spectrometry, respectively. GAPDH was isolated from muscle by immunoprecipitation, and sites of modification of GAPDH were determined by mass spectrometry analysis. 2SC was found, both in vitro and in vivo, about equally at active-site Cys-149 and nucleophilic Cys-244. Inactivation of GAPDH by fumarate in vitro correlated with formation of 2SC. In diabetic compared with control rats, fumarate and 2SC concentration increased approximately fivefold, accompanied by an approximately 25% decrease in GAPDH specific activity. The fractional modification of GAPDH by 2SC was significantly increased in diabetic versus control animals, consistent with the decreased specific activity of GAPDH in muscle of diabetic animals. Fumarate contributes to inactivation of GAPDH in diabetes. 2SC may be a useful biomarker of mitochondrial stress in diabetes. Modification of GAPDH and other enzymes and proteins by fumarate may contribute to the metabolic changes underlying the development of diabetes complications.

  19. Resveratrol plus ethanol counteract the ethanol-induced impairment of energy metabolism: ³¹P NMR study of ATP and sn-glycerol-3-phosphate on isolated and perfused rat liver.

    PubMed

    Gallis, Jean-Louis; Serhan, Nizar; Gin, Henri; Couzigou, Patrice; Beauvieux, Marie-Christine

    2012-03-01

    The effects of trans-resveratrol (RSV) combined with ethanol (EtOH) were evaluated by (31)P NMR on total ATP and sn-glycerol-3-phosphate (sn-G3P) contents measured in real time in isolated and perfused whole liver of the rat. Mitochondrial ATP turnover was assessed by using specific inhibitors of glycolytic and mitochondrial ATP supply (iodacetate and KCN, respectively). In RSV alone, the slight decrease in ATP content (-14±5% of the initial content), sn-G3P content and ATP turnover were similar to those in the Krebs-Henseleit buffer control. Compared to control, EtOH alone (14 or 70 mmol/L) induced a decrease in ATP content (-24.95±2.95% of initial content, p<0.05) and an increase in sn-G3P (+158±22%), whereas ATP turnover tended to be increased. RSV (20 μmol/L) combined with EtOH, (i) maintained ATP content near 100%, (ii) induced a 1.6-fold increase in mitochondrial ATP turnover (p=0.049 and p=0.004 vs EtOH 14 and 70 mmol/L alone, respectively) and (iii) led to an increase in sn-G3P (+49±9% and +81±6% for 14 and 70 mmol/L EtOH, respectively). These improvements were obtained only when glycolysis was efficient at the time of addition of EtOH+RSV. Glycolysis inhibition by iodacetate (IAA) evidenced an almost 21% contribution of this pathway to ATP content. RSV alone or RSV+EtOH prevented the ATP decrease induced by IAA addition (p<0.05 vs control). This is the first demonstration of the combined effects of RSV and EtOH on liver energy metabolism. RSV increased (i) the flux of substrates through ATP producing pathways (glycolysis and phosphorylative oxidation) probably via the activation of AMPkinase, and (ii) maintained the glycolysis deviation to sn-G3P linked to NADH+H⁺ re-oxidation occurring during EtOH detoxication, thus reducing the energy cost due to the latter. Copyright © 2011 Elsevier Ltd. All rights reserved.

  20. Glycerol-3-phosphate acyltransferase-1 upregulation by O-GlcNAcylation of Sp1 protects against hypoxia-induced mouse embryonic stem cell apoptosis via mTOR activation

    PubMed Central

    Lee, H J; Ryu, J M; Jung, Y H; Lee, K H; Kim, D I; Han, H J

    2016-01-01

    Oxygen signaling is critical for stem cell regulation, and oxidative stress-induced stem cell apoptosis decreases the efficiency of stem cell therapy. Hypoxia activates O-linked β-N-acetyl glucosaminylation (O-GlcNAcylation) of stem cells, which contributes to regulation of cellular metabolism, as well as cell fate. Our study investigated the role of O-GlcNAcylation via glucosamine in the protection of hypoxia-induced apoptosis of mouse embryonic stem cells (mESCs). Hypoxia increased mESCs apoptosis in a time-dependent manner. Moreover, hypoxia also slightly increased the O-GlcNAc level. Glucosamine treatment further enhanced the O-GlcNAc level and prevented hypoxia-induced mESC apoptosis, which was suppressed by O-GlcNAc transferase inhibitors. In addition, hypoxia regulated several lipid metabolic enzymes, whereas glucosamine increased expression of glycerol-3-phosphate acyltransferase-1 (GPAT1), a lipid metabolic enzyme producing lysophosphatidic acid (LPA). In addition, glucosamine-increased O-GlcNAcylation of Sp1, which subsequently leads to Sp1 nuclear translocation and GPAT1 expression. Silencing of GPAT1 by gpat1 siRNA transfection reduced glucosamine-mediated anti-apoptosis in mESCs and reduced mammalian target of rapamycin (mTOR) phosphorylation. Indeed, LPA prevented mESCs from undergoing hypoxia-induced apoptosis and increased phosphorylation of mTOR and its substrates (S6K1 and 4EBP1). Moreover, mTOR inactivation by rapamycin (mTOR inhibitor) increased pro-apoptotic proteins expressions and mESC apoptosis. Furthermore, transplantation of non-targeting siRNA and glucosamine-treated mESCs increased cell survival and inhibited flap necrosis in mouse skin flap model. Conversely, silencing of GPAT1 expression reversed those glucosamine effects. In conclusion, enhancing O-GlcNAcylation of Sp1 by glucosamine stimulates GPAT1 expression, which leads to inhibition of hypoxia-induced mESC apoptosis via mTOR activation. PMID:27010859

  1. Participation of analogues of lysophosphatidic acid (LPA): oleoyl-sn-glycero-3-phosphate (L-alpha-LPA) and 1-oleoyl-2-O-methyl-rac-glycerophosphothionate (OMPT) in uterine smooth muscle contractility of the pregnant pigs.

    PubMed

    Markiewicz, W; Kamińska, K; Bogacki, M; Maślanka, T; Jaroszewski, J

    2012-01-01

    Recent studies show that a representative of phospholipids, namely lysophosphatidic acid (LPA) and its receptors (LPA1.3) play a significant role in the reproductive processes, i. a, in the modulation of the uterine contractility. The participation of LPA3 in the reproductive processes has been revealed in mice and has not been studied in gilts. Therefore, in the present study we investigated the role/action of LPA and its receptors LPA1, LPA2 and LPA3 on the contraction activity in the porcine uterus. The study was conducted on an experimental model in which the pig uterus consisted of the one whole uterine horn and a part of the second horn, both connected with the uterine corpus. Uterine strips consisting of the endometrium with the myometrium (ENDO/MYO) and myometrium (MYO) alone were collected on days 12-14 of the estrous cycle (control group; n = 5) or pregnancy (experimental group; n = 5). Two analogues of LPA at increasing doses were used: oleoyl-sn-glycero-3-phosphate (L-alpha-LPA, a selective agonist of LPA1 and LPA2 receptors; 10(-7) M; 10(-6) M and 10(-5) M) and 1-oleoyl-2-O-methyl-rac-glycerophosphothionate (OMPT, a selective agonist of LPA3 receptor; 68 nM; 136 nM and 680 nM). L-alpha-LPA caused an increase in the contraction tension, amplitude and frequency of ENDO/MYO from the uterine horn with the developing embryos. This effect was not observed in MYO in both groups examined. In the ENDO/MYO strips of the uterine horn with developing embryos, OMPT significantly increased the contraction tension at the highest dose (680 nM) and amplitude at all doses examined, while frequency of contractions was decreased at doses of 136 nM and 680 nM. In the MYO strips of the uterine horn with embryos a significant increase in the contraction tension and amplitude after the highest dose of OMPT was observed. The results obtained imply the important role of receptors LPA1, LPA2 and LPA3 in the contraction activity of the porcine uterus during early pregnancy.

  2. Experimental and theoretical studies of Diels-Alder reaction between methyl (Z)-2-nitro-3-(4-nitrophenyl)-2-propenoate and cyclopentadiene.

    PubMed

    Jasiński, Radomir; Kwiatkowska, Magdalena; Sharnin, Valentin; Barański, Andrzej

    The Diels-Alder reaction between methyl (Z)-2-nitro-3-(4-nitrophenyl)-2-propenoate and cyclopentadiene yields a mixture of carbodiene Diels-Alder adducts. B3LYP/6-31G(d) simulations indicate that the conversion of addends into methyl (1R*,2S*,3S*,4R*)-2-nitro-3-(4-nitrophenyl)-bicyclo[2.2.1]hept-5-ene-2-carboxylate occurs via two-stage heterodiene Diels-Alder reaction and (in a second step) skeleton rearrangement of the primary cycloadduct, whereas the reaction leading to methyl (1R*,2R*,3R*,4R*)-2-nitro-3-(4-nitrophenyl)-bicyclo[2.2.1]hept-5-ene-2-carboxylate is a single-step process.

  3. Structural, vibrational and NMR spectroscopic investigations of newly synthesized 3-((ethylthio)(4-nitrophenyl)methyl)-1H-indole

    NASA Astrophysics Data System (ADS)

    Bhat, Sheeraz Ahmad; Dar, Ajaz A.; Ahmad, Shabbir; Khan, Abu T.

    2017-10-01

    The compound 3-((ethylthio)(4-nitrophenyl)methyl)-1H-indole was synthesized at room temperature through one-pot three-component reaction from 1H-indole, 4-nitrobenzaldehyde, and ethanethiol using hydrated ferric sulfate as a Lewis acid catalyst. The structure was characterised by single crystal XRD, FTIR (4000-400 cm-1), FT-Raman (4000-50 cm-1) and 1H and 13C NMR analysis. The compound crystallizes in the monoclinic with volume 3238.3(9) Å3. The experimental vibrational data find the theoretical support through anharmonic frequency calculations using DFT/B3LYP level of theory in combination with 6-31G(d,p) basis set. It is observed that the predicted geometry well reproduces the XRD structural parameters. The experimental 1H and 13C NMR spectra in CDCl3 solvent and the simulated spectra predicted using gauge independent atomic orbital (GIAO) approach are also found in agreement with each other. HOMO-LUMO, MEP, atomic charges and various other thermodynamic and NLO properties of the title molecule are also reported in this paper.

  4. Separation and aquatic toxicity of enantiomers of the organophosphorus insecticide O-ethyl O-4-nitrophenyl phenylphosphonothioate (EPN).

    PubMed

    Sun, Jianqiang; Liu, Jinsong; Tu, Wenqing; Xu, Chao

    2010-11-01

    Enantioselectivity in separation and toxicity of chiral pesticides has become important research areas in environmental science, because these studies give a deeper insight into the environmental effect of chiral pesticides. In this study, enantiomeric separation of the organophosphorus pesticide and acaricide O-ethyl O-4-nitrophenyl phenylphosphonothioate (EPN) was investigated by chiral high-performance liquid chromatography (HPLC) with two chiral stationary phases. The racemate and separated enantiomers of EPN were tested for aquatic toxicities assay using Daphnia magna and zebrafish (Danio rerio) embryo test. The enantiomers of EPN were completely separated on Chiralpak AD and Chiralpak AS columns coupled with a circular dichroism detector at 236 nm. Better separations were achieved with lower temperatures (e.g., 20°C) and lower levels of polar modifiers (e.g., 1%). A significant difference was found between the enantiomers in their acute aquatic toxicity; the (+)-enantiomer was about 10 times more toxic than its antipode. On the contrary, the (-)-enantiomer induced crooked body, yolk sac edema and pericardial edema significantly more than (+)-enantiomer in the zebrafish embryo test. These results suggest that biological toxicity of chiral pesticides should be assessed by using their individual enantiomers with more comprehensive methods.

  5. Crystal and molecular structure of N-(4-nitrophenyl)-β-alanine—Its vibrational spectra and theoretical calculations

    NASA Astrophysics Data System (ADS)

    Marchewka, M. K.; Drozd, M.; Janczak, J.

    2011-08-01

    The N-(4-nitrophenyl)-β-alanine in crystalline form directly by the addition of 4-nitroaniline to the acrylic acid in aqueous solution has been obtained. The title β-alanine derivative crystallizes in the P2 1/ c space group of monoclinic system with four molecules per unit cell. The X-ray geometry of β-alanine derivative molecule has been compared with those obtained by molecular orbital calculations corresponding to the gas phase. In the crystal the molecules related by an inversion center interact via symmetrically equivalent O-H⋯O hydrogen bonds with O⋯O distance of 2.656(2) Å forming a dimeric structure. The dimers of β-alanine derivative weakly interact via N-H⋯O hydrogen bonds between the H atom of β-amine groups and one of O atom of nitro groups. The room temperature powder vibrational (infrared and Raman) measurements are in accordance with the X-ray analysis. In aqueous solution of 4-nitroaniline and acrylic acid, the double C dbnd C bond of vinyl group of acrylic acid breaks as result of 4-nitroaniline addition.

  6. Crystal and molecular structure of N-(4-nitrophenyl)-β-alanine--its vibrational spectra and theoretical calculations.

    PubMed

    Marchewka, M K; Drozd, M; Janczak, J

    2011-08-15

    The N-(4-nitrophenyl)-β-alanine in crystalline form directly by the addition of 4-nitroaniline to the acrylic acid in aqueous solution has been obtained. The title β-alanine derivative crystallizes in the P2(1)/c space group of monoclinic system with four molecules per unit cell. The X-ray geometry of β-alanine derivative molecule has been compared with those obtained by molecular orbital calculations corresponding to the gas phase. In the crystal the molecules related by an inversion center interact via symmetrically equivalent O-H···O hydrogen bonds with O···O distance of 2.656(2) Å forming a dimeric structure. The dimers of β-alanine derivative weakly interact via N-H···O hydrogen bonds between the H atom of β-amine groups and one of O atom of nitro groups. The room temperature powder vibrational (infrared and Raman) measurements are in accordance with the X-ray analysis. In aqueous solution of 4-nitroaniline and acrylic acid, the double CC bond of vinyl group of acrylic acid breaks as result of 4-nitroaniline addition.

  7. Search for possible CNS depressant compounds: Part II. Synthesis of some new 1-(2'-methoxy-4'-nitrophenyl)-2-phenyl-4-substituted arylidene-5-imidazolones as CNS depressants.

    PubMed

    Mukerji, D D; Nautiyal, S R; Prasad, C R

    1981-01-01

    Some new 1-(2'-methoxy-4'-nitrophenyl)-2-phenyl-4-substituted benzylidene, furylidene and naphthylidene-5-imidazolones have been synthesized and tested on their CNS. anticonvulsant and analgesic activities in albino mice. At a dose of 200 mg/kg they displayed CNS depressant properties producing ataxia, decrease in spontaneous motor activity and loss of righting reflex. They also exhibited mild anticonvulsant activity in the pentetrazol test.

  8. The Laforin-Like Dual-Specificity Phosphatase SEX4 from Arabidopsis Hydrolyzes Both C6- and C3-Phosphate Esters Introduced by Starch-Related Dikinases and Thereby Affects Phase Transition of α-Glucans1[W

    PubMed Central

    Hejazi, Mahdi; Fettke, Joerg; Kötting, Oliver; Zeeman, Samuel C.; Steup, Martin

    2010-01-01

    The biochemical function of the Laforin-like dual-specific phosphatase AtSEX4 (EC 3.1.3.48) has been studied. Crystalline maltodextrins representing the A- or the B-type allomorph were prephosphorylated using recombinant glucan, water dikinase (StGWD) or the successive action of both plastidial dikinases (StGWD and AtPWD). AtSEX4 hydrolyzed carbon 6-phosphate esters from both the prephosphorylated A- and B-type allomorphs and the kinetic constants are similar. The phosphatase also acted on prelabeled carbon-3 esters from both crystalline maltodextrins. Similarly, native starch granules prelabeled in either the carbon-6 or carbon-3 position were also dephosphorylated by AtSEX4. The phosphatase did also hydrolyze phosphate esters of both prephosphorylated maltodextrins when the (phospho)glucans had been solubilized by heat treatment. Submillimolar concentrations of nonphosphorylated maltodextrins inhibited AtSEX4 provided they possessed a minimum of length and had been solubilized. As opposed to the soluble phosphomaltodextrins, the AtSEX4-mediated dephosphorylation of the insoluble substrates was incomplete and at least 50% of the phosphate esters were retained in the pelletable (phospho)glucans. The partial dephosphorylation of the insoluble glucans also strongly reduced the release of nonphosphorylated chains into solution. Presumably, this effect reflects fast structural changes that following dephosphorylation occur near the surface of the maltodextrin particles. A model is proposed defining distinct stages within the phosphorylation/dephosphorylation-dependent transition of α-glucans from the insoluble to the soluble state. PMID:20018599

  9. The Laforin-like dual-specificity phosphatase SEX4 from Arabidopsis hydrolyzes both C6- and C3-phosphate esters introduced by starch-related dikinases and thereby affects phase transition of alpha-glucans.

    PubMed

    Hejazi, Mahdi; Fettke, Joerg; Kötting, Oliver; Zeeman, Samuel C; Steup, Martin

    2010-02-01

    The biochemical function of the Laforin-like dual-specific phosphatase AtSEX4 (EC 3.1.3.48) has been studied. Crystalline maltodextrins representing the A- or the B-type allomorph were prephosphorylated using recombinant glucan, water dikinase (StGWD) or the successive action of both plastidial dikinases (StGWD and AtPWD). AtSEX4 hydrolyzed carbon 6-phosphate esters from both the prephosphorylated A- and B-type allomorphs and the kinetic constants are similar. The phosphatase also acted on prelabeled carbon-3 esters from both crystalline maltodextrins. Similarly, native starch granules prelabeled in either the carbon-6 or carbon-3 position were also dephosphorylated by AtSEX4. The phosphatase did also hydrolyze phosphate esters of both prephosphorylated maltodextrins when the (phospho)glucans had been solubilized by heat treatment. Submillimolar concentrations of nonphosphorylated maltodextrins inhibited AtSEX4 provided they possessed a minimum of length and had been solubilized. As opposed to the soluble phosphomaltodextrins, the AtSEX4-mediated dephosphorylation of the insoluble substrates was incomplete and at least 50% of the phosphate esters were retained in the pelletable (phospho)glucans. The partial dephosphorylation of the insoluble glucans also strongly reduced the release of nonphosphorylated chains into solution. Presumably, this effect reflects fast structural changes that following dephosphorylation occur near the surface of the maltodextrin particles. A model is proposed defining distinct stages within the phosphorylation/dephosphorylation-dependent transition of alpha-glucans from the insoluble to the soluble state.

  10. Colorimetric determination of 1-(4'-nitrophenyl)-2-aminopropane-1,3-diol with 2,4,6-trinitrobenzenesulfonic acid in the presence of chloramphenicol.

    PubMed

    Pietta, P G; Agnellini, D; Pace, M

    1979-12-01

    A colorimetric method based on the interaction between the chloramphenicol degradation product 1-(4'-nitrophenyl)-2-aminopropane-1,3-diol and the 2,4,6-trinitrobenzenesulfonic acid reagent was developed. Analytical solutions were reacted with the reagent at pH 9.1 for 20 min at room temperature, and the resulting color was measured at 340 nm. A linear relationship between absorbance and concentration occurred within the 5--25-micrograms/ml range under the conditions studied. Replicate analyses were in good agreement. An average recovery of 99.4 +/- 0.4% was obtained for the synthetic mixtures.

  11. Syntheses of the 3- and 4-thio analogues of 4-nitrophenyl 2-acetamido-2-deoxy-beta-D-gluco- and galactopyranoside.

    PubMed

    Chen, Hong-Ming; Withers, Stephen G

    2007-11-05

    The syntheses of 4-nitrophenyl beta-glycosides of the 3-thio and 4-thio analogues of the two principal 2-acetamido-2-deoxy-hexoses found in living systems, GlcNAc and GalNAc, are described. While synthesis of the 4-thio analogues could be achieved via nucleophilic displacements of sulfonate derivatives with thioacetate, problems with neighbouring group acetamido participation necessitated the use of sulfamidate intermediates for the 3-thio analogues. These 3- and 4-thio analogues are employed in the chemo-enzymatic synthesis of thio-oligosaccharide analogues of structures present in glycosaminoglycans, glycoproteins and glycolipids.

  12. Mixed ligand coordination polymer based on 5-nitroisophthalic acid and 1-(4-nitrophenyl)-1,2,4-triazole: Synthesis, characterization, magnetic and photocatalytic properties

    NASA Astrophysics Data System (ADS)

    Li, Le; Ju, Wen-Wen; Tao, Jian-Qing; Xin, Rong; Wang, Jun; Xu, Xiao-Juan

    2015-09-01

    A new Cu(II) coordination polymer, namely, [Cu(NPT)2(NO2-BDC)]n (1) (NO2-H2BDC = 5-nitro-1,3-benzenedicarboxylic acid, NPT = 4-(4-nitrophenyl)-1,2,4-triazole) has been synthesized under hydrothermal condition and characterized by elemental analysis, and single-crystal X-ray diffraction. Single-crystal X-ray diffraction study reveals that complex 1 features one-dimensional chain structure. The magnetic studies reveal that the antiferromagnetic interactions exist between the adjacent CuII ions. Moreover, complex 1 displays highly photocatalytic degradation activity for the degradation of rhodamine B, methylene blue and methyl orange.

  13. Cellular functions of phosphatidylinositol 3-phosphate and FYVE domain proteins.

    PubMed Central

    Gillooly, D J; Simonsen, A; Stenmark, H

    2001-01-01

    PtdIns3P is a phosphoinositide 3-kinase product that has been strongly implicated in regulating membrane trafficking in both mammalian and yeast cells. PtdIns3P has been shown to be specifically located on membranes associated with the endocytic pathway. Proteins that contain FYVE zinc-finger domains are recruited to PtdIns3P-containing membranes. Structural information is now available concerning the interaction between FYVE domains and PtdIns3P. A number of proteins have been identified which contain a FYVE domain, and in this review we discuss the functions of PtdIns3P and its FYVE-domain-containing effector proteins in membrane trafficking, cytoskeletal regulation and receptor signalling. PMID:11284710

  14. Kinetic studies on the oxidation of aryl methyl sulfides and sulfoxides by dimethyldioxirane; absolute rate constants and activation parameters for 4-nitrophenyl methyl sulfide and sulfoxide.

    PubMed

    Hanson, Peter; Hendrickx, Ramon A A J; Lindsay Smith, John R

    2008-02-21

    The oxidations of methyl 4-nitrophenyl sulfide and sulfoxide by dimethyldioxirane, in acetone and mixtures of acetone with water, methanol, acetonitrile and hexane, have been followed by UV-Vis spectroscopy to monitor the decay of the substrates. The data show that, under all the conditions studied, both oxidations obey second-order kinetics. Grunwald-Winstein and Kamlet-Taft analyses of the influence of solvents on the second-order rate constants have been used to obtain mechanistic information on the two reactions. Activation parameters for the two oxidations in acetone and aqueous acetone have been calculated from rate constants for reactions in the temperature range 283-313 K and compared with those from sulfide and sulfoxide oxidations with other oxidants. For sulfoxide oxidations in acetone and 1-20% v/v water in acetone, the results support a concerted nucleophilic displacement by sulfur of oxygen from dimethyldioxirane with the rate being dependent on the solvent's polarity. Sulfide oxidations in acetone and 1-5% v/v water in acetone also proceed by a concerted mechanism. However, in the most polar solvent system studied, 20% v/v water in acetone, the mechanism changes in favour of a two-step reaction involving a betaine intermediate. Importantly, the sulfide oxidation shows a different solvent dependence to that of the sulfoxide, with the rate of oxidation being determined by the hydrogen bond donor capacity and electron-pair donicity of the solvent.

  15. Use of vibrational spectroscopy to study 2-[4-(N-dodecanoylamino)phenyl]-5-(4-nitrophenyl)-1,3,4-oxadiazole: A combined theoretical and experimental approach

    NASA Astrophysics Data System (ADS)

    Bee, Saba; Agarwal, Parag; Gupta, Archana; Tandon, Poonam

    2013-10-01

    Quantum chemical calculations of geometric structure and vibrational wavenumbers of 2-[4-(N-dodecanoylamino)phenyl]-5-(4-nitrophenyl)-1,3,4-oxadiazole (AF51) were carried out by using density functional theory (DFT/B3LYP/6-311G(d,p) method. The fundamental vibrational modes were characterized depending on their potential energy distribution (PED). In order to predict the reactive sites for electrophilic and nucleophilic attacks of the title molecule, electrostatic potential surface has been plotted. The UV absorption spectrum was examined in chloroform solvent and compared with the calculated one in gas phase as well as in solvent environment using TD-DFT/ PCM approach. The 1H NMR spectra was recorded. Comparison between the experimental and the theoretical results is satisfactory. The thermodynamic properties of the title compound at different temperatures have been calculated. A relationship between molecular structural features, non-linear responses and hyperpolarizability of AF51 has been established using vibrational spectra with emphasis on the role of intramolecular charge transfer mechanism in such organic NLO materials.

  16. Optical properties and device characteristics of 2-(antipyrin-4-ylhydrazono)-2-(4-nitrophenyl)acetonitrile thin films for photodiode applications.

    PubMed

    El-Menyawy, E M; Zedan, I T

    2015-02-25

    2-(Antipyrin-4-ylhydrazono)-2-(4-nitrophenyl)acetonitrile (AHNA) films were deposited via thermal evaporation technique. The optical properties of AHNA films and electrical characteristics of Au/AHNA/n-Si/Au heterojunction diode have been reported. The optical properties of AHNA films were investigated using the spectrophotometric measurements of optical transmittance and reflectance over spectral range 190-2500 nm. The films have indirect allowed optical band gap of 3.6 eV. The refractive index of the films was calculated and the dispersion parameters of the films were determined on the light of the single oscillator model. The electrical properties of Au/AHNA/n-Si/Au heterojunction diode were studied in terms of current-voltage characteristics. The device showed rectification behaviour with a rectification ratio of 100 at ±1 V. The conduction mechanisms and diode parameters such as ideality factor, barrier height and series resistance of the device were determined. The device under illumination showed photovoltaic properties. The short circuit current and open circuit voltage were found to be function of illumination intensity. The device satisfies the conditions to be used as photodiode.

  17. Rate assay of N-acetyl-beta-D-hexosaminidase with 4-nitrophenyl N-acetyl-beta-D-glucosaminide as an artificial substrate.

    PubMed

    Shibata, H; Yagi, T

    1996-07-15

    A rapid and accurate rate assay method for N-acetyl-beta-D-hexosaminidase (EC 3.2.1.52, also known as N-acetyl-beta-D-glucosaminidase, or NAGase) using 4-nitrophenyl N-acetyl-beta-D-glucosaminide (NP-GlcNAc) as an artificial substrate was developed using diethylaminoethyl-alpha-cyclodextrin (DEn-CD, where n is the number of diethylaminoethyl groups introduced to alpha-cyclodextrin), as an additive to ionize 4-nitrophenol to yellow-colored 4-nitrophenoxide at pH near 5, where the enzyme acts optimally. A possible recipe for the rate assay of NAGase is as follows. Prepare a stock solution containing 4.8 mmol/l NP-GlcNAc and 1% DEn-CD (n is preferably near 17) in 0.1 mol/l glycolate buffer, pH 5.50. Introduce the stock solution and a properly diluted sample (urine or other body fluid) to a reaction cell placed in a spectrophotometer at a ratio of 1:1, and monitor the absorbance at 400 or 420 nm. The reaction rate (enzymatic activity) can be conveniently read directly from calibration plots prepared for a given lot of DEn-CD sample, or can be calculated from the rate of the absorbance increase, ionization degree of 4-nitrophenol at pH 5.50, and the millimolar absorbance coefficient of 4-nitrophenoxide in the presence of 0.5% DEn-CD.

  18. Phosphate Triester Hydrolysis Promoted by an N2S (thiolate) Zinc Complex: Mechanistic Implications for the Metal-Dependent Reactivity of Peptide Deformylase

    USDA-ARS?s Scientific Manuscript database

    The zinc(II) complex (PATH)ZnOH, where PATH is an N2S(thiolate) ligand, has been investigated for its ability to promote the hydrolysis of the phosphate triester tris(4-nitrophenyl) phosphate (TNP). The hydrolysis of TNP was examined as a function of PATH-zinc(II) complex concentration, substrate co...

  19. Development of a field kit for use by non-scientists for chemical tracking using 5-(4-nitrophenyl)-2,4-pentadien-1-al.

    PubMed

    Suzuki, Shinichi

    2013-05-10

    5-(4-Nitrophenyl)-2,4-pentadien-1-al (NPPD) can be used for chemical tracking in crime scene investigations. A color test kit for NPPD was developed for use by non-scientists, such as police officers, in the field. However, this kit had problems, including contact with concentrated HCl, and instability of the reagent (naphthoresorcinol methanol solution) used in the first step of color development. To overcome these problems, in the present study, a field kit was developed with the concentrated HCl sealed in a vial so it did not contact the operator. A glass tube with two compartments was used to separate the naphthoresorcinol and methanol before use. When the color test was conducted, a cotton swab was inserted into the tube. Before insertion, the cotton was used to collect a sample from a suspect that had been in contact with a surface sprayed with a 1% NPPD methanol solution. Insertion of the cotton swab broke the thin glass that separated the methanol and naphthoresorcinol, and any NPPD on the swab reacted with the naphthoresorcinol methanol solution. The cotton swab was then pushed further to break the glass separating the concentrated HCl. A red color then developed if NPPD was present on the cotton swab. For testing the kit, NPPD was sprayed in an area where a crime was expected to occur. This kit will be useful for detecting a contact with or near a crime scene, because samples do not require analysis in a forensic science laboratory. Instead, the results can be confirmed at the scene of crime.

  20. Synthesis, vibrational spectroscopic investigations, molecular docking, antibacterial studies and molecular dynamics study of 5-[(4-nitrophenyl)acetamido]-2-(4-tert-butylphenyl)benzoxazole

    NASA Astrophysics Data System (ADS)

    Sheena Mary, Y.; Al-Shehri, Mona M.; Jalaja, K.; Al-Omary, Fatmah A. M.; El-Emam, Ali A.; Yohannan Panicker, C.; Armaković, Stevan; Armaković, Sanja J.; Temiz-Arpaci, Ozlem; Van Alsenoy, C.

    2017-04-01

    Antimicrobial active 5-[(4-nitrophenyl)acetamido]-2-(4-tert-butylphenyl)benzoxazole (NATPB) was synthesized and observed IR, Raman bands are compared with the theoretically predicted wave numbers. In the IR spectrum the NH stretching wave number splits into a doublet with a noted difference and is red shifted from the computed value, which indicates the weakening of NH bond resulting in proton transfer to the neighbouring oxygen atom. The HOMO-LUMO plots reveal the charge transfer in the molecular system through the conjugated paths. The electrophilic and nucleophilic reactive sites are identified from the MEP plot. Mapping of average local ionization energy (ALIE) values to the electron density surface served us as a tool for prediction of molecule sites possibly prone to electrophilic attacks. Other important reactive centres of the title molecule were detected by calculations of Fukui functions. Calculations of bond dissociation energies (BDE) for hydrogen abstraction were used in order to assess whether the NATPB molecules is prone to autoxidation mechanism or not, while BDE of the remaining single acyclic bonds were used in order to determine the weakest bond. Interaction properties with water were investigated by molecular dynamics (MD) simulations and calculations of radial distribution functions (RDFs). The compound possessed broad spectrum activity against all of the tested Gram-positive and Gram-negative bacteria and yeasts, their minimum inhibitory concentrations (MICs) ranging between 32 and 128 μg/ml. The compound exhibited significant antibacterial activity (32 μg/ml) against an antibiotic resistant E. faecalis isolate, at same potency with the compared standard drugs vancomycin and gentamycin sulfate. The molecular docking studies show that the compound might exhibit inhibitory activity against CDK inhibitors.

  1. Neurotoxic and teratogenic effects of an organophosphorus insecticide (phenyl phosphonothioic acid-O-ethyl -O-[4-nitrophenyl] ester) on mallard development

    USGS Publications Warehouse

    Hoffman, D.J.; Sileo, L.

    1984-01-01

    Phenyl phosphonothioic acid-O-ethyl-O-[4-nitrophenyl] ester (EPN) is one of the 10 most frequently used organophosphorus insecticides and causes delayed neurotoxicity in adult chickens and mallards. Small amounts of organophosphorus insecticides placed on birds' eggs are embryotoxic and teratogenic. For this reason, the effects of topical egg application on EPN were examined on mallard (Anas platyrhynchos) embryo development. Mallard eggs were treated topically at 72 hr of incubation with 25 microliter of a nontoxic oil vehicle or with EPN in the vehicle at concentrations of approximately 12, 36, or 108 micrograms/g egg, equivalent to one, three, and nine times the agricultural level of application used to spray crops. Treatment with EPN resulted in 22 to 44% mortality over this dose range by 18 days of development compared with 4 and 5% for untreated and vehicle-treated controls. EPN impaired embryonic growth and was highly teratogenic: 37-42% of the surviving embryos at 18 days were abnormal with cervical and axial scoliosis as well as severe edema. Brain weights were significantly lower in EPN-treated groups at different stages of development including hatchlings. Brain neurotoxic esterase (NTE) activity was inhibited by as much as 91% at 11 days, 81% at 18 days, and 79% in hatchlings. Examination of brain NTE activity during the course of normal development revealed an increase of nearly sixfold from Day 11 through hatching. The most rapid increase occurred between Day 20 and hatching. Brain acetylcholinesterase (AChE) activity was inhibited by as much as 41% at 11 days, 47% at 18 days, and 20% in hatchlings. Plasma cholinesterase and alkaline phosphatase activities were inhibited and plasma aspartate aminotransferase activity was increased at one or more stages of development. Hatchlings from EPN-treated eggs were weaker and slower to right themselves. Histopathological examination did not reveal demyelination and axonopathy of the spinal cord that was

  2. Photoaffinity labeling of the sigma-1 receptor with N-[3-(4-nitrophenyl)propyl]-N-dodecylamine: evidence of receptor dimers.

    PubMed

    Chu, Uyen B; Ramachandran, Subramaniam; Hajipour, Abdol R; Ruoho, Arnold E

    2013-02-05

    The sigma-1 receptor is a ligand-regulated endoplasmic reticulum (ER) resident chaperone involved in the maintenance of cellular homeostasis. Coupling of the sigma-1 receptor with various ER and/or plasma membrane ion channels is associated with its ability to regulate the locomotor activity and cellular proliferation produced in response to sigma-1 receptor ligands. A number of endogenous small molecules bind to the sigma-1 receptor and have been shown to regulate its activity; these include progesterone, N,N-dimethyltryptamine, d-erythro-sphingosine, and/or other endogenous lipids. We previously reported the synthesis of long chain N-alkylamine derivatives and the characterization of the structure-activity relationship between the chain length of N-alkylamine and affinities at the sigma-1 receptor. Here, we present data demonstrating the photoincorporation of one of these N-alkylamine derivatives, N-[3-(4-nitrophenyl)propyl]-N-dodecylamine (4-NPPC12), to the sigma-1 receptor. Matrix-assisted laser desorption ionization time-of-flight and tandem mass spectrometry showed that 4-NPPC12 photoinserted at histidine 154 of the derivatized population of the sigma-1 receptor. Interestingly, light-dependent photoinsertion of 4-NPPC12 resulted in an enhanced electrophoretic mobility of only 50% of the derivatized receptor molecules as assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The proposed binding and reactivity of 4-NPPC12 evoke a ligand binding model for the sigma-1 receptor that likely involves a receptor dimer and/or oligomer.

  3. 1,2-Bis(methylsulfonyl)-1-(2-chloroethyl)-2-[[1-(4-nitrophenyl)ethoxy]carbonyl]hydrazine: An anticancer agent targeting hypoxic cells

    PubMed Central

    Seow, Helen A.; Penketh, Philip G.; Shyam, Krishnamurthy; Rockwell, Sara; Sartorelli, Alan C.

    2005-01-01

    To target malignant cells residing in hypoxic regions of solid tumors, we have designed and synthesized prodrugs generating the cytotoxic alkylating species 1,2-bis(methylsulfonyl)-1-(2-chloroethyl)hydrazine (90CE) after bioreductive activation. We postulate that one of these agents, 1,2-bis(methylsulfonyl)-1-(2-chloroethyl)-2-[[1-(4-nitrophenyl)ethoxy]carbonyl]hydrazine (KS119), requires enzymatic nitro reduction to produce 90CE, whereas another agent, 1,2-bis(methylsulfonyl)-1-(2-chloroethyl)-2-[(4-nitrobenzyloxy)carbonyl]hydrazine (PNBC), can also be activated by nucleophilic attack by thiols such as glutathione (GSH)/GST. We demonstrated that these agents selectively kill hypoxic EMT6 mouse mammary carcinoma and CHO cells. In hypoxia, 50 μM KS119 produced 5 logs of kill of EMT6 cells without discernable cytotoxicity in air; similar effects were observed with CHO cells. PNBC was less efficacious against hypoxic tumor cells and also had some toxicity to aerobic cells, presumably because of GST/thiol activation, making PNBC less interesting as a selective hypoxic-cell cytotoxin. BALB/c mice with established EMT6 solid tumors were used to demonstrate that KS119 could reach and kill hypoxic cells in solid tumors. To gain information on bioreductive enzymes involved in the activation of KS119, cytotoxicity was measured in CHO cell lines overexpressing NADH:cytochrome b5 reductase (NBR), NADPH:cytochrome P450 reductase (NPR), or NAD(P)H: quinone oxidoreductase 1 (NQO1). Increased cytotoxicity occurred in cells overexpressing NBR and NPR, whereas overexpressed NQO1 had no effect. These findings were supported by enzymatic studies using purified NPR and xanthine oxidase to activate KS119. KS119 has significant potential as a hypoxia-selective tumor-cell cytotoxin and is unlikely to cause major toxicity to well oxygenated normal tissues. PMID:15964988

  4. Fast and Facile Synthesis of 4-Nitrophenyl 2-Azidoethylcarbamate Derivatives from N-Fmoc-Protected α-Amino Acids as Activated Building Blocks for Urea Moiety-Containing Compound Library.

    PubMed

    Chen, Ying-Ying; Chang, Li-Te; Chen, Hung-Wei; Yang, Chia-Ying; Hsin, Ling-Wei

    2017-03-13

    A fast and facile synthesis of a series of 4-nitrophenyl 2-azidoethylcarbamate derivatives as activated urea building blocks was developed. The N-Fmoc-protected 2-aminoethyl mesylates derived from various commercially available N-Fmoc-protected α-amino acids, including those having functionalized side chains with acid-labile protective groups, were directly transformed into 4-nitrophenyl 2-azidoethylcarbamate derivatives in 1 h via a one-pot two-step reaction. These urea building blocks were utilized for the preparation of a series of urea moiety-containing mitoxantrone-amino acid conjugates in 75-92% yields and parallel solution-phase synthesis of a urea compound library consisted of 30 members in 38-70% total yields.

  5. Synthesis, crystal structure and spectroscopy properties of Na{sub 3} AZr(PO{sub 4}){sub 3} (A=Mg, Ni) and Li{sub 2.6}Na{sub 0.4}NiZr(PO{sub 4}){sub 3} phosphates

    SciTech Connect

    Chakir, M. . E-mail: fachakir@yahoo.fr; El Jazouli, A.; Waal, D. de

    2006-06-15

    Na{sub 3} AZr(PO{sub 4}){sub 3} (A=Mg, Ni) phosphates were prepared at 750 deg. C by coprecipitation route. Their crystal structures have been refined at room temperature from X-ray powder diffraction data using Rietveld method. Li{sub 2.6}Na{sub 0.4}NiZr(PO{sub 4}){sub 3} was synthesized through ion exchange from the sodium analog. These materials belong to the Nasicon-type structure. Raman spectra of Na{sub 3} AZr(PO{sub 4}){sub 3} (A=Mg, Ni) phosphates present broad peaks in favor of the statistical distribution in the sites around PO{sub 4} tetrahedra. Diffuse reflectance spectra indicate the presence of octahedrally coordinated Ni{sup 2+} ions. - Graphical abstract: Structure of Na{sub 3} AZr(PO{sub 4}){sub 3} (A=Mg, Ni) phosphates. Display Omitted.

  6. 1,2-Bis(methylsulfonyl)-1-(2-chloroethyl)-2-[[1-(4-nitrophenyl)ethoxy]carbonyl]hydrazine (KS119): a Cytotoxic Prodrug with Two Stable Conformations Differing in Biological and Physical Properties

    PubMed Central

    Penketh, Philip G.; Baumann, Raymond P.; Shyam, Krishnamurthy; Williamson, Hugh S.; Ishiguro, Kimiko; Zhu, Rui; Eriksson, Emma S. E.; Eriksson, Leif A.; Sartorelli, Alan C.

    2011-01-01

    The anticancer prodrug 1,2-bis(methylsulfonyl)-1-(2-chloroethyl)-2-[[1-(4-nitrophenyl)ethoxy]carbonyl]hydrazine (KS119) selectively releases a short-lived cytotoxin following enzymatic reduction in hypoxic environments found in solid tumors. KS119, in addition to two enantiomers, has two stable atropisomers (conformers differing in structure owing to hindered bond rotation) that interconvert at 37 °C in aqueous solution by first order kinetics with t1/2 values of ~50 and ~64 hours. The atropisomers differ in physical properties such as partition coefficients that allow their chromatographic separation on non-chiral columns. A striking difference in the rate of metabolism of the two atropisomers occurs in intact EMT6 murine mammary carcinoma cells under oxygen deficient conditions. A structurally related molecule, 1,2-bis(methylsulfonyl)-1-(2-chloroethyl)-2-[[1-(3-hydroxy-4-nitrophenyl)ethoxy]carbonyl]hydrazine (KS119WOH), was also found to exist in similar stable atropisomers. The ratio of the atropisomers of KS119 and structurally related agents has the potential to impact the bioavailability, activation and therapeutic activity. Thus, thermally stable atropisomers/conformers in small molecules can result in chemically and enantiomerically pure compounds having differences in biological activities. PMID:21777394

  7. Rapid hydrolysis of model phosphate diesters by alkaline-earth cations in aqueous DMSO: speciation and kinetics.

    PubMed

    Taran, Olga; Medrano, Felipe; Yatsimirsky, Anatoly K

    2008-12-14

    Kinetics of the cleavage of two phosphate diesters, bis(4-nitrophenyl) phosphate and 2-hydroxypropyl 4-nitrophenyl phosphate and a triester, 4-nitrophenyl diphenyl phosphate, in the presence of Mg(II), Ca(II) and Sr(II) were studied in 90% vol. DMSO at 37 degrees C. The alkaline hydrolysis of the triester was inhibited by all cations, but with both phosphodiesters strong catalytic effects were observed. Potentiometric titrations of metal perchlorates by Bu4N(OH) revealed formation of M2(OH)3+, M(OH)+, M(OH)2 and M2(OH)5- species. Rate constants for phosphodiester cleavage by individual species were obtained from analysis of rate-concentration profiles. Observed first-order rate constants in the presence of 1-2 mM Mg(II) or Ca(II) in neutral and weakly basic solutions were 10(8)-10(11) times higher than those for background hydrolysis at the same pH while in water additions of up to 50 mM metal produced <100-fold accelerations. Possible structures of DMSO solvated catalyst-substrate complexes were modeled by DFT calculations with Mg(II). The increased catalytic activity in 90% DMSO is attributed to stronger association of hydroxide ions and anionic phosphodiesters with metal ions and to preferable solvation of cations by DMSO, which creates favorable for reaction anhydrous microenvironment in the coordination sphere of the catalyst.

  8. Synthesis and crystal structure of N-[(dimethylamino)methylidene]-4-[1-(4-nitrophenyl)-1H-tetrazol-5-yl]-benzenesulfonamide: Molecular docking and bioassay studies as cyclooxygenase-2 inhibitor

    NASA Astrophysics Data System (ADS)

    Jawabrah Al-Hourani, Baker; El-Barghouthi, Musa I.; McDonald, Robert; Al-Awaida, Wajdy; Sharma, Sai Kiran; Wuest, Frank

    2016-09-01

    The synthesis of N-[(dimethylamino)methylidene]-4-[1-(4-nitrophenyl)-1H-tetrazol-5-yl]benzenesulfonamide (3) has been easily approached and the structure has been determined by X-ray crystallography. Tetrazole 3 crystallizes in the monoclinic space group C2/c, with the cell parameters determined as a = 35.5408 (18) Å, b = 7.6972 (4) Å, c = 13.0700 (7) Å3, β = 96.8598 (6)°, V = 3549.9 (3) Å3, and Z = 8. Its structure refines to R1 = 0.0341 (for 2986 observed reflections [I ≥ 2σ(I)]) and wR2 = 0.0990 (for all 3637 unique reflections). The aryl rings at the 1- and 5-positions show no conjugation to the tetrazole group, and the [(Dimethylamino)methylene]aminosulfonyl (Me2NCHNSO2) group is disordered, with the two disorder conformers being related by a pseudo mirror plane. In the crystal, intermolecular interactions between adjacent molecule of 3 are dominated by weak (2.4-2.7 Å) CeH…O and CeH…N hydrogen bonds. The molecular docking studies were carried out to understand the interaction of compound 3 within the active site of the cyclooxygenase-2 enzyme, followed by a comparison study with the celecoxib drug as a reference compound. The in vitro bioassay studies of tetrazole 3 toward cyclooxygenase-1 and cyclooxygenase-2 enzymes showed that compound 3 has no inhibition potency for either enzyme.

  9. AC conductivity and dielectric properties of 2-(2,3-dihydro-1,5-dimethyl-3-oxo-2-phenyl-1 H-pyrazol-4-ylimino)-2-(4-nitrophenyl)acetonitrile thin films

    NASA Astrophysics Data System (ADS)

    El-Menyawy, E. M.; Zeyada, H. M.; El-Nahass, M. M.

    2010-12-01

    The dark AC conductivity and dielectric properties of thermally evaporated 2-(2,3-dihydro-1,5-dimethyl-3-oxo-2-phenyl-1 H-pyrazol-4-ylimino)-2-(4-nitrophenyl)acetonitrile (DOPNA) thin films in sandwich structure employing symmetrical gold ohmic contacts have been investigated as function of temperature (303-443 K) and frequency (100 Hz-5 MHz). The AC conductivity, σAC( ω), is found to obey Jonscher's universal power law, σAC( ω)= Aω s ( ω is the angular frequency). The AC conductivity of DOPNA thin films has been analyzed with reference to various theoretical models. The correlated barrier hopping is found to be the dominant conduction mechanism for charge carrier transport; the maximum barrier height, hopping length and the density of localized states are estimated. The temperature dependence of the AC conductivity shows Arrhenius type with two thermal activation energies. The activation energies are determined as a function of frequency. The behavior of the real and imaginary parts of the dielectric constant as a function of both temperature and frequency is discussed.

  10. Characterization of the optical non-linear response of the (E)-4-(4-dimetylaminophenyl) but-3-en-2-one and (E)-4-(4-nitrophenyl) but-3-en-2-one by Z-Scan

    NASA Astrophysics Data System (ADS)

    Rodriguez, K.; Pérez, A.; Racedo, F.

    2017-01-01

    We presents the study carried out by the technique Z-Scan, to analyse the nonlinear optical properties of (E)-4-(4-dimetylaminophenyl) but-3-en-2-one and (E)-4-(4-nitrophenyl) but-3-en-2-one, diluted in Ethyl Acetate with concentration levels of [0.02M, 0.08M, 0.23M] and [0.0047M, 0.013M, 0.041M] respectively. The measurements were performed using a Nd:YAG laser emitting at 532nm, for samples with Leff =1mm thickness, and a automated scanning of 10cm symmetric to the lens focus, the iris diameter was 1mm, the samples were also characterized by an UV-Vis Spectroscopy. We calculated the nonlinear refractive index (η2), the nonlinear absorption coefficient (β) and the the third-order nonlinear optical susceptibility (χ3) of the two researched compounds. The results show a combination of thermal response and nonlinear self-defocusing and self-focusing, which make their application interesting as new optoelectronic materials.

  11. Spectroscopic investigation (FT-IR and FT-Raman), vibrational assignments, HOMO-LUMO analysis and molecular docking study of 2-(Adamantan-1-yl)-5-(4-nitrophenyl)-1,3,4-oxadiazole

    NASA Astrophysics Data System (ADS)

    Haress, Nadia G.; Al-Omary, Fatmah; El-Emam, Ali A.; Mary, Y. Sheena; Panicker, C. Yohannan; Al-Saadi, Abdulaziz A.; War, Javeed Ahmad; Van Alsenoy, Christian

    2015-01-01

    FT-IR and FT-Raman spectra of 2-(Adamantan-1-yl)-5-(4-nitrophenyl)-1,3,4-oxadiazole were recorded and analyzed. The vibrational wavenumbers were computed using DFT quantum chemical calculations. The data obtained from wavenumber calculations are used to assign vibrational bands obtained experimentally. The energy barriers of the internal rotations about the Csbnd C bonds connecting the oxadiazole to the adamantane and benzene rings are reported. The geometrical parameters (DFT) of the title compound are in agreement with the XRD results. The calculated HOMO and LUMO energies allow the calculations of atomic and molecular properties and they also showed that charge transfer occurs in the molecule. A detailed molecular picture of the title compound and its interactions were obtained from NBO analysis. As can be seen from the MEP map of the title compound, which regions having the negative potential are over the electro negative atoms, the region having the positive potential are over the phenyl and adamantine rings and the remaining species are surrounded by zero potential. The molecular docking studies reveal that the adamantyl derivative may exhibit C-South African HIV-proteas inhibitory activity.

  12. Spectroscopic investigation (FT-IR and FT-Raman), vibrational assignments, HOMO-LUMO analysis and molecular docking study of 2-(Adamantan-1-yl)-5-(4-nitrophenyl)-1,3,4-oxadiazole.

    PubMed

    Haress, Nadia G; Al-Omary, Fatmah; El-Emam, Ali A; Mary, Y Sheena; Panicker, C Yohannan; Al-Saadi, Abdulaziz A; War, Javeed Ahmad; Van Alsenoy, Christian

    2015-01-25

    FT-IR and FT-Raman spectra of 2-(Adamantan-1-yl)-5-(4-nitrophenyl)-1,3,4-oxadiazole were recorded and analyzed. The vibrational wavenumbers were computed using DFT quantum chemical calculations. The data obtained from wavenumber calculations are used to assign vibrational bands obtained experimentally. The energy barriers of the internal rotations about the C-C bonds connecting the oxadiazole to the adamantane and benzene rings are reported. The geometrical parameters (DFT) of the title compound are in agreement with the XRD results. The calculated HOMO and LUMO energies allow the calculations of atomic and molecular properties and they also showed that charge transfer occurs in the molecule. A detailed molecular picture of the title compound and its interactions were obtained from NBO analysis. As can be seen from the MEP map of the title compound, which regions having the negative potential are over the electro negative atoms, the region having the positive potential are over the phenyl and adamantine rings and the remaining species are surrounded by zero potential. The molecular docking studies reveal that the adamantyl derivative may exhibit C-South African HIV-proteas inhibitory activity.

  13. Evidence for a catalytic six-membered cyclic transition state in aminolysis of 4-nitrophenyl 3,5-dinitrobenzoate in acetonitrile: comparative brønsted-type plot, entropy of activation, and deuterium kinetic isotope effects.

    PubMed

    Um, Ik-Hwan; Kim, Min-Young; Bae, Ae-Ri; Dust, Julian M; Buncel, Erwin

    2015-01-02

    A kinetic study for reactions of 4-nitrophenyl 3,5-dinitrobenzoate (1a) with a series of cyclic secondary amines in acetonitrile is reported. Plots of the pseudo-first-order rate constant (kobsd) vs [amine] curve upward, while those of kobsd /[amine] vs [amine] exhibit excellent linear correlations with positive intercepts, indicating that the reaction proceeds through both uncatalyzed and catalyzed routes. Brønsted-type plots for uncatalyzed and catalyzed reactions are linear with βnuc = 1.03 and 0.69, respectively. The ΔH(⧧) and ΔS(⧧) values measured for the catalytic reaction with morpholine are -0.80 kcal/mol and -61.7 cal/(mol K), respectively. The negative ΔH(⧧) with a large negative ΔS(⧧) suggests that the reaction proceeds through a highly ordered transition state (i.e., a six-membered cyclic transition state, which includes a second amine molecule that accepts a proton from the aminium moiety of the zwitterionic tetrahedral intermediate and simultaneously donates a proton to the aryloxyl oxygen of the nucleofuge with concomitant C-OAr bond scission). This proposal is consistent with the smaller βnuc value for the catalyzed reaction as compared to the uncatalyzed reaction. An inverse deuterium kinetic isotope effect (DKIE) value of 0.93 and a contrasting normal primary DKIE value of 3.23 for the uncatalyzed and catalyzed routes, respectively, also support the proposed cyclic transition state.

  14. The influence of steric hindrance on kinetics and isotope effects in the reaction of 2,2-bis(4-dimethylaminophenyl)-1-nitro-1-(4-nitrophenyl)ethane with DBU base in acetonitrile

    NASA Astrophysics Data System (ADS)

    Nowak, Iwona; Jarczewski, Arnold

    2014-11-01

    The pKa value for 2,2-bis(4-dimethylaminophenyl)-1-nitro-1-(4-nitrophenyl)ethane, (dmap)2 (pKa = 25.11) has been measured spectrophotometrically using buffer solutions of a few strong amine bases: 1,8-diazabicyclo[5.4.0]undec-7-ene, (DBU); 1,1,3,3-tetramethylguanidine, (TMG); 1,5,7-triazabicyclo[4.4.0]dec-5-ene, (TBD); 7-methyl-1,5,7-triazabicyclo[4.4.0]dec-5-ene, (MTBD) and their salts. The low energy conformers of nitrophenyl nitroalkanes have been determined using the semiempirical PM6 methods, (B3-LYP) density functional theory (DFT) together with the 6-31G(d,p) basis set. The participation of the low energy conformer in the proton transfer reaction to DBU base has been discussed. The kinetic data for proton transfer reactions between (dmap)2 and DBU in acetonitrile (MeCN) at pseudo-first order conditions have been presented. The influence of steric hindrance brought by reacting C-acid and organic base on the stability of the transition state has been discussed. The rates of second-order rate constants for series of nitrophenyl nitroalkanes, NO2PhCHRNO2 (R = Me; Et; iPr; dimethylaminophenyl = (dmap)2) are presented and discussed.

  15. Small-angle neutron scattering study of halophilic glyceraldehyde 3-phosphate dehydrogenase (hGAPDH)

    NASA Astrophysics Data System (ADS)

    Ebel, Christine; Krishnan, Gomathi; Altekar, Wijaya; Zaccai, Giuseppe

    1991-10-01

    hGAPDH has a radius of gyration of 32±0.5 Å in high salt concentration, and a shape similar to an oblate ellipsoid of axial ratio 0.5 but with a much larger specific surface. The enzyme dissociates and unfolds in low salt solutions. These results are discussed in terms of the halophilic character of the protein.

  16. HUMAN GLYCERALDEHYDE 3-PHOSPHATE DEHYDROGENASE-2 (GAPD2) GENE IS EXPRESSED SPECIFICALLY IN SPERMATOGENIC CELLS

    EPA Science Inventory

    Although the process of glycolysis is highly conserved in eukaryotes, several glycolytic enzymes have unique structural or functional features in spermatogenic cells. We previously identified and characterized the mouse complementary DNA (cDNA) and a gene for 1 of these enzymes, ...

  17. Enhanced enzymatic activity of glycerol-3-phosphate dehydrogenase from the cryophilic Saccharomyces kudriavzevii.

    PubMed

    Oliveira, Bruno M; Barrio, Eladio; Querol, Amparo; Pérez-Torrado, Roberto

    2014-01-01

    During the evolution of the different species classified within the Saccharomyces genus, each one has adapted to live in different environments. One of the most important parameters that have influenced the evolution of Saccharomyces species is the temperature. Here we have focused on the study of the ability of certain species as Saccharomyces kudriavzevii to grow at low temperatures, in contrast to Saccharomyces cerevisiae. We observed that S. kudriavzevii strains isolated from several regions are able to synthesize higher amounts of glycerol, a molecule that has been shown to accumulate in response to freeze and cold stress. To explain this observation at the molecular level we studied the expression of glycerol biosynthetic pathway genes and we observed a higher expression of GPD1 gene in S. kudriavzevii compared to S. cerevisiae in micro-vinification conditions. We observed higher enzymatic activity of Gpd1p in S. kudriavzevii in response to osmotic and cold stress. Also, we determined that S. kudriavzevii Gpd1p enzyme presents increased catalytic properties that will contribute to increase glycerol production. Finally, we evaluated the glycerol production with S. cerevisiae, S. kudriavzevii or a recombinant Gpd1p variant in the same background and observed that the S. kudriavzevii enzyme produced increased glycerol levels at 12 or 28°C. This suggests that glycerol is increased in S. kudriavzevii mainly due to increased V max of the Gpd1p enzyme. All these differences indicate that S. kudriavzevii has changed the metabolism to promote the branch of the glycolytic pathway involved in glycerol production to adapt to low temperature environments and maintain the NAD(+)/NADH ratio in alcoholic fermentations. This knowledge is industrially relevant due to the potential use, for example, of S. cerevisiae-S. kudriavzevii hybrids in the wine industry where glycerol content is an important quality parameter.

  18. Immunoprotection of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from Lactococcus garvieae against Lactococcosis in tilapia.

    PubMed

    Tsai, Ming-An; Wang, Pei-Chi; Cao, Thanh-Trung; Liao, Pei-Chih; Liaw, Li-Ling; Chen, Shih-Chu

    2013-01-01

    In this study, the gene encoding 40 kDa GAPDH of L. garvieae was determined and overexpressed by using the Escherichia coli expression system. Analysis results indicated that the sequences of GAPDH of L. garvieae nucleotide and its amino acid are highly homologous (80.4-100%) to several products of GAPDH from L. garvieae and other Streptococcus-related bacteria. According to Western blotting results, rabbit antiserum and tilapia infection serum reacted strongly to the recombinant GAPDH protein. In another experiment, tilapia were immunized intraperitoneally with formalin-killed L. garvieae whole cells, recombinant GAPDH (50 μg fish(-1)) from L. garvieae or both. ISA 763A was used as an adjuvant for vaccine and saline was used as a negative control. The fish challenged at 4 weeks after immunization with GAPDH+WC+ISA had the highest survival rate at 100%, followed by fish immunized with WC+ISA or GAPDH+ISA, which had RPS values of 87.5% and 50%, respectively. Additionally, specific antibody responses against L. garvieae whole cells and GAPDH were based on enzyme-linked immunosorbent assay. Following 4 weeks of immunization, the specific antibody level of all vaccine groups significantly increased, except for antibody responses against L. garvieae GAPDH of those immunized with formalin-killed L. garvieae whole cells. Our results further demonstrated that GAPDH from L. garvieae protected tilapia from experimental L. garvieae infection, implying the potential use of L. garvieae GAPDH as a vaccine against L. garvieae.

  19. Glyceradehyde-3-phosphate dehydrogenase as a suitable vaccine candidate for protection against bacterial and parasitic diseases.

    PubMed

    Perez-Casal, Jose; Potter, Andrew A

    2016-02-17

    The enzyme glyceraldehyde-3-P-dehydrogenase (GAPDH) has been identified as having other properties in addition to its key role in glycolysis. The ability of GAPDH to bind to numerous extracellular matrices, modulation of host-immune responses, a role in virulence and surface location has prompted numerous investigators to postulate that GAPDH may be a good vaccine candidate for protection against numerous pathogens. Although immune responses against GAPDH have been described for many microorganisms, vaccines containing GAPDH have been successfully tested in few cases including those against the trematode-Schistosoma mansoni, the helminth-Enchinococcus multilocularis; the nematode filaria- Litomosoides sigmodontis; fish pathogens such as Aeromonas spp., Vibrio spp., Edwarsiella spp., and Streptococcus iniae; and environmental streptococci, namely, Streptococcus uberis and Streptococcus dysgalactiae. Before GAPDH-based vaccines are considered viable options for protection against numerous pathogens, we need to take into account the homology between the host and pathogen GAPDH proteins to prevent potential autoimmune reactions, thus protective GAPDH epitopes unique to the pathogen protein must be identified.

  20. Identification of tissue transglutaminase-reactive lysine residues in glyceraldehyde-3-phosphate dehydrogenase.

    PubMed

    Orru, Stefania; Ruoppolo, Margherita; Francese, Simona; Vitagliano, Luigi; Marino, Gennaro; Esposito, Carla

    2002-01-01

    Polyglutamine domains are excellent substrates for tissue transglutaminase resulting in the formation of cross-links with polypeptides containing lysyl residues. This finding suggests that tissue transglutaminase may play a role in the pathology of neurodegenerative diseases associated with polyglutamine expansion. The glycolytic enzyme GAPDH previously was shown to tightly bind several proteins involved in such diseases. The present study confirms that GAPDH is an in vitro lysyl donor substrate of tissue transglutaminase. A dansylated glutamine-containing peptide was used as probe for labeling the amino-donor sites. SDS gel electrophoresis of a time-course reaction mixture revealed the presence of both fluorescent GAPDH monomers and high molecular weight polymers. Western blot analysis performed using antitransglutaminase antibodies reveals that tissue transglutaminase takes part in the formation of heteropolymers. The reactive amino-donor sites were identified using mass spectrometry. Here, we report that of the 26 lysines present in GAPDH, K191, K268, and K331 were the only amino-donor residues modified by tissue transglutaminase.

  1. Enhanced Enzymatic Activity of Glycerol-3-Phosphate Dehydrogenase from the Cryophilic Saccharomyces kudriavzevii

    PubMed Central

    Oliveira, Bruno M.; Barrio, Eladio; Querol, Amparo; Pérez-Torrado, Roberto

    2014-01-01

    During the evolution of the different species classified within the Saccharomyces genus, each one has adapted to live in different environments. One of the most important parameters that have influenced the evolution of Saccharomyces species is the temperature. Here we have focused on the study of the ability of certain species as Saccharomyces kudriavzevii to grow at low temperatures, in contrast to Saccharomyces cerevisiae. We observed that S. kudriavzevii strains isolated from several regions are able to synthesize higher amounts of glycerol, a molecule that has been shown to accumulate in response to freeze and cold stress. To explain this observation at the molecular level we studied the expression of glycerol biosynthetic pathway genes and we observed a higher expression of GPD1 gene in S. kudriavzevii compared to S. cerevisiae in micro-vinification conditions. We observed higher enzymatic activity of Gpd1p in S. kudriavzevii in response to osmotic and cold stress. Also, we determined that S. kudriavzevii Gpd1p enzyme presents increased catalytic properties that will contribute to increase glycerol production. Finally, we evaluated the glycerol production with S. cerevisiae, S. kudriavzevii or a recombinant Gpd1p variant in the same background and observed that the S. kudriavzevii enzyme produced increased glycerol levels at 12 or 28°C. This suggests that glycerol is increased in S. kudriavzevii mainly due to increased Vmax of the Gpd1p enzyme. All these differences indicate that S. kudriavzevii has changed the metabolism to promote the branch of the glycolytic pathway involved in glycerol production to adapt to low temperature environments and maintain the NAD+/NADH ratio in alcoholic fermentations. This knowledge is industrially relevant due to the potential use, for example, of S. cerevisiae-S. kudriavzevii hybrids in the wine industry where glycerol content is an important quality parameter. PMID:24498063

  2. HUMAN GLYCERALDEHYDE 3-PHOSPHATE DEHYDROGENASE-2 (GAPD2) GENE IS EXPRESSED SPECIFICALLY IN SPERMATOGENIC CELLS

    EPA Science Inventory

    Although the process of glycolysis is highly conserved in eukaryotes, several glycolytic enzymes have unique structural or functional features in spermatogenic cells. We previously identified and characterized the mouse complementary DNA (cDNA) and a gene for 1 of these enzymes, ...

  3. Photoinduced anisotropy of push-pull chromophores incorporating 5-(1,3-benzodithiol-2-ylidene)-1-(4-nitrophenyl) penta-1,3-diene embedded into photopolymer oligoetheracrylate matrices

    NASA Astrophysics Data System (ADS)

    Sahraoui, B.; Kityk, I. V.; Kasperczyk, J.; Salle, M.; Nguyen, T. T.

    2000-04-01

    Photoinduced (PI) birefringence (Δ n) and phototransparency ( T) have been studied for a wavelength of 633 nm (He-Ne laser) under photoillumination by doubled-frequency YAG:Nd pulse laser polarized beam ( λ=0.53 μm; pulse duration τ=10-50 ps). The push-pull chromophore 5-(1,3-benzodithiol-2-ylidene)-1-(4-nitrophenyl) penta-1,3-diene was incorporated into an oligoetheracrylate photopolymer matrix in a concentration of about 2.5 wt.%. Special properties of the molecule are connected with substantial role played by electron-vibration interactions in the observed PI effects. The Δ n decreases with decreasing temperature. Optical density D depends non-linearly on the PI beam power. With increasing photoinducing power (up to 0.41 GW/cm 2), the PI birefringence Δ n and the optical density D approaches (1.4-1.7)×10 -3 and 1.5%, respectively. The intensity dependence of intramolecular (669 cm -1) and intermolecular (89.5 cm -1) vibration mode has a maximum and a broad minimum (at a PI power of about 0.45 GW/cm 2), respectively. Such behavior correlates well with the PI dependencies of the Δ n and the optical density D. Description of the obtained results is based on quantum chemical calculations with taking into account an electron-vibration anharmonicity of the chromophore. Essential role of the PI electrostatic potential redistribution under influence of the PI polarized light beam has been demonstrated. Possible mechanisms of the observed phenomena are discussed within a phenomenological as well as microscopic approach.

  4. Involvement of p38 MAPK and ATF-2 signaling pathway in anti-inflammatory effect of a novel compound bis[(5-methyl)2-furyl](4-nitrophenyl)methane on lipopolysaccharide-stimulated macrophages.

    PubMed

    Udompong, Sarinporn; Mankhong, Sakulrat; Jaratjaroonphong, Jaray; Srisook, Klaokwan

    2017-09-01

    Activated macrophages produce various pro-inflammatory mediators such as inducible nitric oxide synthase (iNOS)-derived nitric oxide (NO) and cyclooxygenase (COX)-2-derived prostaglandin E2 (PGE2) during inflammatory response. However, overproduction of NO and PGE2 appears to be involved in pathogenesis of various inflammatory diseases. Therefore, inhibition of NO and PGE2 production might be useful for the treatment of inflammatory-related diseases. In this study, the bis[(5-methyl)2-furyl](4-nitrophenyl)methane or BFNM was evaluated for the anti-inflammatory activity and mechanism of action in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophage. BFNM inhibited NO and PGE2 production in a concentration-dependent manner and down-regulated the expression of iNOS and COX-2 at mRNA and protein levels. BFNM suppressed nuclear translocation of NF-κB p65 subunit only very slightly, and failed to decrease NF-κB DNA binding activity. In contrast, the compound significantly reduced phosphorylation of p38 MAPK and ATF-2, a component of AP-1 known to be involved in the transcriptional regulation of iNOS and COX-2, in a dose-dependent manner in LPS-induced cells. Collectively, these results suggest that BFNM has an anti-inflammatory effect in RAW 264.7 macrophages, at least in part, by suppression of NO and PGE2 production. The inhibitory effect of BFNM is mediated mainly via the p38 MAPK/ATF-2 signaling pathway. Thus, BFNM would be a lead compound for the development of novel anti-inflammatory agents. Copyright © 2017 Elsevier B.V. All rights reserved.

  5. Analysis of the structure and the FT-IR and Raman spectra of 2-(4-nitrophenyl)-4H-3,1-benzoxazin-4-one. Comparisons with the chlorinated and methylated derivatives

    NASA Astrophysics Data System (ADS)

    Castillo, María V.; Rudyk, Roxana A.; Davies, Lilian; Brandán, Silvia Antonia

    2017-07-01

    In this work, the structural, topological and vibrational properties of the monomer and three dimers of the 2-(4-nitrophenyl)-4H-3,1-benzoxazin-4-one (NPB) derivative were studied combining the experimental FTIR and FT-Raman spectra in the solid phase with DFT calculations. Here, Natural Bond Orbital (NBO), Atoms in Molecules (AIM) and HOMO and LUMO calculations were performed by using the hybrid B3LYP/6-31G*and B3LYP/6-311++G** methods in order to compute those properties and to predict their reactivities. The comparisons with the properties reported for the chlorinated (Cl-PB) and methylated (CH3-PB) derivatives at the same levels of theory can be clearly justified by the activating (CH3) and deactivating (NO2 and Cl) characteristics of the different groups linked to oxaxin rings. The NBO and AIM studies evidence the following stability orders: Cl-PB > NO2-PB > CH3-PB in very good concordance with the f(νC23-X26) force constants values. The frontier orbitals analyses reveal that the Cl-PB and NO2-PB derivatives have good stabilities and high chemical hardness while CH3-PB has a higher chemical reactivity. On the other hand, the complete vibrational assignments for monomer and dimers species of NPB were presented. The presence of the IR bands at 1574 and 1037 cm-1 and, of the Raman bands at 1571 and 1038 cm-1 support clearly the presence of the different dimeric species proposed for NPB.

  6. Effects of the specific α4β2 nAChR antagonist, 2-fluoro-3-(4-nitrophenyl) deschloroepibatidine, on nicotine reward-related behaviors in rats and mice

    PubMed Central

    Tobey, K. M.; Wiley, J. L.; Carroll, F. I.; Damaj, M. I.; Azar, M. R.; Koob, G. F.; George, O.; Harris, L. S.; Vann, R. E.

    2012-01-01

    Rationale Alleviating addiction to tobacco products could prevent millions of deaths. Investigating novel compounds selectively targeting α4β2 nAChRs hypothesized to have a key role in the rewarding effects of nicotine may be a useful approach for future treatment. Objectives The present study was designed to evaluate 2-fluoro-3-(4-nitrophenyl) deschloroepibatidine (4-nitro-PFEB), a potent competitive antagonist of neuronal α4β2 nAChRs, in several animal models related to nicotine reward: drug discrimination, intracranial self-stimulation (ICSS), conditioned place preference, and limited access to self-administration. Methods Long Evans rats were trained in a two-lever discrimination procedure to discriminate 0.4 mg/kg nicotine (s.c.) from saline. Male Sprague–Dawley rats were stereotaxically implanted with electrodes and trained to respond for direct electrical stimulation of the medial forebrain bundle. ICR mice were evaluated using an unbiased place preference paradigm, and finally, male Wistar rats were implanted with intrajugular catheters and tested for nicotine self-administration under limited access (1 h/day). Results 4-Nitro-PFEB attenuated the discriminative stimulus effects of nicotine, but alone did not produce nicotine-like discriminative stimulus effects. Nicotine-induced facilitation of ICSS reward thresholds was reversed by 4-nitro-PFEB, which alone had no effect on thresholds. 4-Nitro-PFEB also blocked the conditioned place preference produced by nicotine, but alone had no effect on conditioned place preference. Finally, 4-nitro-PFEB dose-dependently decreased nicotine self-administration. Conclusions These results support the hypothesis that neuronal α4β2 nAChRs play a key role in mediating the rewarding effects of nicotine and further suggest that targeting α4β2 nAChRs may yield a potential candidate for the treatment of nicotine dependence. PMID:22526534

  7. Phosphate ester hydrolysis by hydroxo complexes of trivalent lanthanides stabilized by 4-imidazolecarboxylate.

    PubMed

    Aguilar-Pérez, Francisco; Gómez-Tagle, Paola; Collado-Fregoso, Elisa; Yatsimirsky, Anatoly K

    2006-11-13

    The anion of 4-imidazolecarboxylic acid (HL) stabilizes hydroxo complexes of trivalent lanthanides of the type ML(OH)+ (M = La, Pr) and M2L(n)(OH)(6-n) (M = La, n = 2; M = Pr, n = 2, 3; M = Nd, Eu, Dy, n = 1-3). Compositions and stability constants of the complexes have been determined by potentiometric titrations. Spectrophotometric and (1)H NMR titrations with Nd(III) support the reaction model for the formation of hydroxo complexes proposed on the basis of potentiometric results. Kinetics of the hydrolysis of two phosphate diesters, bis(4-nitrophenyl) phosphate (BNPP) and 2-hydroxypropyl 4-nitrophenyl phosphate (HPNPP), and a triester, 4-nitrophenyl diphenyl phosphate (NPDPP), in the presence of hydroxo complexes of five lanthanides were studied as a function of pH and metal and ligand concentrations. With all lanthanides and all substrates, complexes with the smallest n, that is M2L2(OH)4 for La and Pr and M2L(OH)5 for Nd, Eu, and Dy, exhibited the highest catalytic activity. Strong inhibitory effects by simple anions (Cl-, NO3-, (EtO)2PO2-, AcO-) were observed indicating high affinity of neutral hydroxo complexes toward anionic species. The catalytic activity decreased in the order La > Pr > Nd > Eu > Dy for both diester substrates and was practically independent of the nature of cation for a triester substrate. The efficiency of catalysis, expressed as the ratio of the second-order rate constant for the ester cleavage by the hydroxo complex to the second-order rate constant for the alkaline hydrolysis of the respective substrate, varied from ca. 1 for NPDPP to 10(2) for HPNPP and to 10(5) for BNPP. The proposed mechanism of catalytic hydrolysis involves reversible bridging complexation of a phosphodiester to the binuclear active species followed by attack on the phosphoryl group by bridging hydroxide (BNPP) or by the alkoxide group of the deprotonated substrate (HPNPP).

  8. Improved Manganese Phosphate Coatings

    DTIC Science & Technology

    1975-04-01

    Conversion coatings 3 . Phosphating bath 20 AGrjC onln odd*. ta It .. c..soMV midP 1J.,alft. by block noc.mb) Work was conducted to determine the mechanism by...34 TABULAR DATA Table I Analyses of Solution and Coating for Phosphating Baths 4 of Di-ferlng Compositions 11 Atomic Absorption...manganese and iron phosphate coating: k * a. Mn(H 2PO4) 2 Nn-P0 4 + H3PO0 k2 k) b. 3MnHPO4 - Mn3 (P04) 2 + H3i’O4 k4 k5 c. Fe(H 2PO4) 2 -01 FeHPO4

  9. The pentose phosphate pathway of glucose metabolism. Measurement of the non-oxidative reactions of the cycle

    PubMed Central

    Novello, F.; McLean, Patricia

    1968-01-01

    Methods for the quantitative determination of ribose 5-phosphate isomerase, ribulose 5-phosphate 3-epimerase, transketolase and transaldolase in tissue extracts are described. The determinations depend on the measurement of glyceraldehyde 3-phosphate by using the coupled system triose phosphate isomerase, α-glycero-phosphate dehydrogenase and NADH. By using additional purified enzymes transketolase, ribose 5-phosphate isomerase and ribulose 5-phosphate epimerase conditions could be arranged so that each enzyme in turn was made rate-limiting in the overall system. Transaldolase was measured with fructose 6-phosphate and erythrose 4-phosphate as substrates, and again glyceraldehyde 3-phosphate was measured by using the same coupled system. Measurements of the activities of the non-oxidative reactions of the pentose phosphate pathway were made in a variety of tissues and the values compared with those of the two oxidative steps catalysed by glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase. PMID:16742603

  10. π-stacking and C-X...D (X = H, NO2; D = O, π) interactions in the crystal network of both C-H...N and π-stacked dimers of 1,2-bis(4-bromophenyl)-1H-benzimidazole and 2-(4-bromophenyl)-1-(4-nitrophenyl)-1H-benzimidazole.

    PubMed

    González-Padilla, Jazmin E; Rosales-Hernández, Martha C; Padilla-Martínez, Itzia I; García-Báez, Efren V; Rojas-Lima, Susana; Salazar-Pereda, Veronica

    2014-01-01

    Molecules of 1,2-bis(4-bromophenyl)-1H-benzimidazole, C19H12Br2N2, (I), and 2-(4-bromophenyl)-1-(4-nitrophenyl)-1H-benzimidazole, C19H12BrN3O2, (II), are arranged in dimeric units through C-H...N and parallel-displaced π-stacking interactions favoured by the appropriate disposition of N- and C-bonded phenyl rings with respect to the mean benzimidazole plane. The molecular packing of the dimers of (I) and (II) arises by the concurrence of a diverse set of weak intermolecular C-X...D (X = H, NO2; D = O, π) interactions.

  11. Distinguishing between weedy Amaranthus species based on intron one sequences from the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS)gene

    USDA-ARS?s Scientific Manuscript database

    Hybridization between Amaranthus species and the potential for herbicide resistance to be transferred by hybridization are of growing concern in the weed science community. It is important to confirm suspect hybrid populations early to develop an effective control strategy. With this in mind, a PC...

  12. Characterization of Arabidopsis lines deficient in GAPC-1, a cytosolic NAD-dependent glyceraldehyde-3-phosphate dehydrogenase.

    PubMed

    Rius, Sebastián P; Casati, Paula; Iglesias, Alberto A; Gomez-Casati, Diego F

    2008-11-01

    Phosphorylating glyceraldehyde-3-P dehydrogenase (GAPC-1) is a highly conserved cytosolic enzyme that catalyzes the conversion of glyceraldehyde-3-P to 1,3-bis-phosphoglycerate; besides its participation in glycolysis, it is thought to be involved in additional cellular functions. To reach an integrative view on the many roles played by this enzyme, we characterized a homozygous gapc-1 null mutant and an as-GAPC1 line of Arabidopsis (Arabidopsis thaliana). Both mutant plant lines show a delay in growth, morphological alterations in siliques, and low seed number. Embryo development was altered, showing abortions and empty embryonic sacs in basal and apical siliques, respectively. The gapc-1 line shows a decrease in ATP levels and reduced respiratory rate. Furthermore, both lines exhibit a decrease in the expression and activity of aconitase and succinate dehydrogenase and reduced levels of pyruvate and several Krebs cycle intermediates, as well as increased reactive oxygen species levels. Transcriptome analysis of the gapc-1 mutants unveils a differential accumulation of transcripts encoding for enzymes involved in carbon partitioning. According to these studies, some enzymes involved in carbon flux decreased (phosphoenolpyruvate carboxylase, NAD-malic enzyme, glucose-6-P dehydrogenase) or increased (NAD-malate dehydrogenase) their activities compared to the wild-type line. Taken together, our data indicate that a deficiency in the cytosolic GAPC activity results in modifications of carbon flux and mitochondrial dysfunction, leading to an alteration of plant and embryo development with decreased number of seeds, indicating that GAPC-1 is essential for normal fertility in Arabidopsis plants.

  13. Separate physiological roles for two isozymes of pyridine nucleotide-linked glycerol-3-phosphate dehydrogenase in chicken.

    NASA Technical Reports Server (NTRS)

    White, H. B., III; Kaplan, N. O.

    1972-01-01

    The isozymes considered are designated 'liver type' and 'muscle type' based on the tissue of highest concentration. Electrophoretic analysis shows that the liver type is found in small amounts or is undetectable in all tissues studied except liver. The muscle type is found in skeletal muscles and kidney. Presumptive hybrid enzymes occur at low levels in chicken liver and kidney. The tissue distribution of glyceron-3-P dehydrogenase in several birds capable of sustained flight is different than in chicken.

  14. A Novel Naturally Occurring Class I 5-Enolpyruvylshikimate-3-Phosphate Synthase from Janibacter sp. Confers High Glyphosate Tolerance to Rice

    PubMed Central

    Yi, Shu-yuan; Cui, Ying; Zhao, Yan; Liu, Zi-duo; Lin, Yong-jun; Zhou, Fei

    2016-01-01

    As glyphosate is a broad spectrum herbicide extensively used in agriculture worldwide, identification of new aroA genes with high level of glyphosate tolerance is essential for the development and breeding of transgenic glyphosate-tolerant crops. In this study, an aroA gene was cloned from a Janibacter sp. strain isolated from marine sediment (designated as aroAJ. sp). The purified aroAJ. sp enzyme has a Km value of 30 μM for PEP and 83 μM for S3P, and a significantly higher Ki value for glyphosate (373 μM) than aroAE. coli. AroAJ. sp is characterized as a novel and naturally occurring class I aroA enzyme with glyphosate tolerance. Furthermore, we show that aroAJ. sp can be used as an effective selectable marker in both japonica and indica rice cultivar. Transgenic rice lines were tested by herbicide bioassay and it was confirmed that they could tolerate up to 3360 g/ha glyphosate, a dosage four-fold that of the recommended agricultural application level. To our knowledge, it is the first report of a naturally occurring novel class I aroA gene which can be efficiently utilized to study and develop transgenic glyphosate-tolerant crops, and can facilitate a more economical and simplified weed control system. PMID:26754957

  15. Separate physiological roles for two isozymes of pyridine nucleotide-linked glycerol-3-phosphate dehydrogenase in chicken.

    NASA Technical Reports Server (NTRS)

    White, H. B., III; Kaplan, N. O.

    1972-01-01

    The isozymes considered are designated 'liver type' and 'muscle type' based on the tissue of highest concentration. Electrophoretic analysis shows that the liver type is found in small amounts or is undetectable in all tissues studied except liver. The muscle type is found in skeletal muscles and kidney. Presumptive hybrid enzymes occur at low levels in chicken liver and kidney. The tissue distribution of glyceron-3-P dehydrogenase in several birds capable of sustained flight is different than in chicken.

  16. Physical Mapping of Amplified Copies of the 5-Enolpyruvylshikimate-3-Phosphate Synthase Gene in Glyphosate-Resistant Amaranthus tuberculatus.

    PubMed

    Dillon, Andrew; Varanasi, Vijay K; Danilova, Tatiana V; Koo, Dal-Hoe; Nakka, Sridevi; Peterson, Dallas E; Tranel, Patrick J; Friebe, Bernd; Gill, Bikram S; Jugulam, Mithila

    2017-02-01

    Recent and rapid evolution of resistance to glyphosate, the most widely used herbicides, in several weed species, including common waterhemp (Amaranthus tuberculatus), poses a serious threat to sustained crop production. We report that glyphosate resistance in A tuberculatus was due to amplification of the 5-enolpyruvylshikimate-3-P synthase (EPSPS) gene, which encodes the molecular target of glyphosate. There was a positive correlation between EPSPS gene copies and its transcript expression. We analyzed the distribution of EPSPS copies in the genome of A tuberculatus using fluorescence in situ hybridization on mitotic metaphase chromosomes and interphase nuclei. Fluorescence in situ hybridization analysis mapped the EPSPS gene to pericentromeric regions of two homologous chromosomes in glyphosate sensitive A tuberculatus In glyphosate-resistant plants, a cluster of EPSPS genes on the pericentromeric region on one pair of homologous chromosomes was detected. Intriguingly, two highly glyphosate-resistant plants harbored an additional chromosome with several EPSPS copies besides the native chromosome pair with EPSPS copies. These results suggest that the initial event of EPSPS gene duplication may have occurred because of unequal recombination mediated by repetitive DNA. Subsequently, gene amplification may have resulted via several other mechanisms, such as chromosomal rearrangements, deletion/insertion, transposon-mediated dispersion, or possibly by interspecific hybridization. This report illustrates the physical mapping of amplified EPSPS copies in A tuberculatus.

  17. How phosphoinositide 3-phosphate controls growth downstream of amino acids and autophagy downstream of amino acid withdrawal.

    PubMed

    Ktistakis, Nicholas T; Manifava, Maria; Schoenfelder, Priya; Rotondo, Sergio

    2012-02-01

    The simple phosphoinositide PtdIns3P has been shown to control cell growth downstream of amino acid signalling and autophagy downstream of amino acid withdrawal. These opposing effects depend in part on the existence of distinct complexes of Vps34 (vacuolar protein sorting 34), the kinase responsible for the majority of PtdIns3P synthesis in cells: one complex is activated after amino acid withdrawal to induce autophagy and another regulates mTORC1 (mammalian target of rapamycin complex 1) activation when amino acids are present. However, lipid-dependent signalling almost always exhibits a spatial dimension, related to the site of formation of the lipid signal. In the case of PtdIns3P-regulated autophagy induction, recent data suggest that PtdIns3P accumulates in a membrane compartment dynamically connected to the endoplasmic reticulum that constitutes a platform for the formation of some autophagosomes. For PtdIns3P-regulated mTORC1 activity, a spatial context is not yet known: several possibilities can be envisaged based on the known effects of PtdIns3P on the endocytic system and on recent data suggesting that activation of mTORC1 depends on its localization on lysosomes.

  18. Expression, purification, enzymatic characterization and crystallization of glyceraldehyde-3-phosphate dehydrogenase from Naegleria gruberi, the first one from phylum Percolozoa.

    PubMed

    Machado, Agnes Thiane Pereira; Silva, Marcio; Iulek, Jorge

    2016-11-01

    Naegleria gruberi had its genome sequenced by Fritz-Laylin and collaborators in 2010. It is not pathogenic, but has characteristics similar to those of Naegleria fowleri, opportunistic pathogen that can cause fatal encephalitis in humans. N. gruberi genome has contributed to a better understanding of the primitive eukaryotic metabolism and revealed the complexity of several metabolic pathways. In this paper we describe the expression, purification, enzyme characterization and crystallization of N. gruberi GAPDH, the first one for an organism belonging to phylum Percolozoa. The results indicated that 10 mM, 8.0 and 25 °C are the optimum arsenate concentration, pH and temperature, respectively. The enzyme presents allosteric positive cooperativity for substrates NAD(+) and G3P as indicated by the Hill coefficients. The phylogenetic proximity between N. fowleri and N. gruberi suggests that contributions from the study of the latter might provide information to assist the search for treatments of Primary Amebic Meningoencephalitis, especially, in this work, taking into account that GAPDH is identified as a therapeutic target.

  19. Protective Efficacy of Coccidial Common Antigen Glyceraldehyde 3-Phosphate Dehydrogenase (GAPDH) against Challenge with Three Eimeria Species

    PubMed Central

    Tian, Lu; Li, Wenyu; Huang, Xinmei; Tian, Di; Liu, Jianhua; Yang, Xinchao; Liu, Lianrui; Yan, Ruofeng; Xu, Lixin; Li, Xiangrui; Song, Xiaokai

    2017-01-01

    Coccidiosis is an intestinal disorder of poultry and often caused by simultaneous infections of several Eimeria species. GAPDH is one of the immunogenic common antigens among Eimeria tenella, E. acervulina, and E. maxima identified in our previous study. The present study was performed to further evaluate its immunogenicity and protective efficacy. The genes of GAPDH cloned from E. acervulina and E. maxima were named as EaGAPDH and EmGAPDH, respectively. The immunogenicity of recombinant proteins of EaGAPDH and EmGAPDH were analyzed by Western blot. The transcription and expression of pVAX-EaGAPDH and pVAX-EmGAPDH in the injected muscles were detected by reverse transcription PCR (RT-PCR) and Western blot, respectively. GAPDH-induced changes of T lymphocytes subpopulation, cytokines production, and antibody were determined using flow cytometry, quantitative real-time PCR (qPCR), and ELISA, respectively. Finally, the protective efficacies of pVAX-EaGAPDH and pVAX-EmGAPDH were evaluated by vaccination and challenge experiments. The results revealed that the recombinant GAPDH proteins reacted with the corresponding chicken antisera. The EaGAPDH genes were successfully transcribed and expressed in the injected muscles. Vaccination with pVAX-EaGAPDH and pVAX-EmGAPDH significantly increased the proportion of CD4+ and CD8+ T lymphocytes, the cytokines productions of IFN-γ, IL-2, IL-4 et al., and IgG antibody levels compared to controls. The vaccination increased the weight gains, decreased the oocyst outputs, alleviate the enteric lesions compared to controls, and induced moderate anti-coccidial index (ACI). In conclusion, the coccidial common antigen of GAPDH induced significant humoral and cellular immune response and effective protection against E. tenella, E. acervulina, E. maxima, and mixed infection of the three Eimeria species. PMID:28769877

  20. A Novel Naturally Occurring Class I 5-Enolpyruvylshikimate-3-Phosphate Synthase from Janibacter sp. Confers High Glyphosate Tolerance to Rice.

    PubMed

    Yi, Shu-yuan; Cui, Ying; Zhao, Yan; Liu, Zi-duo; Lin, Yong-jun; Zhou, Fei

    2016-01-12

    As glyphosate is a broad spectrum herbicide extensively used in agriculture worldwide, identification of new aroA genes with high level of glyphosate tolerance is essential for the development and breeding of transgenic glyphosate-tolerant crops. In this study, an aroA gene was cloned from a Janibacter sp. strain isolated from marine sediment (designated as aroAJ. sp). The purified aroAJ. sp enzyme has a Km value of 30 μM for PEP and 83 μM for S3P, and a significantly higher Ki value for glyphosate (373 μM) than aroAE. coli. AroAJ. sp is characterized as a novel and naturally occurring class I aroA enzyme with glyphosate tolerance. Furthermore, we show that aroAJ. sp can be used as an effective selectable marker in both japonica and indica rice cultivar. Transgenic rice lines were tested by herbicide bioassay and it was confirmed that they could tolerate up to 3360 g/ha glyphosate, a dosage four-fold that of the recommended agricultural application level. To our knowledge, it is the first report of a naturally occurring novel class I aroA gene which can be efficiently utilized to study and develop transgenic glyphosate-tolerant crops, and can facilitate a more economical and simplified weed control system.

  1. Glyphosate-Resistant Goosegrass. Identification of a Mutation in the Target Enzyme 5-enolpyruvylshikimate-3-phosphate Synthase

    USDA-ARS?s Scientific Manuscript database

    The spontaneous occurrence of resistance to the herbicide glyphosate in weed species has been an extremely infrequent event, despite over 20 years of extensive use. Recently, a glyphosate-resistant biotype of goosegrass (Eleusine indica) was identified in Malaysia exhibiting an LD50 value approxima...

  2. Overexpression of Arabidopsis thaliana PTEN caused accumulation of autophagic bodies in pollen tubes by disrupting phosphatidylinositol 3-phosphate dynamics

    USDA-ARS?s Scientific Manuscript database

    Autophagy is a pathway in eukaryotes by which nutrient remobilization occurs through bulk protein and organelle turnover. Autophagy not only aides cells in coping with harsh environments but also plays a key role in many physiological processes that include pollen germination and tube growth. Most a...

  3. Phosphate salts

    MedlinePlus

    ... taken by mouth or used as enemas. Indigestion. Aluminum phosphate and calcium phosphate are FDA-permitted ingredients ... Phosphate salts containing sodium, potassium, aluminum, or calcium are LIKELY SAFE for most people when taken by mouth short-term, when sodium phosphate is inserted into the ...

  4. The role of the liver in the production of free radicals during halothane anaesthesia in the rat. Quantification of N-tert-butyl-alpha-(4- nitrophenyl)nitrone (PBN)-trapped adducts in bile from halothane as compared with carbon tetrachloride.

    PubMed Central

    Hughes, H M; George, I M; Evans, J C; Rowlands, C C; Powell, G M; Curtis, C G

    1991-01-01

    Halothane or CCl4 was co-administered with the spin trap N-tert-butyl-alpha-(4-nitrophenyl)nitrone (PBN) to rats fitted with bile duct cannuli or to isolated perfused liver preparations. Rats maintained under halothane anaesthesia generated significant amounts of free radicals, and 5-9 nmol was excreted in bile over 1 h. No adducts were detected in urine or plasma. The hepatic origin of these free radicals was confirmed by studies on isolated perfused livers where the addition of halothane to the perfusate resulted in the biliary elimination of the same PBN-trapped radical adducts. Similarly, following CCl4 administration, the same radical species were eliminated in bile in the whole animal and the perfused liver preparation. In the perfused liver, over 3 h the total biliary elimination of radicals derived from halothane or CCl4 (administered at equimolar concentrations) was approximately the same (5-7 nmol); however, the elimination of halothane-derived radicals was more rapid over the first 1 h. PMID:1651704

  5. Crystal structure, vibrational and magnetic properties of the monohydrated cobalt (II) complex with 1-(4-Nitrophenyl)-1H-imidazolium cation, (C9H8N3O2)2CoCl4·H2O

    NASA Astrophysics Data System (ADS)

    Amamou, W.; Chniba-Boudjada, N.; Zouari, F.

    2017-01-01

    Single crystals of organic-inorganic hybrid compound Bis(1-(4-Nitrophenyl)-1H-imidazolium) tetrachlorocobaltate monohydrate, was obtained by slow evaporation of an aqueous solution at room temperature and characterized by a single-crystal X-ray diffraction, an elemental and thermal analysis, UV-Vis spectra, FT-IR and FT-Raman spectroscopies as well as magnetic measurements. The entitled compound crystallizes into triclinic system of P-1 space group. The Co(II) ion of the [CoCl4]2- anion shows a tetrahedral coordinating geometry. The atomic arrangement can be described as an alternation of organic and inorganic layers along the c-axis. The different components are connected by Nsbnd H⋯Cl, Osbnd H⋯Cl and Osbnd H⋯O hydrogen bonds. The differential scanning calorimetry (DSC) of the title compound revealed an endothermic peak at 52 °C related with a phase transformation caused by a slight deformation of the inorganic group. The room temperature IR and Raman spectra were recorded and analyzed on the basis of literary data to gain more information about the entitled compound. The magnetic susceptibility measurements in the temperature range 2-100 K shows that the complex displays a weak antiferromagnetic exchange interaction at very low temperatures.

  6. Acute Oral Toxicity Potential of 4-Nitrophenyl Methkyl Phenyl Phosphinate.

    DTIC Science & Technology

    1982-09-01

    methyl phenyl phosphinate Chemical Abstract Service Registry No.: None Molecular structure: C Ii NO P 13 12 4 .0 0~ NO., CH3 o o...C 56.33 56.17 H 4.36 4.28 N 5.05 5.14 P 11.17 11.25 2. Chemical Name: Polysorbate 80 (Tween 80) Chemical Abstract Service Registry No.: 9005-65-6...administration particularly in chronic toxicity studies in experimental data. 3. Chemical Name: Citric Acid, monohydrate Chemical Abstract Service

  7. Mechanism of substrate specificity of phosphatidylinositol phosphate kinases

    PubMed Central

    Muftuoglu, Yagmur; Xue, Yi; Gao, Xiang; Wu, Dianqing; Ha, Ya

    2016-01-01

    The phosphatidylinositol phosphate kinase (PIPK) family of enzymes is primarily responsible for converting singly phosphorylated phosphatidylinositol derivatives to phosphatidylinositol bisphosphates. As such, these kinases are central to many signaling and membrane trafficking processes in the eukaryotic cell. The three types of phosphatidylinositol phosphate kinases are homologous in sequence but differ in catalytic activities and biological functions. Type I and type II kinases generate phosphatidylinositol 4,5-bisphosphate from phosphatidylinositol 4-phosphate and phosphatidylinositol 5-phosphate, respectively, whereas the type III kinase produces phosphatidylinositol 3,5-bisphosphate from phosphatidylinositol 3-phosphate. Based on crystallographic analysis of the zebrafish type I kinase PIP5Kα, we identified a structural motif unique to the kinase family that serves to recognize the monophosphate on the substrate. Our data indicate that the complex pattern of substrate recognition and phosphorylation results from the interplay between the monophosphate binding site and the specificity loop: the specificity loop functions to recognize different orientations of the inositol ring, whereas residues flanking the phosphate binding Arg244 determine whether phosphatidylinositol 3-phosphate is exclusively bound and phosphorylated at the 5-position. This work provides a thorough picture of how PIPKs achieve their exquisite substrate specificity. PMID:27439870

  8. A specific process to purify 2-methyl-D-erythritol-4-phosphate enzymatically converted from D-glyceraldehyde-3-phosphate and pyruvate.

    PubMed

    Yang, Shao-Qing; Deng, Jian; Wu, Qian-Qian; Li, Heng; Gao, Wen-Yun

    2015-02-01

    A one-pot enzymatic cascade was established to synthesize MEP, one of the key intermediates in the MEP terpenoid biosynthetic pathway. D-GAP and sodium pyruvate were converted to MEP in a reaction catalyzed by DXP synthase and DXP reductoisomerase (DXR) in the presence of the coenzymes ThPP, NADPH, and Mg2+. The product was then isolated by using a specific two-step purification process and MEP was obtained in a yield of nearly 60% and high purity. Importantly, MEP prepared by this way was totally free from contamination by minor amounts of DXP that was not completely convertible by DXR.

  9. Dual Mechanisms of Metabolite Acquisition by the Obligate Intracytosolic Pathogen Rickettsia prowazekii Reveal Novel Aspects of Triose Phosphate Transport

    PubMed Central

    Frohlich, Kyla M.

    2013-01-01

    Rickettsia prowazekii is an obligate intracytosolic pathogen and the causative agent of epidemic typhus fever in humans. As an evolutionary model of intracellular pathogenesis, rickettsiae are notorious for their use of transport systems that parasitize eukaryotic host cell biochemical pathways. Rickettsial transport systems for substrates found only in eukaryotic cell cytoplasm are uncommon among free-living microorganisms and often possess distinctive mechanisms. We previously reported that R. prowazekii acquires triose phosphates for phospholipid biosynthesis via the coordinated activities of a novel dihydroxyacetone phosphate transport system and an sn-glycerol-3-phosphate dehydrogenase (K. M. Frohlich et al., J. Bacteriol. 192:4281–4288, 2010). In the present study, we have determined that R. prowazekii utilizes a second, independent triose phosphate acquisition pathway whereby sn-glycerol-3-phosphate is directly transported and incorporated into phospholipids. Herein we describe the sn-glycerol-3-phosphate and dihydroxyacetone phosphate transport systems in isolated R. prowazekii with respect to kinetics, energy coupling, transport mechanisms, and substrate specificity. These data suggest the existence of multiple rickettsial triose phosphate transport systems. Furthermore, the R. prowazekii dihydroxyacetone phosphate transport systems displayed unexpected mechanistic properties compared to well-characterized triose phosphate transport systems from plant plastids. Questions regarding possible roles for dual-substrate acquisition pathways as metabolic virulence factors in the context of a pathogen undergoing reductive evolution are discussed. PMID:23772074

  10. Experimental and theoretical (FT-IR, FT-Raman, UV-vis, NMR) spectroscopic analysis and first order hyperpolarizability studies of non-linear optical material: (2E)-3-[4-(methylsulfanyl) phenyl]-1-(4-nitrophenyl) prop-2-en-1-one using density functional theory.

    PubMed

    Kumar, Amit; Deval, Vipin; Tandon, Poonam; Gupta, Archana; Deepak D'silva, E

    2014-09-15

    A combined experimental and theoretical investigation on FT-IR, FT-Raman, NMR, UV-vis spectra of a chalcone derivative (2E)-3-[4-(methylsulfanyl) phenyl]-1-(4-nitrophenyl) prop-2-en-1-one (4N4MSP) has been reported. 4N4MSP has two planar rings connected through conjugated double bond and it provides a necessary configuration to show non-linear optical (NLO) response. The molecular structure, fundamental vibrational frequencies and intensity of the vibrational bands are interpreted with the aid of structure optimizations and normal coordinate force field calculations based on density functional theory (DFT) with B3LYP functional and 6-311++G(d,p) basis set combination. The analysis of the fundamental modes was made with the help of potential energy distribution (PED). Molecular electrostatic potential (MEP) surface was plotted over the geometry primarily for predicting sites and relative reactivities towards electrophilic and nucleophilic attack. The delocalization of electron density of various constituents of the molecule has been discussed with the aid of NBO analysis. The electronic properties, such as excitation energies, oscillator strength, wavelengths, HOMO and LUMO energies, were calculated by time-dependent density functional theory (TD-DFT) and the results complement the experimental findings. The recorded and calculated 1H chemical shifts in gas phase and MeOD solution are gathered for reliable calculations of magnetic properties. Thermodynamic properties like heat capacity (C°p,m), entropy (S°m), enthalpy (H°m) have been calculated for the molecule at the different temperatures. Based on the finite-field approach, the non-linear optical (NLO) parameters such as dipole moment, mean polarizability, anisotropy of polarizability and first order hyperpolarizability of 4N4MSP molecule are calculated. The predicted first hyperpolarizability shows that the molecule has a reasonably good nonlinear optical (NLO) behavior.

  11. Reductive activation of the prodrug 1,2-bis(methylsulfonyl)-1-(2-chloroethyl)-2-[[1-(4-nitrophenyl)ethoxy]carbonyl]hydrazine (KS119) selectively occurs in oxygen-deficient cells and overcomes O6-alkylguanine-DNA alkyltransferase mediated KS119 tumor cell resistance

    PubMed Central

    Baumann, Raymond P.; Penketh, Philip G.; Ishiguro, Kimiko; Shyam, Krishnamurthy; Zhu, Yong L.; Sartorelli, Alan C.

    2010-01-01

    1,2-Bis(methylsulfonyl)-1-(2-chloroethyl)-2-[[1-(4-nitrophenyl)ethoxy]carbonyl]hydrazine (KS119) is a prodrug of the 1,2-bis(sulfonyl)hydrazine class of antineoplastic agents designed to exploit the oxygen-deficient regions of cancerous tissue. Thus, under reductive conditions in hypoxic cells this agent decomposes to produce the reactive intermediate 1,2-bis(methylsulfonyl)-1-(2-chloroethyl)hydrazine (90CE), which in turn generates products that alkylate the O6-position of guanine in DNA. Comparison of the cytotoxicity of KS119 in cultured cells lacking O6-alkylguanine-DNA alkyltransferase (AGT) to an agent such as Onrigin™, which through base catalyzed activation produces the same critical DNA G-C cross-link lesions by the generation of 90CE, indicates that KS119 is substantially more potent than Onrigin™ under conditions of oxygen deficiency, despite being incompletely activated. In cell lines expressing relatively large amounts of AGT, the design of the prodrug KS119, which requires intracellular activation by reductase enzymes to produce a cytotoxic effect, results in an ability to overcome resistance derived from the expression of AGT. This appears to derive from the ability of a small portion of the chloroethylating species produced by the activation of KS119 to slip through the cellular protection afforded by AGT to generate the few DNA G-C cross-links that are required for tumor cell lethality. The findings also demonstrate that activation of KS119 under oxygen-deficient conditions is ubiquitous, occurring in all of the cell lines tested thus far, suggesting that the enzymes required for reductive activation of this agent are widely distributed in many different tumor types. PMID:20005211

  12. Experimental and theoretical (FT-IR, FT-Raman, UV-vis, NMR) spectroscopic analysis and first order hyperpolarizability studies of non-linear optical material: (2E)-3-[4-(methylsulfanyl) phenyl]-1-(4-nitrophenyl) prop-2-en-1-one using density functional theory

    NASA Astrophysics Data System (ADS)

    Kumar, Amit; Deval, Vipin; Tandon, Poonam; Gupta, Archana; Deepak D'silva, E.

    2014-09-01

    A combined experimental and theoretical investigation on FT-IR, FT-Raman, NMR, UV-vis spectra of a chalcone derivative (2E)-3-[4-(methylsulfanyl) phenyl]-1-(4-nitrophenyl) prop-2-en-1-one (4N4MSP) has been reported. 4N4MSP has two planar rings connected through conjugated double bond and it provides a necessary configuration to show non-linear optical (NLO) response. The molecular structure, fundamental vibrational frequencies and intensity of the vibrational bands are interpreted with the aid of structure optimizations and normal coordinate force field calculations based on density functional theory (DFT) with B3LYP functional and 6-311++G(d,p) basis set combination. The analysis of the fundamental modes was made with the help of potential energy distribution (PED). Molecular electrostatic potential (MEP) surface was plotted over the geometry primarily for predicting sites and relative reactivities towards electrophilic and nucleophilic attack. The delocalization of electron density of various constituents of the molecule has been discussed with the aid of NBO analysis. The electronic properties, such as excitation energies, oscillator strength, wavelengths, HOMO and LUM