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Sample records for erythrocyte glucose 6-phosphate

  1. 21 CFR 864.7360 - Erythrocytic glucose-6-phosphate dehydrogenase assay.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Erythrocytic glucose-6-phosphate dehydrogenase... § 864.7360 Erythrocytic glucose-6-phosphate dehydrogenase assay. (a) Identification. An erythrocytic glucose-6-phosphate dehydrogenase assay is a device used to measure the activity of the enzyme...

  2. 21 CFR 864.7360 - Erythrocytic glucose-6-phosphate dehydrogenase assay.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Erythrocytic glucose-6-phosphate dehydrogenase... § 864.7360 Erythrocytic glucose-6-phosphate dehydrogenase assay. (a) Identification. An erythrocytic glucose-6-phosphate dehydrogenase assay is a device used to measure the activity of the enzyme...

  3. 21 CFR 864.7360 - Erythrocytic glucose-6-phosphate dehydrogenase assay.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Erythrocytic glucose-6-phosphate dehydrogenase... § 864.7360 Erythrocytic glucose-6-phosphate dehydrogenase assay. (a) Identification. An erythrocytic glucose-6-phosphate dehydrogenase assay is a device used to measure the activity of the enzyme...

  4. 21 CFR 864.7360 - Erythrocytic glucose-6-phosphate dehydrogenase assay.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Erythrocytic glucose-6-phosphate dehydrogenase... § 864.7360 Erythrocytic glucose-6-phosphate dehydrogenase assay. (a) Identification. An erythrocytic glucose-6-phosphate dehydrogenase assay is a device used to measure the activity of the enzyme...

  5. 21 CFR 864.7360 - Erythrocytic glucose-6-phosphate dehydrogenase assay.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Erythrocytic glucose-6-phosphate dehydrogenase... § 864.7360 Erythrocytic glucose-6-phosphate dehydrogenase assay. (a) Identification. An erythrocytic glucose-6-phosphate dehydrogenase assay is a device used to measure the activity of the enzyme...

  6. Glucose-6-phosphate dehydrogenase

    MedlinePlus

    ... this page: //medlineplus.gov/ency/article/003671.htm Glucose-6-phosphate dehydrogenase test To use the sharing features on this page, please enable JavaScript. Glucose-6-phosphate dehydrogenase (G6PD) is a type of ...

  7. Pharmacological targeting of glucose-6-phosphate dehydrogenase in human erythrocytes by Bay 11-7082, parthenolide and dimethyl fumarate.

    PubMed

    Ghashghaeinia, Mehrdad; Giustarini, Daniela; Koralkova, Pavla; Köberle, Martin; Alzoubi, Kousi; Bissinger, Rosi; Hosseinzadeh, Zohreh; Dreischer, Peter; Bernhardt, Ingolf; Lang, Florian; Toulany, Mahmoud; Wieder, Thomas; Mojzikova, Renata; Rossi, Ranieri; Mrowietz, Ulrich

    2016-01-01

    In mature erythrocytes, glucose-6-phosphate dehydrogenase (G6PDH) and 6-phosphogluconate dehydrogenase (6PGDH) yield NADPH, a crucial cofactor of the enzyme glutathione reductase (GR) converting glutathione disulfide (GSSG) into its reduced state (GSH). GSH is essential for detoxification processes in and survival of erythrocytes. We explored whether the anti-inflammatory compounds Bay 11-7082, parthenolide and dimethyl fumarate (DMF) were able to completely deplete a common target (GSH), and to impair the function of upstream enzymes of GSH recycling and replenishment. Treatment of erythrocytes with Bay 11-7082, parthenolide or DMF led to concentration-dependent eryptosis resulting from complete depletion of GSH. GSH depletion was due to strong inhibition of G6PDH activity. Bay 11-7082 and DMF, but not parthenolide, were able to inhibit the GR activity. This approach "Inhibitors, Detection of their common target that is completely depleted or inactivated when pharmacologically relevant concentrations of each single inhibitor are applied, Subsequent functional analysis of upstream enzymes for this target" (IDS), can be applied to a broad range of inhibitors and cell types according to the selected target. The specific G6PDH inhibitory effect of these compounds may be exploited for the treatment of human diseases with high NADPH and GSH consumption rates, including malaria, trypanosomiasis, cancer or obesity. PMID:27353740

  8. Pharmacological targeting of glucose-6-phosphate dehydrogenase in human erythrocytes by Bay 11–7082, parthenolide and dimethyl fumarate

    PubMed Central

    Ghashghaeinia, Mehrdad; Giustarini, Daniela; Koralkova, Pavla; Köberle, Martin; Alzoubi, Kousi; Bissinger, Rosi; Hosseinzadeh, Zohreh; Dreischer, Peter; Bernhardt, Ingolf; Lang, Florian; Toulany, Mahmoud; Wieder, Thomas; Mojzikova, Renata; Rossi, Ranieri; Mrowietz, Ulrich

    2016-01-01

    In mature erythrocytes, glucose-6-phosphate dehydrogenase (G6PDH) and 6-phosphogluconate dehydrogenase (6PGDH) yield NADPH, a crucial cofactor of the enzyme glutathione reductase (GR) converting glutathione disulfide (GSSG) into its reduced state (GSH). GSH is essential for detoxification processes in and survival of erythrocytes. We explored whether the anti-inflammatory compounds Bay 11–7082, parthenolide and dimethyl fumarate (DMF) were able to completely deplete a common target (GSH), and to impair the function of upstream enzymes of GSH recycling and replenishment. Treatment of erythrocytes with Bay 11–7082, parthenolide or DMF led to concentration-dependent eryptosis resulting from complete depletion of GSH. GSH depletion was due to strong inhibition of G6PDH activity. Bay 11–7082 and DMF, but not parthenolide, were able to inhibit the GR activity. This approach “Inhibitors, Detection of their common target that is completely depleted or inactivated when pharmacologically relevant concentrations of each single inhibitor are applied, Subsequent functional analysis of upstream enzymes for this target” (IDS), can be applied to a broad range of inhibitors and cell types according to the selected target. The specific G6PDH inhibitory effect of these compounds may be exploited for the treatment of human diseases with high NADPH and GSH consumption rates, including malaria, trypanosomiasis, cancer or obesity. PMID:27353740

  9. Canine malignant hyperthermia susceptibility: erythrocytic defects--osmotic fragility, glucose-6-phosphate dehydrogenase deficiency and abnormal Ca2+ homeostasis.

    PubMed Central

    O'Brien, P J; Forsyth, G W; Olexson, D W; Thatte, H S; Addis, P B

    1984-01-01

    Two dogs were diagnosed as malignant hyperthermia susceptible based on increased susceptibility (P less than 0.001) of biopsied muscle to caffeine-induced contracture. Erythrocytes from malignant hyperthermia and normal dogs were then examined for an antioxidant system deficiency. Values for serum muscle enzymes, reticulocytes and corpuscular hemoglobin were mildly elevated. Osmotic fragility was increased: hemolysis occurred at a NaCl concentration 10 mM higher than for normal dogs (P less than 0.001). A 35% glucose-6-phosphate dehydrogenase deficiency (P less than 0.001) with a 40% compensatory increase (P less than 0.01) in 6-phosphogluconate dehydrogenase activity was found. The membrane Ca2+-activated ATPase activity was abnormal: 100% increased with a 40% decreased Arrhenius activation energy (P less than 0.005) and increased thermostability. A 40% increased intracellular accumulation of total Ca2+ occurred in response to in vitro energy depletion in erythrocytes from one malignant hyperthermia dog (P less than 0.01). The multifactorial pattern of inheritance and the broad spectrum of malignant hyperthermia susceptibility are proposed to result from an antioxidant system deficit unmasking or aggravating an intrinsic muscle membrane anomaly. An individual from a family with a history of malignant hyperthermia or unexplained anesthetic death should be considered malignant hyperthermia susceptible if erythrocyte osmotic fragility is abnormal and there is a mild, unexplained elevation in serum creatine kinase. PMID:6150753

  10. N-acetyl cysteine, L-cysteine, and beta-mercaptoethanol augment selenium-glutathione peroxidase activity in glucose-6-phosphate dehydrogenase-deficient human erythrocytes.

    PubMed

    Alicigüzel, Y; Aslan, M

    2004-09-01

    In glucose-6-phosphate dehydrogenase (G6PD)-deficient erythrocytes, failure to maintain normal levels of reduced glutathione (GSH) due to decreased NADPH regeneration in the hexose monophosphate pathway results in acute hemolytic anemia following exposure to oxidative insults, such as ingestion of Vicia fava beans or use of certain drugs. GSH is a source of protection against oxidative attack, used by the selenium-dependent glutathione peroxidase (Se-GSH-Px)/reductase (GR) system to detoxify hydrogen peroxide and organic peroxides, provided that sufficient GSH is made available. In this study, Se-GSH-Px activity was analyzed in G6PD-deficient patients in the presence of reducing agents such as N-Acetyl cysteine, L-cysteine, and beta-mercaptoethanol. Se-GSH-Px activity was decreased in G6PD-deficient red blood cells (RBCs). N-Acetyl cysteine, L-cysteine, and beta-mercaptoethanol increased Se-GSH-Px activity in G6PD-deficient human erythrocytes, indicating that other reducing agents can be utilized to complement Se-GSH-Px activity in G6PD deficiency. Based on the increased susceptibility of G6PD-deficient patients to oxidative stress, the reported increase in Se-GSH-Px activity can facilitate the detoxification of reactive oxygen species. PMID:15598086

  11. Producing Glucose 6-Phosphate from Cellulosic Biomass

    PubMed Central

    Bacik, John-Paul; Klesmith, Justin R.; Whitehead, Timothy A.; Jarboe, Laura R.; Unkefer, Clifford J.; Mark, Brian L.; Michalczyk, Ryszard

    2015-01-01

    The most abundant carbohydrate product of cellulosic biomass pyrolysis is the anhydrosugar levoglucosan (1,6-anhydro-β-d-glucopyranose), which can be converted to glucose 6-phosphate by levoglucosan kinase (LGK). In addition to the canonical kinase phosphotransfer reaction, the conversion requires cleavage of the 1,6-anhydro ring to allow ATP-dependent phosphorylation of the sugar O6 atom. Using x-ray crystallography, we show that LGK binds two magnesium ions in the active site that are additionally coordinated with the nucleotide and water molecules to result in ideal octahedral coordination. To further verify the metal binding sites, we co-crystallized LGK in the presence of manganese instead of magnesium and solved the structure de novo using the anomalous signal from four manganese atoms in the dimeric structure. The first metal is required for catalysis, whereas our work suggests that the second is either required or significantly promotes the catalytic rate. Although the enzyme binds its sugar substrate in a similar orientation to the structurally related 1,6-anhydro-N-acetylmuramic acid kinase (AnmK), it forms markedly fewer bonding interactions with the substrate. In this orientation, the sugar is in an optimal position to couple phosphorylation with ring cleavage. We also observed a second alternate binding orientation for levoglucosan, and in these structures, ADP was found to bind with lower affinity. These combined observations provide an explanation for the high Km of LGK for levoglucosan. Greater knowledge of the factors that contribute to the catalytic efficiency of LGK can be used to improve applications of this enzyme for levoglucosan-derived biofuel production. PMID:26354439

  12. Inhibitory effect of a fava bean component on the in vitro development of Plasmodium falciparum in normal and glucose-6-phosphate dehydrogenase deficient erythrocytes.

    PubMed

    Golenser, J; Miller, J; Spira, D T; Navok, T; Chevion, M

    1983-03-01

    We examined the hypothesis that G-6-PD deficiency associated with fava bean ingestion confers resistance to malaria by studying the in vitro interactions between malaria parasites (Plasmodium falciparum), human erythrocytes with varying degrees of G-6-PD deficiency, and isouramil (IU), a fava bean extract that is known to cause oxidant stress and hemolysis of G-6-PD-deficient erythrocytes. Untreated G-6-PD-deficient and normal erythrocytes supported the in vitro growth of P. falciparum equally well. However, after pretreatment with IU, G-6-PD-deficient erythrocytes did not support parasite growth in vitro, whereas growth remained high in normal erythrocytes. Parasite growth was proportional to the G-6-PD activity of the IU-treated erythrocytes. In contrast, when parasitized erythrocytes were exposed to IU, parasites even in normal erythrocytes were destroyed. Ring forms were much less sensitive than late trophozoites and schizonts. The results suggest that there are two modes by which IU affects the development of P. falciparum and demonstrate in vitro that G-6-PD deficiency confers resistance against malaria under conditions of fava-bean-associated oxidant stress.

  13. Glucose and fructose 6-phosphate cycle in humans

    SciTech Connect

    Karlander, S.; Roovete, A.; Vranic, M.; Efendic, S.

    1986-11-01

    We have determined the rate of glucose cycling by comparing turnovers of (2-/sup 3/H)- and (6-/sup 3/H)glucose under basal conditions and during a glucose infusion. Moreover, the activity of the fructose 6-phosphate cycle was assessed by comparing (3-/sup 3/H)- and (6-/sup 3/H)glucose. The study included eight lean subjects with normal glucose tolerance. They participated in two randomly performed investigations. In one experiment (2-/sup 3/H)- and (6-/sup 3/H)glucose were given simultaneously, while in the other only (3-/sup 3/H)glucose was given. The basal rate of glucose cycling was 0.32 +/- 0.08 mg X kg-1 X min-1 or 17% of basal glucose production (P less than 0.005). During glucose infusion the activity of endogenous glucose cycling did not change but since glucose production was suppressed it amounted to 130% of glucose production. The basal fructose 6-phosphate cycle could be detected only in three subjects and was suppressed during glucose infusion. In conclusion, the glucose cycle is active in healthy humans both in basal conditions and during moderate hyperglycemia. In some subjects, the fructose 6-phosphate cycle also appears to be active. Thus it is preferable to use (6-/sup 3/H)glucose rather than (3-/sup 3/H)glucose when measuring glucose production and particularly when assessing glucose cycle.

  14. Structural basis for glucose-6-phosphate activation of glycogen synthase

    SciTech Connect

    Baskaran, Sulochanadevi; Roach, Peter J.; DePaoli-Roach, Anna A.; Hurley, Thomas D.

    2010-11-22

    Regulation of the storage of glycogen, one of the major energy reserves, is of utmost metabolic importance. In eukaryotes, this regulation is accomplished through glucose-6-phosphate levels and protein phosphorylation. Glycogen synthase homologs in bacteria and archaea lack regulation, while the eukaryotic enzymes are inhibited by protein kinase mediated phosphorylation and activated by protein phosphatases and glucose-6-phosphate binding. We determined the crystal structures corresponding to the basal activity state and glucose-6-phosphate activated state of yeast glycogen synthase-2. The enzyme is assembled into an unusual tetramer by an insertion unique to the eukaryotic enzymes, and this subunit interface is rearranged by the binding of glucose-6-phosphate, which frees the active site cleft and facilitates catalysis. Using both mutagenesis and intein-mediated phospho-peptide ligation experiments, we demonstrate that the enzyme's response to glucose-6-phosphate is controlled by Arg583 and Arg587, while four additional arginine residues present within the same regulatory helix regulate the response to phosphorylation.

  15. Glucose-6-phosphate dehydrogenase deficiency: the added value of cytology.

    PubMed

    Roelens, Marie; Dossier, Claire; Fenneteau, Odile; Couque, Nathalie; Da Costa, Lydie

    2016-06-01

    We report the case of a 2 year-old boy hospitalized into the emergency room for influenza pneumonia infection. The evolution was marked by a respiratory distress syndrome, a severe hemolytic anemia, associated with thrombocytopenia and kidney failure. First, a diagnosis of hemolytic uremic syndrome (HUS) has been judiciously suggested due to the classical triad: kidney failure, hemolytic anemia and thrombocytopenia. But, strikingly, blood smears do not exhibit schizocytes, but instead ghosts and hemighosts, some characteristic features of a glucose-6-phosphate dehydrogenase deficiency. Our hypothesis has been confirmed by enzymatic dosage and molecular biology. The unusual initial aplastic feature of this anemia could be the result of a transient erythroblastopenia due to the viral agent, at the origin of the G6PD crisis on a background of a major erythrocyte anti-oxydant enzyme defect. This case of G6PD defect points out the continuously importance of the cytology, which was able to redirect the diagnosis by the hemighost and ghost detection.

  16. Glucose-6-phosphate dehydrogenase deficiency: the added value of cytology.

    PubMed

    Roelens, Marie; Dossier, Claire; Fenneteau, Odile; Couque, Nathalie; Da Costa, Lydie

    2016-06-01

    We report the case of a 2 year-old boy hospitalized into the emergency room for influenza pneumonia infection. The evolution was marked by a respiratory distress syndrome, a severe hemolytic anemia, associated with thrombocytopenia and kidney failure. First, a diagnosis of hemolytic uremic syndrome (HUS) has been judiciously suggested due to the classical triad: kidney failure, hemolytic anemia and thrombocytopenia. But, strikingly, blood smears do not exhibit schizocytes, but instead ghosts and hemighosts, some characteristic features of a glucose-6-phosphate dehydrogenase deficiency. Our hypothesis has been confirmed by enzymatic dosage and molecular biology. The unusual initial aplastic feature of this anemia could be the result of a transient erythroblastopenia due to the viral agent, at the origin of the G6PD crisis on a background of a major erythrocyte anti-oxydant enzyme defect. This case of G6PD defect points out the continuously importance of the cytology, which was able to redirect the diagnosis by the hemighost and ghost detection. PMID:27101632

  17. Human mutations in glucose 6-phosphate dehydrogenase reflect evolutionary history.

    PubMed

    Notaro, R; Afolayan, A; Luzzatto, L

    2000-03-01

    Glucose 6-phosphate dehydrogenase (G6PD) is a cytosolic enzyme encoded by a housekeeping X-linked gene whose main function is to produce NADPH, a key electron donor in the defense against oxidizing agents and in reductive biosynthetic reactions. Inherited G6PD deficiency is associated with either episodic hemolytic anemia (triggered by fava beans or other agents) or life-long hemolytic anemia. We show here that an evolutionary analysis is a key to understanding the biology of a housekeeping gene. From the alignment of the amino acid (aa) sequence of 52 glucose 6-phosphate dehydrogenase (G6PD) species from 42 different organisms, we found a striking correlation between the aa replacements that cause G6PD deficiency in humans and the sequence conservation of G6PD: two-thirds of such replacements are in highly and moderately conserved (50-99%) aa; relatively few are in fully conserved aa (where they might be lethal) or in poorly conserved aa, where presumably they simply would not cause G6PD deficiency. This is consistent with the notion that all human mutants have residual enzyme activity and that null mutations are lethal at some stage of development. Comparing the distribution of mutations in a human housekeeping gene with evolutionary conservation is a useful tool for pinpointing amino acid residues important for the stability or the function of the corresponding protein. In view of the current explosive increase in full genome sequencing projects, this tool will become rapidly available for numerous other genes.

  18. The glucose/glucose-6-phosphate cycle in the periportal and perivenous zone of rat liver.

    PubMed

    Jungermann, K; Heilbronn, R; Katz, N; Sasse, D

    1982-04-01

    Periportal and perivenous hepatocytes contain different activities (V) of antagonistic key enzymes such as glucokinase and glucose-6-phosphatase. In order to get an insight into the metabolism of the periportal and perivenous area the flux rates (v) of the glucose/glucose-6-phosphate cycle were calculated on the basis of the Michaelis-Menten equation using the measured zonal concentrations of glucose and glucose 6-phosphate, the zonal activities of glucokinase and glucose-6-phosphatase previously reported and the half-saturating substrate concentrations (Km) of the two enzymes found in the literature. The concentrations of glucose were obtained as a first approximation by measuring the concentrations in portal (= periportal) and hepatovenous (= perivenous) blood; those of glucose 6-phosphate were calculated from the levels determined in microdissected periportal and perivenous liver tissue. The calculations showed (a) that the overall cycling rates agreed remarkably well with those reported for intact animals and (b) that during a normal feeding rhythm the periportal zone should catalyze net glucose output and the perivenous zone should mediate net glucose uptake, as proposed by the model of 'metabolic zonation'.

  19. Amelioration by glucose-6-phosphate and NADP of potato glycoalkaloid inhibition in cell, enzyme and liposome assays.

    PubMed

    Roddick, J G; Leonard, A L

    1999-05-01

    Lysis of human erythrocytes by 20 microM chaconine was reduced by 0.5 mM glucose-6-phosphate (G6P) and NADP. Both compounds caused approximately 50% inhibition of haemolysis at 1 mM. Glucose, glucose-1-phosphate, rhamnose, galactose and galactose-6-phosphate were ineffective; NAD was effective, although not to the extent of NADP. Of the tested sugars, only G6P reduced solanine-induced haemolysis. G6P also reduced the synergistic haemolytic action of solanine and chaconine in combination. G6P and NADP at or above 5 mM antagonised chaconine-induced betanin loss from excised red beet root discs; NADP was more effective than G6P. Disruption of PC/cholesterol liposomes by chaconine and inhibition of acetylcholinesterase by chaconine or solanine, were unaffected by up to 10 mM NADP or 50 mM G6P.

  20. Glucose-6-phosphate dehydrogenase mutations and haplotypes in Mexican Mestizos.

    PubMed

    Arámbula, E; Aguilar L, J C; Vaca, G

    2000-08-01

    In a screening for glucose-6-phosphate dehydrogenase (G-6-PD) deficiency in 1985 unrelated male subjects from the general population (Groups A and B) belonging to four states of the Pacific coast, 21 G-6-PD-deficient subjects were detected. Screening for mutations at the G-6-PD gene by PCR-restriction enzyme in these 21 G-6-PD-deficient subjects as well as in 14 G-6-PD-deficient patients with hemolytic anemia belonging to several states of Mexico showed two common G-6-PD variants: G-6-PD A-(202A/376G) (19 cases) and G-6-PD A-(376G/968C) (9 cases). In 7 individuals the mutations responsible for the enzyme deficiency remain to be determined. Furthermore, four silent polymorphic sites at the G-6-PD gene (PvuII, PstI, 1311, and NlaIII) were investigated in the 28 individuals with G-6-PD A- variants and in 137 G-6-PD normal subjects. As expected, only 10 different haplotypes were observed. To date, in our project aiming to determine the molecular basis of G-6-PD deficiency in Mexico, 60 unrelated G-6-PD-deficient Mexican males-25 in previous studies and 35 in the present work-have been studied. More than 75% of these individuals are from states of the Pacific coast (Sinaloa, Nayarit, Jalisco, Michoacán, Guerrero, Oaxaca, and Chiapas). The results show that although G-6-PD deficiency is heterogeneous at the DNA level in Mexico, only three polymorphic variants have been observed: G-6-PD A-(202A/376G) (36 cases), G-6-PD A-(376G/968C) (13 cases), and G-6-PD Seattle(844C) (2 cases). G-6-PD A- variants are relatively distributed homogeneously and both variants explain 82% of the overall prevalence of G-6-PD deficiency. The variant G-6-PD A-(202A/376G) represents 73% of the G-6-PD A- alleles. Our data also show that the variant G-6-PD A-(376G/968C)-which has been observed in Mexico in the context of two different haplotypes-is more common than previously supposed. The three polymorphic variants that we observed in Mexico are on the same haplotypes as found in subjects from

  1. Glucose-6-phosphate dehydrogenase from Plasmodium berghei: kinetic and electrophoretic characterization.

    PubMed

    Buckwitz, D; Jacobasch, G; Kuckelkorn, U; Plonka, A; Gerth, C

    1990-04-01

    Evidence is given for the existence of a parasite-specific glucose-6-phosphate dehydrogenase (G6PD) in Plasmodium berghei by characterization of its kinetic and electrophoretic properties. From infected rat erythrocytes the parasites were isolated, washed, and lysed. G6PD was purified by affinity chromatography with 2'5'-ADP-Sepharose 4B, although the separation of the malaria-specific enzyme from that of the host cell was not complete. Malarial G6PD significantly differed from the red cell enzyme with respect to its electrophoretic properties. In cellulose acetate electrophoresis, a band with catodic mobility was observed in addition to the anodically mobile host cell enzyme at pH 7.0. The subunits of the parasite-specific G6PD have a molecular weight of 55 kDa in contrast to 59 kDa of red cell G6PD subunits. The enzyme from P. berghei shows no cross-reactivity with polyclonal antibodies against G6PD from rat erythrocytes. Thus, a close evolutionary relationship between both proteins and the presence of proteolytic modifications could be excluded. The Km value for G6P of malarial G6PD is increased by one order of magnitude compared with the host cell enzyme.

  2. Glucose-6-phosphate dehydrogenase deficiency presented with convulsion: a rare case.

    PubMed

    Merdin, Alparslan; Avci, Fatma; Guzelay, Nihal

    2014-01-29

    Red blood cells carry oxygen in the body and Glucose-6-Phosphate Dehydrogenase protects these cells from oxidative chemicals. If there is a lack of Glucose-6-Phosphate Dehydrogenase, red blood cells can go acute hemolysis. Convulsion is a rare presentation for acute hemolysis due to Glucose-6-Phosphate Dehydrogenase deficiency. Herein, we report a case report of a Glucose-6-Phosphate Dehydrogenase deficiency diagnosed patient after presentation with convulsion. A 70 year-old woman patient had been hospitalized because of convulsion and fatigue. She has not had similar symptoms before. She had ingested fava beans in the last two days. Her hypophyseal and brain magnetic resonance imaging were normal. Blood transfusion was performed and the patient recovered.

  3. Glucose-6-phosphate dehydrogenase deficiency presented with convulsion: a rare case.

    PubMed

    Merdin, Alparslan; Avci, Fatma; Guzelay, Nihal

    2014-01-29

    Red blood cells carry oxygen in the body and Glucose-6-Phosphate Dehydrogenase protects these cells from oxidative chemicals. If there is a lack of Glucose-6-Phosphate Dehydrogenase, red blood cells can go acute hemolysis. Convulsion is a rare presentation for acute hemolysis due to Glucose-6-Phosphate Dehydrogenase deficiency. Herein, we report a case report of a Glucose-6-Phosphate Dehydrogenase deficiency diagnosed patient after presentation with convulsion. A 70 year-old woman patient had been hospitalized because of convulsion and fatigue. She has not had similar symptoms before. She had ingested fava beans in the last two days. Her hypophyseal and brain magnetic resonance imaging were normal. Blood transfusion was performed and the patient recovered. PMID:24711919

  4. Quantification of glucose cycling and the extent of equilibration of glucose 6-phosphate with fructose 6-phosphate in islets from ob/ob mice.

    PubMed Central

    Chandramouli, V; Khan, A; Ostenson, C G; Berggren, P O; Löw, H; Landau, B R; Efendić, S E

    1991-01-01

    Pancreatic islets from fed and fasted obese hyperglycaemic (ob/ob) mice were incubated with [1-14C]glucose at 5.5 mM and 16.7 mM, [1-14C]mannose at 16.7 mM, and 3H2O. Yields of 14CO2 and 14C-labelled lactate, and amounts of 14C from [1-14C]mannose incorporated into glucose and of 3H bound to C-2 of glucose, were measured. Glucose utilization was determined from yields of 3H2O from [5-3H]glucose. From the results using 14C, 32-43% of the hexoses phosphorylated to hexose 6-phosphate were estimated to have been dephosphorylated, i.e. cycled. Estimates of hexose cycling from 3H incorporation into glucose were similar. Differences in the ratios of 14C yields in CO2 and lactate indicated incomplete isotopic equilibration between glucose 6-phosphate and fructose 6-phosphate, making the estimates of cycling semi-quantitative. In the fasted state, a larger percentage of the hexose utilized went to lactate than in the fed state. Thus conversion of mannose into glucose in islets indicates the occurrence of glucose cycling in islets. Yields of 14C from [1-14C]mannose, compared with from [1-14C]glucose, provide an approach for quantifying the extent of this cycling. These data provide further evidence for extensive glucose cycling occurring in ob/ob islets in both the fed and the fasted state. PMID:1898326

  5. Dental Considerations in Children with Glucose-6-phosphate Dehydrogenase Deficiency (Favism): A Review of the Literature and Case Report.

    PubMed

    Hernández-Pérez, Daniela; Butrón-Téllez Girón, Claudia; Ruiz-Rodríguez, Socorro; Garrocho-Rangel, Arturo; Pozos-Guillén, Amaury

    2015-01-01

    Glucose-6-phosphate dehydrogenase (G6PD) deficiency is an uncommon inherited enzyme deficiency characterized by hemolytic anemia, caused by the inability of erythrocytes to detoxify oxidizing agents such as drugs, infectious diseases, or fava bean ingestion. In this later case, the disorder is known as favism. The aim of the present report was to present a review of the literature in this disease, to describe a case report concerning an affected 9-year-old male, and to review the main implications and precautions in pediatric dental management.

  6. Dental Considerations in Children with Glucose-6-phosphate Dehydrogenase Deficiency (Favism): A Review of the Literature and Case Report.

    PubMed

    Hernández-Pérez, Daniela; Butrón-Téllez Girón, Claudia; Ruiz-Rodríguez, Socorro; Garrocho-Rangel, Arturo; Pozos-Guillén, Amaury

    2015-01-01

    Glucose-6-phosphate dehydrogenase (G6PD) deficiency is an uncommon inherited enzyme deficiency characterized by hemolytic anemia, caused by the inability of erythrocytes to detoxify oxidizing agents such as drugs, infectious diseases, or fava bean ingestion. In this later case, the disorder is known as favism. The aim of the present report was to present a review of the literature in this disease, to describe a case report concerning an affected 9-year-old male, and to review the main implications and precautions in pediatric dental management. PMID:26435857

  7. Dental Considerations in Children with Glucose-6-phosphate Dehydrogenase Deficiency (Favism): A Review of the Literature and Case Report

    PubMed Central

    Hernández-Pérez, Daniela; Butrón-Téllez Girón, Claudia; Ruiz-Rodríguez, Socorro; Garrocho-Rangel, Arturo; Pozos-Guillén, Amaury

    2015-01-01

    Glucose-6-phosphate dehydrogenase (G6PD) deficiency is an uncommon inherited enzyme deficiency characterized by hemolytic anemia, caused by the inability of erythrocytes to detoxify oxidizing agents such as drugs, infectious diseases, or fava bean ingestion. In this later case, the disorder is known as favism. The aim of the present report was to present a review of the literature in this disease, to describe a case report concerning an affected 9-year-old male, and to review the main implications and precautions in pediatric dental management. PMID:26435857

  8. Cryopreservation of glucose-6-phosphate dehydrogenase activity inside red blood cells: developing a specimen repository in support of development and evaluation of glucose-6-phosphate dehydrogenase deficiency tests

    PubMed Central

    2013-01-01

    Background Glucose-6-phosphate dehydrogenase (G6PD) deficiency is a common human enzyme deficiency. It is characterized by abnormally low levels of G6PD activity. Individuals with G6PD deficiency are at risk of undergoing acute haemolysis when exposed to 8‒aminoquinoline-based drugs, such as primaquine. For this reason it is imperative to identify individuals with G6PD deficiency prior to administering these anti-malarial drugs. There is a need for the development and evaluation of point-of-care G6PD deficiency screening tests suitable for areas of the developing world where malarial treatments are frequently administered. The development and evaluation of new G6PD tests will be greatly assisted with the availability of specimen repositories. Methods Cryopreservation of erythrocytes was evaluated as a means to preserve G6PD activity. Blood specimens from 31 patients including ten specimens with normal G6PD activity, three with intermediate activity, and 18 with deficient activity were cryopreserved for up to six months. Results Good correlation in G6PD activity between fresh and cryopreserved specimens (R2 = 0.95). The cryopreserved specimens show an overall small drop in mean G6PD activity of 0.23 U/g Hb (P=0.23). Cytochemical staining showed that intracellular G6PD activity distribution within the red blood cell populations is preserved during cryopreservation. Furthermore, the mosaic composition of red blood cells in heterozygous women is also preserved for six months or more. The fluorescent spot and the BinaxNOW qualitative tests for G6PD deficiency also showed high concordance in G6PD status determination between cryopreserved specimens and fresh specimens. Conclusions A methodology for establishing a specimen panel for evaluation of G6PD tests is described. The approach is similar to that used in several malaria research facilities for the cryopreservation of parasites in clinical specimens and axenic cultures. Specimens stored in this manner will aid

  9. Multiple transcripts encode glucose 6-phosphate dehydrogenase in the southern cattle tick, Rhipicephalus (Boophilus) microplus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Glucose 6-phosphate dehydrogenase (G6PDH) is an enzyme that plays a critical role in the production of NADPH. Here we describe the identification of four transcripts (G6PDH-A, -B, -C, and -D) that putatively encode the enzyme in the southern cattle tick, Rhipicephalus (Boophilus) microplus. The geno...

  10. Glucose-6-phosphate dehydrogenase-derived NADPH fuels superoxide production in the failing heart

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In the failing heart, NADPH oxidase and uncoupled NO synthase utilize cytosolic NADPH to form superoxide. NADPH is supplied principally by the pentose phosphate pathway, whose rate-limiting enzyme is glucose 6-phosphate dehydrogenase (G6PD). Therefore, we hypothesized that cardiac G6PD activation dr...

  11. Glucose-6-phosphate dehydrogenase deficiency and sulfadimidin acetylation phenotypes in Egyptian oases.

    PubMed

    Hussein, L; Yamamah, G; Saleh, A

    1992-04-01

    Screening of 1315 males from two Egyptian oases for glucose-6-phosphate dehydrogenase deficiency (G-6PD) found an incidence of 5.9%. The rate of acetylation of sulfadimidin was also studied, and a bimodal distribution was found with 73% rapid acetylators. There is a correlation between high frequency of G-6PD deficiency and high frequency of slow acetylation rate.

  12. Appearance of Novel Glucose-6-Phosphate Dehydrogenase Isoforms in Chlamydomonas reinhardtii during Growth on Nitrate.

    PubMed Central

    Huppe, H. C.; Turpin, D. H.

    1996-01-01

    Extractable glucose-6-phosphate dehydrogenase activity is higher from N-limited Chlamydomonas reinhardtii cells than from N-sufficient cells. Native gels reveal that the isoform complexity varies depending on the form of N supplied. The isoforms associated with NO3- growth appear within 2 h of switching cells from NH4+ to NO3-. PMID:12226271

  13. Glucose-6-phosphate dehydrogenase alloenzymes and their relationship to pigmentation in Serratia marcescens.

    PubMed

    Gargallo, D; Lorén, J G; Guinea, J; Viñas, M

    1987-08-01

    A comparative study of environmental and clinical isolates of Serratia marcescens was undertaken with regard to glucose-6-phosphate dehydrogenase (G6PD) electrophoretic mobility and the production of prodigiosin. Two electromorphs of G6PD with electrophoretic mobilities of 0.22 and 0.30 were detected. G6PD electrophoretic type showed a good correlation with the ability to produce prodigiosin.

  14. Glucose-6-phosphate mediates activation of the carbohydrate responsive binding protein (ChREBP)

    SciTech Connect

    Li, Ming V.; Chen, Weiqin; Harmancey, Romain N.; Nuotio-Antar, Alli M.; Imamura, Minako; Saha, Pradip; Taegtmeyer, Heinrich; Chan, Lawrence

    2010-05-07

    Carbohydrate response element binding protein (ChREBP) is a Mondo family transcription factor that activates a number of glycolytic and lipogenic genes in response to glucose stimulation. We have previously reported that high glucose can activate the transcriptional activity of ChREBP independent of the protein phosphatase 2A (PP2A)-mediated increase in nuclear entry and DNA binding. Here, we found that formation of glucose-6-phosphate (G-6-P) is essential for glucose activation of ChREBP. The glucose response of GAL4-ChREBP is attenuated by D-mannoheptulose, a potent hexokinase inhibitor, as well as over-expression of glucose-6-phosphatase (G6Pase); kinetics of activation of GAL4-ChREBP can be modified by exogenously expressed GCK. Further metabolism of G-6-P through the two major glucose metabolic pathways, glycolysis and pentose-phosphate pathway, is not required for activation of ChREBP; over-expression of glucose-6-phosphate dehydrogenase (G6PD) diminishes, whereas RNAi knockdown of the enzyme enhances, the glucose response of GAL4-ChREBP, respectively. Moreover, the glucose analogue 2-deoxyglucose (2-DG), which is phosphorylated by hexokinase, but not further metabolized, effectively upregulates the transcription activity of ChREBP. In addition, over-expression of phosphofructokinase (PFK) 1 and 2, synergistically diminishes the glucose response of GAL4-ChREBP. These multiple lines of evidence support the conclusion that G-6-P mediates the activation of ChREBP.

  15. Identification of the binding domain for NADP sup + of human glucose-6-phosphate dehydrogenase by sequence analysis of mutants

    SciTech Connect

    Hirono, A.; Kuhl, W.; Gelbart, T.; Forman, L.; Beutler, E. ); Fairbanks, V.F. )

    1989-12-01

    Human erythrocyte glucose-6-phosphate is normally quite stable in the presence of 10 {mu}M NADP{sup +}. Certain glucose-6-phosphate dehydrogenase variants lose virtually all their activity at this concentration of NADP{sup +} but are reactivated by 200 {mu}M NADP{sup +}. Such variants presumably have a defect in their NADP{sup +}-binding site. The authors analyzed the sequence of cDNA or genomic DNA from seven unrelated patients with hemolytic anemia due to the inheritance of variants that are reactivated by NADP{sup +}. Six patients had substitutions of one of three adjacent amino acids, and the seventh patient had another amino acid substitution 23 residues downstream. These amino acids are highly conserved, all being present in rat and all but one being found also in Drosophila. The anomalous electrophoretic behavior of some of the variants can be explained by their loss of ability to bind NADP{sup +}. The conclude that the region in which these mutations occur defines the binding domain for NADP{sup +} and that binding NADP{sup +} that has been designated as structural and as catalytic probably occurs at the same site.

  16. NMR studies on mechanism of isomerisation of fructose 6-phosphate to glucose 6-phosphate catalysed by phosphoglucose isomerase from Thermococcus kodakarensis.

    PubMed

    Abbas, Shahzada Nadeem; Mok, Kenneth Hun; Rashid, Naeem; Xie, Yongjing; Ruether, Manuel; O'Brien, John; Akhtar, Muhammad

    2016-06-01

    The fate of hydrogen atoms at C-2 of glucose 6-phosphate (G6P) and C-1 of fructose 6-phosphate (F6P) was studied in the reaction catalysed by phosphoglucose isomerase from Thermococcus kodakarensis (TkPGI) through 1D and 2D NMR methods. When the reaction was performed in (2)H2O the hydrogen atoms in the aforementioned positions were exchanged with deuterons indicating that the isomerization occurred by a cis-enediol intermediate involving C-1 pro-R hydrogen of F6P. These features are similar to those described for phosphoglucose isomerases from rabbit muscle and Pyrococcus furiosus.

  17. Stenotrophomonas Infection in a Patient with Glucose-6-Phosphate Dehydrogenase Deficiency

    PubMed Central

    Harthan, Aaron A.; Heger, Margaret L

    2013-01-01

    The drug of choice for treatment of Stenotrophomonas maltophilia is sulfamethoxazole/trimethoprim, and second-line therapy usually consists of a fluoroquinolone. However, in patients with glucose-6-phosphate dehydrogenase deficiency, neither sulfamethoxazole/trimethoprim nor a fluoroquinolone is a preferred option as it may result in hemolysis. Currently, there is a paucity of data regarding treatment of S maltophilia infection in these patients. This case report presents a patient who was successfully treated with doxycycline and inhaled colistimethate. PMID:23798908

  18. [Polymorphism of Glucose-6-phosphate Dehydrogenase in the Chronically Irradiated Scots Pine Populations].

    PubMed

    Kazakova, E A; Volkova, P Yu; Geras'kin, S A; Pomelova, D O

    2015-01-01

    Polymorphism of glucose-6-phosphate dehydrogenase enzyme was studied in the Scots pine populations growing in the sites of Bryansk region which were radioactively contaminated as a result of the Chernobyl accident. It was revealed that the frequency of mutations in isozyme loci increased along with the level of a dose rate (7-130 mGy/year) in the sites under the study. Significant changes in the activity of this enzyme did not depend on the level of radiation exposure.

  19. Androgen-estrogen synergy in rat levator ani muscle Glucose-6-phosphate dehydrogenase

    NASA Technical Reports Server (NTRS)

    Max, S. R.

    1984-01-01

    The effects of castration and hormone administration on the activity of glucose-6-phosphate dehydrogenase in the rat levator ani muscle were studied. Castration caused a decrease in enzyme activity and in wet weight of the levator ani muscle. Chronic administration of testosterone propionate increased glucose-6-phosphate dehydrogenase activity in the levator ani muscle of castrated rats; the magnitude of the recovery of enzyme activity was related to the length of time of exposure to testosterone propionate after castration as well as to the length of time the animals were castrated. The longer the period of castration before exposure to testosterone propionate, the greater the effect. This result may be related to previously reported castration-mediated increases in androgen receptor binding in muscle. Dihydrotestosterone was less effective than testosterone propionate in enhancing glucose-6-phosphate dehydrogenase activity in the levator ani muscle from castrated rats; estradiol-17-beta alone was ineffective. Combined treatment with estradiol-17-beta and dihydrotestosterone, however, was as effective as testosterone alone. Thus, androgens and estrogens may exert synergistic effects on levator ani muscle.

  20. Beta glucosidase from Bacillus polymyxa is activated by glucose-6-phosphate.

    PubMed

    Weiss, Paulo H E; Álvares, Alice C M; Gomes, Anderson A; Miletti, Luiz C; Skoronski, Everton; da Silva, Gustavo F; de Freitas, Sonia M; Magalhães, Maria L B

    2015-08-15

    Optimization of cellulose enzymatic hydrolysis is crucial for cost effective bioethanol production from lignocellulosic biomass. Enzymes involved in cellulose hydrolysis are often inhibited by their end-products, cellobiose and glucose. Efforts have been made to produce more efficient enzyme variants that are highly tolerant to product accumulation; however, further improvements are still necessary. Based on an alternative approach we initially investigated whether recently formed glucose could be phosphorylated into glucose-6-phosphate to circumvent glucose accumulation and avoid inhibition of beta-glucosidase from Bacillus polymyxa (BGLA). The kinetic properties and structural analysis of BGLA in the presence of glucose-6-phosphate (G6P) were investigated. Kinetic studies demonstrated that enzyme was not inhibited by G6P. In contrast, the presence of G6P activated the enzyme, prevented beta glucosidase feedback inhibition by glucose accumulation and improved protein stability. G6P binding was investigated by fluorescence quenching experiments and the respective association constant indicated high affinity binding of G6P to BGLA. Data reported here are of great impact for future design strategies for second-generation bioethanol production.

  1. Identification of protein components of the microsomal glucose 6-phosphate transporter by photoaffinity labelling.

    PubMed

    Kramer, W; Burger, H J; Arion, W J; Corsiero, D; Girbig, F; Weyland, C; Hemmerle, H; Petry, S; Habermann, P; Herling, A

    1999-05-01

    The glucose-6-phosphatase system catalyses the terminal step of hepatic glucose production from both gluconeogenesis and glycogenolysis and is thus a key regulatory factor of blood glucose homoeostasis. To identify the glucose 6-phosphate transporter T1, we have performed photoaffinity labelling of human and rat liver microsomes by using the specific photoreactive glucose-6-phosphate translocase inhibitors S 0957 and S 1743. Membrane proteins of molecular mass 70, 55, 33 and 31 kDa were labelled in human microsomes by [3H]S 0957, whereas in rat liver microsomes bands at 95, 70, 57, 54, 50, 41, 33 and 31 kDa were detectable. The photoprobe [3H]S 1743 led to the predominant labelling of a 57 kDa and a 50 kDa protein in the rat. Stripping of microsomes with 0.3% CHAPS retains the specific binding of T1 inhibitors; photoaffinity labelling of such CHAPS-treated microsomes resulted in the labelling of membrane proteins of molecular mass 55, 33 and 31 kDa in human liver and 50, 33 and 31 kDa in rat liver. Photoaffinity labelling of human liver tissue samples from a healthy individual and from liver samples of patients with a diagnosed glycogen-storage disease type 1b (GSD type 1b; von Gierke's disease) revealed the absence of the 55 kDa protein from one of the patients with GSD type 1. These findings support the identity of the glucose 6-phosphate transporter T1, with endoplasmic reticulum protein of molecular mass 50 kDa in rat liver and 55 kDa in human liver. PMID:10215602

  2. Changing kinetic properties of glucose-6-phosphate dehydrogenase from pea chloroplasts during photosynthetic induction

    SciTech Connect

    Yuan, X.; Anderson, L.E.

    1987-04-01

    The first enzyme of the oxidative pentose phosphate pathway, glucose-6-P dehydrogenase (EC 1.1.1.49), is inactivated when pea chloroplasts are irradiated. They have examined the kinetics of light inactivation of glucose-6-P dehydrogenase in intact chloroplasts during photosynthetic induction and the kinetic parameters of the active (dark) and less active (light) form of the dehydrogenase. Light inactivation of the dehydrogenase is rapid and occurs before photosynthetic O/sub 2/ evolution is measureable in intact chloroplasts. Likewise dark activation is quite rapid. The major change in the kinetic parameters of glucose-6-phosphate dehydrogenase is in maximal velocity. This light inactivation probably prevents operation of a futile cycle involving glucose-6-P, NADPH and oxidative and reductive pentose phosphate pathway enzymes.

  3. Complete Deficiency of Leukocyte Glucose-6-Phosphate Dehydrogenase with Defective Bactericidal Activity

    PubMed Central

    Cooper, M. Robert; DeChatelet, Lawrence R.; McCall, Charles E.; La Via, Mariano F.; Spurr, Charles L.; Baehner, Robert L.

    1972-01-01

    A 52 yr old Caucasian female (F. E.) had hemolytic anemia, a leukemoid reaction, and fatal sepsis due to Escherichia coli. Her leukocytes ingested bacteria normally but did not kill catalase positive Staphylococcus aureus, Escherichia coli, and Serratia marcescens. An H2O2-producing bacterium, Streptococcus faecalis, was killed normally. Granule myeloperoxidase, acid and alkaline phosphatase, and beta glucuronidase activities were normal, and these enzymes shifted normally to the phagocyte vacuole (light and electron microscopy). Intravacuolar reduction of nitroblue tetrazolium did not occur. Moreover, only minimal quantities of H2O2 were generated, and the hexose monophosphate shunt (HMPS) was not stimulated during phagocytosis. These observations suggested the diagnosis of chronic granulomatous disease. However, in contrast to control and chronic granulomatous disease leukocytes, glucose-6-phosphate dehydrogenase activity was completely absent in F. E. leukocytes whereas NADH oxidase and NADPH oxidase activities were both normal. Unlike chronic granulomatous disease, methylene blue did not stimulate the hexose monophosphate shunt in F. E. cells. Thus, F. E. and chronic granulomatous disease leukocytes appear to share certain metabolic and bactericidal defects, but the metabolic basis of the abnormality differs. Chronic granulomatous disease cells lack oxidase activity which produces H2O2; F. E. cells had normal levels of oxidase activity but failed to produce NADPH due to complete glucose-6-phosphate dehydrogenase deficiency. These data indicate that a complete absence of leukocyte glucose-6-phosphate dehydrogenase with defective hexose monophosphate shunt activity is associated with low H2O2 production and inadequate bactericidal activity, and further suggest an important role for NADPH in the production of H2O2 in human granulocytes. Images PMID:4401271

  4. GD (--) Aachen, a new variant of deficient glucose-6-phosphate dehydrogenase. Clinical, genetic, biochemical aspects.

    PubMed

    Kahn, A; Esters, A; Habedank, M

    1976-05-19

    A deficient G-6PD variant was discovered in 4 males of one family from northwestern Germany. Five generations of this family could be studied. The deficient G-6PD was a new variant, called "Gd (--) Aachen". Its main characteristics are the following: severe enzyme deficiency in erythrocytes (3% of normal), contrasting with an almost normal activity in leukocytes; normal molecular specific activity (i.e., normal ratio enzyme activity/cross-reacting material); slow mobility in starch gel electrophoresis (92-94% of normal); increased Michaelis constant for glucoes-6-phosphate (60-70 muM) and NADP+ (20-25 muM); decreased inhibition constant by NADPH with respect to NADP+ (7 muM); increased inhibition by ATP; normal utilization of the substrate analogues; slightly biphasic pH curve; thermal instability, and normal activation energy of the enzymatic reaction. The relationships between the hematologic disorders (severe and frequent hemolytic crises) and the unfavorable kinetic modifications are discussed.

  5. Gd(-) Muret and gd(-) Colomiers, two new variants of glucose-6-phosphate dehydrogenase associated with favism.

    PubMed

    Vergnes, H; Ribet, A; Bommelaer, G; Amadieu, J; Brun, H

    1981-01-01

    Two males subjects are described with hitherto undescribed glucose-6-phosphate dehydrogenase (G6PD) variants. The first is of French ancestry, the second of Sicilian extraction. Each subject suffered from acute hemolytic anemia following ingestion of broad beans (Vicia fava). In both cases the hemolytic crisis occurred in a late period of life (29 and 58 years). No previous hemolytic crisis was recorded. The electrophoretic and kinetic properties of the mutant enzymes examined after purification from the red cells allowed each to be distinguished from other G6PD variants reported until now. The first variant was named Gd(-) Muret, the other Gd(-) Colomiers. PMID:7250973

  6. Gas Phase Spectra and Structural Determination of Glucose 6 Phosphate Using Cryogenic Ion Vibrational Spectroscopy

    NASA Astrophysics Data System (ADS)

    Kregel, Steven J.; Voss, Jonathan; Marsh, Brett; Garand, Etienne

    2014-06-01

    Glucose-6-Phosphate (G6P) is one member of a class of simple phosphorylated sugars that are relevant in biological processes. We have acquired a gas phase infrared spectrum of G6P- using cryogenic ion vibrational spectroscopy (CIVS) in a home-built spectrometer. The experimental spectrum was compared with calculated vibrational spectra from a systematic conformer search. For both of the α and β anomers, results show that only the lowest energy conformers are present in the gas phase. If spectral signatures for similar sugars could be cataloged, it would allow for conformer-specific determination of mixture composition, for example, for glycolyzation processes.

  7. Fed-Batch Production of Glucose 6-Phosphate Dehydrogenase Using Recombinant Saccharomyces cerevisiae

    NASA Astrophysics Data System (ADS)

    Das Neves, Luiz Carlos Martins; Pessoa, Adalberto; Vitolo, Michele

    The strain Saccharomyces cerevisiae W303-181, having the plasmid YEpPGK-G6P (built by coupling the vector YEPLAC 181 with the promoter phosphoglycerate kinase 1), was cultured by fed-batch process in order to evaluate its capability in the formation of glucose 6-phosphate dehydrogenase (EC.1.1.1.49). Two liters of culture medium (10.0 g/L glucose, 3.7 g/L yeast nitrogen broth (YNB), 0.02 g/L l-tryptophan, 0.02 g/L l-histidine, 0.02 g/L uracil, and 0.02 g/L adenine) were inoculated with 1.5 g dry cell/L and left fermenting in the batch mode at pH 5.7, aeration of 2.2 vvm, 30°C, and agitation of 400 rpm. After glucose concentration in the medium was lower than 1.0 g/L, the cell culture was fed with a solution of glucose (10.0 g/L) or micronutrients (l-tryptophan, l-histidine, uracil, and adenine each one at a concentration of 0.02 g/L) following the constant, linear, or exponential mode. The volume of the culture medium in the fed-batch process was varied from 2 L up to 3 L during 5 h. The highest glucose 6-phosphate dehydrogenase activity (350 U/L; 1 U=1 μmol of NADP/min) occurred when the glucose solution was fed into the fermenter through the decreasing linear mode.

  8. Effect of divalent metals on fungal and bacterial glucose-6-phosphate dehydrogenases

    SciTech Connect

    Jiang, W.; Niehaus, W.G.

    1986-05-01

    The authors have studied the effect of Zn/sup 2 +/ and Mg/sup 2 +/ on glucose-6-phosphate dehydrogenase purified from the fungi Aspergillus parasiticus, Alternaria alternata, Aphanomyces astaci, Saccharomyces cerevesiae, and Torula utilis, and from the bacteria Escherichia coli, Leuconostoc mesenteroides, and Bacillus stearothermophilus. Zn/sup 2 +/ reversibly inhibited the enzymes from A. parasiticus, S. cerevesiae, and T. utilis. Inhibition was competitive versus glucose-6-phosphate, with Ki = 25 ..mu..M, 75 ..mu..M, 25 ..mu..M, respectively. Zn/sup 2 +/ at 100 or 500 ..mu..M did not affect Vmax or Vmax/Km for the enzymes from A. alternata, A. astaci, L. mesenteroides, or B. stearothermophilus. Zn/sup 2 +/ caused loss of activity of the E. coli enzyme, which was not reversed by EDTA. Mg/sup 2 +/ stimulated both Vmax and Vmax/Km for all enzymes except that from A. astaci, on which it had no effect. Maximum stimulation occurred between 1 and 15 mM Mg/sup 2 +/ and ranged from 2 to 6-fold. For the enzymes from A. parasiticus, S. cerevesiae, and T. utilis, inclusion of 5 mM Mg/sup 2 +/ reversed the inhibition caused by 100 ..mu..M Zn/sup 2 +/.

  9. Cloning, expression, purification and characterization of his-tagged human glucose-6-phosphate dehydrogenase: a simplified method for protein yield.

    PubMed

    Gómez-Manzo, Saúl; Terrón-Hernández, Jessica; de la Mora-de la Mora, Ignacio; García-Torres, Itzhel; López-Velázquez, Gabriel; Reyes-Vivas, Horacio; Oria-Hernández, Jesús

    2013-10-01

    Glucose-6-phosphate dehydrogenase (G6PD) catalyzes the first step of the pentose phosphate pathway. In erythrocytes, the functionality of the pathway is crucial to protect these cells against oxidative damage. G6PD deficiency is the most frequent enzymopathy in humans with a global prevalence of 4.9 %. The clinical picture is characterized by chronic or acute hemolysis in response to oxidative stress, which is related to the low cellular activity of G6PD in red blood cells. The disease is heterogeneous at genetic level with around 160 mutations described, mostly point mutations causing single amino acid substitutions. The biochemical studies aimed to describe the detrimental effects of mutations on the functional and structural properties of human G6PD are indispensable to understand the molecular physiopathology of this disease. Therefore, reliable systems for efficient expression and purification of the protein are highly desirable. In this work, human G6PD was heterologously expressed in Escherichia coli and purified by immobilized metal affinity chromatography in a single chromatographic step. The structural and functional characterization indicates that His-tagged G6PD resembles previous preparations of recombinant G6PD. In contrast with previous protein yield systems, our method is based on commonly available resources and fully accessible laboratory equipment; therefore, it can be readily implemented.

  10. Sickle cell disease in Bahrain: coexistence and interaction with glucose-6-phosphate dehydrogenase (G6PD) deficiency.

    PubMed

    Mohammad, A M; Ardatl, K O; Bajakian, K M

    1998-04-01

    The object was to determine the frequency of glucose-6-phosphate dehydrogenase in Bahraini individuals with HbS as compared to those without HbS. Haemolysates of erythrocytes from 310 Bahraini individuals attending Health Centres were obtained, electrophoresed on cellulose acetate at PH 8.2-8.6, and stained for G6PD. HbS was present in 125 individuals (study group) and in 185 only HbA was present (control group). G6PD deficiency (very low to undetectable) was identified in 59 samples (47 per cent) of the study group and 35 (19 per cent) of the control group. A positive correlation between G6PD deficiency and HbS is present in Bahraini individuals tested. This is similar to the situation in the Eastern Province of Saudi Arabia. We speculate that the observation could be explained on the basis of historic endemicity of Falciparum malaria in both regions on the East coast of the Saudi Peninsula.

  11. Irradiation shortens the survival time of red cells deficient in glucose-6-phosphate dehydrogenasee

    SciTech Connect

    Westerman, M.P.; Wald, N.; Diloy-Puray, M.

    1980-03-01

    X radiation of glucose-6-phosphate dehydrogenase (G6PD)-deficient red cells causes distinct shortening of their survival time. This is accompanied by significant lowering of reduced glutathione content and is not observed in similarly prepared and treated normal cells. The damage is most likely related to irradiation-induced formation of activated oxygen products and to their subsequent effects on the cells. Neither methemoglobin increases nor Heinz body formation were observed, suggesting that hemolysis occurred prior to these changes. The study provides a model for examining the effects of irradiation and activated oxygen on red cells and suggests that patients with G6PD deficiency who receive irradiation could develop severe hemolysis in certain clinical settings.

  12. Glucose-6-phosphate dehydrogenase deficiency enhances human coronavirus 229E infection.

    PubMed

    Wu, Yi-Hsuan; Tseng, Ching-Ping; Cheng, Mei-Ling; Ho, Hung-Yao; Shih, Shin-Ru; Chiu, Daniel Tsun-Yee

    2008-03-15

    The host cellular environment is a key determinant of pathogen infectivity. Viral gene expression and viral particle production of glucose-6-phosphate dehydrogenase (G6PD)-deficient and G6PD-knockdown cells were much higher than their counterparts when human coronavirus (HCoV) 229E was applied at 0.1 multiplicity of infection. These phenomena were correlated with increased oxidant production. Accordingly, ectopic expression of G6PD in G6PD-deficient cells or addition of antioxidant (such as alpha-lipoic acid) to G6PD-knockdown cells attenuated the increased susceptibility to HCoV 229E infection. All experimental data indicated that oxidative stress in host cells is an important factor in HCoV 229E infectivity. PMID:18269318

  13. Molecular analysis of glucose-6-phosphate dehydrogenase variants in the Solomon Islands

    SciTech Connect

    Hirono, A.; Ishii, A.; Hirono, K.; Miwa, S.; Kere, N.; Fujii, H.

    1995-05-01

    Glucose-6-phosphate dehydrogenase (G6PD) deficiency is one of the most prevalent genetic disorders, and >100 million people are considered to have mutant genes. G6PD deficiency is frequent in the area where plasmodium falciparum infection is endemic, probably because the G6PD-deficient subjects are resistant to the parasite. Falciparum and vivax malarias have been highly endemic in the Solomon Islands, and a high frequency of G6PD deficiency has also been expected. A recent investigation showed that the frequency of G6PD deficiency in the Solomon Islands was 8.4%-14.4%. Although >80 G6PD variants from various populations have been molecularly analyzed, little is known about those in Melanesians. G6PD Maewo, which was originally found in Vanuatu, has so far been the only Melanesian variant whose structural abnormality was determined. 14 refs., 1 fig.

  14. Glucose-6-Phosphate Dehydrogenase of Trypanosomatids: Characterization, Target Validation, and Drug Discovery

    PubMed Central

    Gupta, Shreedhara; Igoillo-Esteve, Mariana; Michels, Paul A. M.; Cordeiro, Artur T.

    2011-01-01

    In trypanosomatids, glucose-6-phosphate dehydrogenase (G6PDH), the first enzyme of the pentosephosphate pathway, is essential for the defense of the parasite against oxidative stress. Trypanosoma brucei, Trypanosoma cruzi, and Leishmania mexicana G6PDHs have been characterized. The parasites' G6PDHs contain a unique 37 amino acid long N-terminal extension that in T. cruzi seems to regulate the enzyme activity in a redox-state-dependent manner. T. brucei and T. cruzi G6PDHs, but not their Leishmania spp. counterpart, are inhibited, in an uncompetitive way, by steroids such as dehydroepiandrosterone and derivatives. The Trypanosoma enzymes are more susceptible to inhibition by these compounds than the human G6PDH. The steroids also effectively kill cultured trypanosomes but not Leishmania and are presently considered as promising leads for the development of new parasite-selective chemotherapeutic agents. PMID:22091394

  15. Bilateral pulmonary edema after endoscopic sympathectomy in a patient with glucose-6-phosphate dehydrogenase deficiency.

    PubMed

    Lan, C J; Luk, H N; Wu, C T; Chang, W K; Tsou, M Y; Lui, P W; Lee, T Y

    2001-01-01

    Transaxillary endoscopic sympathectomy of thoracic ganglia (T2-T3) has recently gained wider acceptance as the treatment of choice for palmar hyperhidrosis. It requires one-lung ventilation to facilitate the surgery. One-lung ventilation, however, is not without complications, among which acute pulmonary edema has been reported. In this case report, we present a patient with palmar hyperhidrosis complicated by glucose-6-phosphate dehydrogenase (G-6-PD) deficiency, who received bilateral endoscopic sympathectomy under alternate one-lung anesthesia, and developed acute pulmonary edema immediately after recruitment of the successive collapsed lung. The effects of hypoxemia, G-6-PD deficiency and sympathectomy might all add to the development of acute pulmonary edema secondary to reexpansion of each individual lung after alternate one-lung ventilation. The possibilities of the inferred causes are herein discussed. PMID:11152024

  16. Production of glucose-6-phosphate by glucokinase coupled with an ATP regeneration system.

    PubMed

    Yan, Bingkun; Ding, Qingbao; Ou, Ling; Zou, Zhi

    2014-03-01

    A process of glucose-6-phosphate (G-6-P) production coupled with an adenosine triphosphate (ATP) regeneration system was constructed that utilized acetyl phosphate (ACP) via acetate kinase (ACKase). The genes glk and ack from Escherichia coli K12 were amplified and cloned into pET-28a(+), then transformed into E. coli BL21 (DE3) and the recombinant strains were named pGLK and pACK respectively. Glucokinase (glkase) in pGLK and ACKase in pACK were both overexpressed in soluble form. G-6-P was efficiently produced from glucose and ACP using a very small amount of ATP. The conversion yield was greater than 97 % when the reaction solution containing 10 mM glucose, 20 mM ACP-Na₂, 0.5 mM ATP, 5 mM Mg²⁺, 50 mM potassium phosphate buffer (pH 7.0), 4.856 U glkase and 3.632 U ACKase were put into 37 °C water bath for 1 h. PMID:24165747

  17. Metabolism of tritiated D-glucose in rat erythrocytes

    SciTech Connect

    Manuel y Keenoy, B.; Malaisse-Lagae, F.; Malaisse, W.J. )

    1991-09-01

    The metabolism of D-(U-14C)glucose, D-(1-14C)glucose, D-(6-14C)glucose, D-(1-3H)glucose, D-(2-3H)glucose, D-(3-3H)glucose, D-(3,4-3H)glucose, D-(5-3H)glucose, and D-(6-3H)glucose was examined in rat erythrocytes. There was a fair agreement between the rate of 3HOH production from either D-(3-3H)glucose and D-(5-3H)glucose, the decrease in the 2,3-diphosphoglycerate pool, its fractional turnover rate, the production of 14C-labeled lactate from D-(U-14C)glucose, and the total lactate output. The generation of both 3HOH and tritiated acidic metabolites from D-(3,4-3H)glucose indicated incomplete detritiation of the C4 during interconversion of fructose-1,6-bisphosphate and triose phosphates. Erythrocytes unexpectedly generated 3HOH from D-(6-3H)glucose, a phenomenon possibly attributable to the detritiation of (3-3H)pyruvate in the reaction catalyzed by glutamate pyruvate transaminase. The production of 3HOH from D-(2-3H)glucose was lower than that from D-(5-3H)glucose, suggesting enzyme-to-enzyme tunneling of glycolytic intermediates in the hexokinase/phosphoglucoisomerase/phosphofructokinase sequence. The production of 3HOH from D-(1-3H)glucose largely exceeded that of 14CO2 from D-(1-14C)glucose, a situation tentatively ascribed to the generation of 3HOH in the phosphomannoisomerase reaction. It is further speculated that the adjustment in specific radioactivity of D-(1-3H)glucose-6-phosphate cannot simultaneously match the vastly different degrees of isotopic discrimination in velocity at the levels of the reactions catalyzed by either glucose-6-phosphate dehydrogenase or phosphoglucoisomerase. The interpretation of the present findings thus raises a number of questions, which are proposed as a scope for further investigations.

  18. An enzymatic fluorimetric assay for glucose-6-phosphate: application in an in vitro Warburg-like effect

    PubMed Central

    Zhu, Aiping; Romero, Roberto; Petty, Howard R.

    2009-01-01

    Recently, there has been a resurgence of interest in the regulatory role of cell metabolism in tumor biology and immunology. To assess changes in metabolite levels in cell populations and tissues, especially from small clinical samples, highly sensitive assays are required. Based upon glucose 6-phosphate’s reaction and the diaphorase-resazurin amplifying system, we have developed a fluorescence methodology to measure glucose 6-phosphate concentrations in cell extracts. In this approach, glucose 6-phosphate is oxidized by glucose-6-phosphate dehydrogenase in the presence of NADP+, and the stoichiometrically generated NADPH is then amplified by the diaphorase-cycling system to produce a highly fluorescent molecule - resorufin. The limit of detection (LOD) of the assay is 10 pmol. The assay has a Z′ factor of 0.81. Its usefulness is demonstrated by experiments in which the pyruvate kinase inhibitor, phenylalanine, is added to cells. After 2 hours incubation at 37°C, glucose-6-phosphate levels rose by 20%, thus illustrating an in vitro Warburg-like effect on cell metabolism. PMID:19454216

  19. Perioperative management of the glucose-6-phosphate dehydrogenase deficient patient: a review of literature.

    PubMed

    Elyassi, Ali R; Rowshan, Henry H

    2009-01-01

    Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common enzymatic disorder of red blood cells in humans. It is estimated that about 400 million people are affected by this deficiency. The G6PD enzyme catalyzes the first step in the pentose phosphate pathway, leading to antioxidants that protect cells against oxidative damage. A G6PD-deficient patient, therefore, lacks the ability to protect red blood cells against oxidative stresses from certain drugs, metabolic conditions, infections, and ingestion of fava beans. The following is a literature review, including disease background, pathophysiology, and clinical implications, to help guide the clinician in management of the G6PD-deficient patient. A literature search was conducted in the following databases: PubMed, The Cochrane Library, Web of Science, OMIM, and Google; this was supplemented by a search for selected authors. Keywords used were glucose-6-phosphate dehydrogenase (G6PD) deficiency, anesthesia, analgesia, anxiolysis, management, favism, hemolytic anemia, benzodiazepines, codeine, codeine derivatives, ketamine, barbiturates, propofol, opioids, fentanyl, and inhalation anesthetics. Based on titles and abstracts, 23 papers and 1 website were identified. The highest prevalence of G6PD is reported in Africa, southern Europe, the Middle East, Southeast Asia, and the central and southern Pacific islands; however, G6PD deficiency has now migrated to become a worldwide disease. Numerous drugs, infections, and metabolic conditions have been shown to cause acute hemolysis of red blood cells in the G6PD-deficient patient, with the rare need for blood transfusion. Benzodiazepines, codeine/codeine derivatives, propofol, fentanyl, and ketamine were not found to cause hemolytic crises in the G6PD-deficient patient. The most effective management strategy is to prevent hemolysis by avoiding oxidative stressors. Thus, management for pain and anxiety should include medications that are safe and have not been

  20. Glucose-6-Phosphate Dehydrogenase Deficiency Improves Insulin Resistance With Reduced Adipose Tissue Inflammation in Obesity.

    PubMed

    Ham, Mira; Choe, Sung Sik; Shin, Kyung Cheul; Choi, Goun; Kim, Ji-Won; Noh, Jung-Ran; Kim, Yong-Hoon; Ryu, Je-Won; Yoon, Kun-Ho; Lee, Chul-Ho; Kim, Jae Bum

    2016-09-01

    Glucose-6-phosphate dehydrogenase (G6PD), a rate-limiting enzyme of the pentose phosphate pathway, plays important roles in redox regulation and de novo lipogenesis. It was recently demonstrated that aberrant upregulation of G6PD in obese adipose tissue mediates insulin resistance as a result of imbalanced energy metabolism and oxidative stress. It remains elusive, however, whether inhibition of G6PD in vivo may relieve obesity-induced insulin resistance. In this study we showed that a hematopoietic G6PD defect alleviates insulin resistance in obesity, accompanied by reduced adipose tissue inflammation. Compared with wild-type littermates, G6PD-deficient mutant (G6PD(mut)) mice were glucose tolerant upon high-fat-diet (HFD) feeding. Intriguingly, the expression of NADPH oxidase genes to produce reactive oxygen species was alleviated, whereas that of antioxidant genes was enhanced in the adipose tissue of HFD-fed G6PD(mut) mice. In diet-induced obesity (DIO), the adipose tissue of G6PD(mut) mice decreased the expression of inflammatory cytokines, accompanied by downregulated proinflammatory macrophages. Accordingly, macrophages from G6PD(mut) mice greatly suppressed lipopolysaccharide-induced proinflammatory signaling cascades, leading to enhanced insulin sensitivity in adipocytes and hepatocytes. Furthermore, adoptive transfer of G6PD(mut) bone marrow to wild-type mice attenuated adipose tissue inflammation and improved glucose tolerance in DIO. Collectively, these data suggest that inhibition of macrophage G6PD would ameliorate insulin resistance in obesity through suppression of proinflammatory responses. PMID:27284106

  1. Trehalose Phosphate Synthesis in Streptomyces hygroscopicus: Purification of Guanosine Diphosphate d-Glucose: d-Glucose-6-Phosphate 1-Glucosyl-Transferase

    PubMed Central

    Elbein, Alan D.

    1968-01-01

    Guanosine diphosphate d-glucose:d-glucose-6-phosphate 1-glucosyl-transferase was purified approximately 100-fold from extracts of Streptomyces hygroscopicus. The purified enzyme catalyzed the transfer of glucose from guanosine diphosphate-d-glucose to glucose-6-phosphate to form trehalose phosphate and guanosine diphosphate. The enzyme was specific for these two substrates and was stimulated by the addition of magnesium ions. The product was characterized as α-α-trehalose-6-phosphate by its physical and chemical properties. The enzyme was present in a large number of Streptomyces species, suggesting that this group of organisms synthesized trehalose phosphate in a unique manner. This enzyme was not detected in fungi, since these organisms utilized uridine diphosphate-d-glucose as the glucosyl donor. PMID:5726304

  2. Four transcripts encode glucose 6-phosphate dehydrogenase (G6PDH) in the Southern cattle tick, Rhipicephalus (Boophilus) microplus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Glucose 6-phosphate dehydrogenase (G6PDH) is an enzyme that plays a critical role in the production of NADPH. Here we describe the characterization of four transcripts (G6PDH-A, -B, -C, and -D) that putatively encode the enzyme in the southern cattle tick, Rhipicephalus (Boophilus) microplus. The ...

  3. Resistance of glucose-6-phosphate dehydrogenase deficiency to malaria: effects of fava bean hydroxypyrimidine glucosides on Plasmodium falciparum growth in culture and on the phagocytosis of infected cells.

    PubMed

    Ginsburg, H; Atamna, H; Shalmiev, G; Kanaani, J; Krugliak, M

    1996-07-01

    The balanced polymorphism of glucose-6-phosphate dehydrogenase deficiency (G6PD-) is believed to have evolved through the selective pressure of malarial combined with consumption of fava beans. The implicated fava bean constituents are the hydroxypyrimidine glucosides vicine and convicine, which upon hydrolysis of their beta-O-glucosidic bond, became protein pro-oxidants. In this work we show that the glucosides inhibit the growth of Plasmodium falciparum, increase the hexose-monophosphate shunt activity and the phagocytosis of malaria-infected erythrocytes. These activities are exacerbated in the presence of beta-glucosidase, implicating their pro-oxidant aglycones in the toxic effect, and are more pronounced in infected G6PD- erythrocytes. These results suggest that G6PD- infected erythrocytes are more susceptible to phagocytic cells, and that fava bean pro-oxidants are more efficiently suppressing parasite propagation in G6PD- erythrocytes, either by directly affecting parasite growth, or by means of enhanced phagocytic elimination of infected cells. The present findings could account for the relative resistance of G6PD- bearers to falciparum malaria, and establish a link between dietary habits and malaria in the selection of the G6PD- genotype.

  4. Resistance of glucose-6-phosphate dehydrogenase deficiency to malaria: effects of fava bean hydroxypyrimidine glucosides on Plasmodium falciparum growth in culture and on the phagocytosis of infected cells.

    PubMed

    Ginsburg, H; Atamna, H; Shalmiev, G; Kanaani, J; Krugliak, M

    1996-07-01

    The balanced polymorphism of glucose-6-phosphate dehydrogenase deficiency (G6PD-) is believed to have evolved through the selective pressure of malarial combined with consumption of fava beans. The implicated fava bean constituents are the hydroxypyrimidine glucosides vicine and convicine, which upon hydrolysis of their beta-O-glucosidic bond, became protein pro-oxidants. In this work we show that the glucosides inhibit the growth of Plasmodium falciparum, increase the hexose-monophosphate shunt activity and the phagocytosis of malaria-infected erythrocytes. These activities are exacerbated in the presence of beta-glucosidase, implicating their pro-oxidant aglycones in the toxic effect, and are more pronounced in infected G6PD- erythrocytes. These results suggest that G6PD- infected erythrocytes are more susceptible to phagocytic cells, and that fava bean pro-oxidants are more efficiently suppressing parasite propagation in G6PD- erythrocytes, either by directly affecting parasite growth, or by means of enhanced phagocytic elimination of infected cells. The present findings could account for the relative resistance of G6PD- bearers to falciparum malaria, and establish a link between dietary habits and malaria in the selection of the G6PD- genotype. PMID:8710417

  5. Detection of Occult Acute Kidney Injury in Glucose-6-Phosphate Dehydrogenase Deficiency Anemia

    PubMed Central

    Abdel Hakeem, Gehan Lotfy; Abdel Naeem, Emad Allam; Swelam, Salwa Hussein; El Morsi Aboul Fotoh, Laila; El Mazary, Abdel Azeem Mohamed; Abdel Fadil, Ashraf Mohamed; Abdel Hafez, Asmaa Hosny

    2016-01-01

    Background Glucose-6-phosphate dehydrogenase (G6PD) deficiency anemia is associated with intravascular hemolysis. The freely filtered hemoglobin can damage the kidney. We aimed to assess any subclinical renal injury in G6PD children. Methods Sixty children were included. Thirty G6PD deficiency anemia children were enrolled during the acute hemolytic crisis and after the hemolytic episode had elapsed. Another thirty healthy children were included as controls. Serum cystatin C, creatinine levels, and urinary albumin/creatinine (A/C) ratio were measured, and the glomerular filtration rate (GFR) was calculated. Results Significantly higher urinary A/C ratio (p=0.001,0.002 respectively) and lower GFR (p=0.001 for both) were found during hemolysis and after the hemolytic episode compared to the controls. Also, significant higher serum cystatin C (p=0.001), creatinine (p=0.05) and A/C (p= 0.001) ratio and insignificant lower GFR (p=0.3) during acute hemolytic crisis compared to the same children after the hemolytic episode subsided. Conclusions G6PD deficiency anemia is associated with a variable degree of acute renal injury during acute hemolytic episodes which may persist after elapsing of the hemolytic crises. PMID:27648201

  6. Molecular characterization of a German variant of glucose-6-phosphate dehydrogenase deficiency (G6PD Aachen).

    PubMed

    Efferth, T; Osieka, R; Beutler, E

    2000-02-01

    Glucose-6-phosphate dehydrogenase (G6PD) deficiency is an X-chromosome-linked hereditary disorder. Clinically, patients with G6PD deficiency often present with drug- or food-induced hemolytic crises or neonatal jaundice. G6PD is involved in the generation of NADPH and reduced glutathione. In contrast to American, Mediterranean, and African ancestries, only few variants are known from Middle and Northern Europe. We describe the molecular characterization of a distinct variant from the northwestern area of Germany, G6PD Aachen. The sequence of the G6PD gene from three afflicted males was found to be hemizygous at cDNA residue 1089 for a C-->G mutation with a predicted amino acid change of Asn363Lys. The 1089 C-->G point mutation is unique, but produces the identical amino acid change found in a Mexican variant of G6PD deficiency, G6PD Loma Linda. This G6PD-deficient variant is caused by a 1089 C-->A mutation. The 363-amino-acid replacement is located outside a known mutation cluster region between amino acid residues 380 and 450, but may disrupt or weaken dimer interactions of G6PD enzyme subunits.

  7. Glucose-6-Phosphate Dehydrogenase Deficiency among Male Blood Donors in Sana’a City, Yemen

    PubMed Central

    Al-Nood, Hafiz A.; Bazara, Fakiha A.; Al-Absi, Rashad; Habori, Molham AL

    2012-01-01

    Objectives To determine the prevalence of Glucose-6-phosphate dehydrogenase (G-6-PD) deficiency among Yemeni people from different regions of the country living in the capital city, Sana’a, giving an indication of its overall prevalence in Yemen. Methods A cross-sectional study was conducted among Yemeni male blood donors attending the Department of Blood Bank at the National Centre of the Public Health Laboratories in the capital city, Sana’a, Yemen. Fluorescent spot method was used for screening, spectrophotometeric estimation of G-6-PD activity and separation by electrophoresis was done to determine the G-6-PD phenotype. Results Of the total 508 male blood donors recruited into the study, 36 were G-6-PD deficient, giving a likely G-6-PD deficiency prevalence of 7.1%. None of these deficient donors had history of anemia or jaundice. Thirty-five of these deficient cases (97.2%) showed severe G-6-PD deficiency class II (<10% of normal activity), and their phenotyping presumptively revealed a G-6-PD-Mediterranean variant. Conclusion The results showed a significant presence of G-6-PD deficiency with predominance of a severe G-6-PD deficiency type in these blood donors in Sana’a City, which could represent an important health problem through occurrence of hemolytic anemia under oxidative stress. A larger sample size is needed to determine the overall prevalence of G-6-PD deficiency, and should be extended to include DNA analysis to identify its variants in Yemen. PMID:22359725

  8. Aldosterone impairs vascular reactivity by decreasing glucose-6-phosphate dehydrogenase activity

    PubMed Central

    Leopold, Jane A.; Dam, Aamir; Maron, Bradley A.; Scribner, Anne W.; Liao, Ronglih; Handy, Diane E.; Stanton, Robert C.; Pitt, Bertram; Loscalzo, Joseph

    2013-01-01

    Hyperaldosteronism is associated with impaired vascular reactivity; however, the mechanism by which aldosterone promotes endothelial dysfunction remains unknown. Glucose-6-phosphate dehydrogenase (G6pd), the principal source of Nadph, modulates vascular function by limiting oxidant stress to preserve bioavailable nitric oxide (NO•). In these studies, we show that aldosterone (10−9-10−7 mol/l) decreases endothelial G6pd expression and activity in vitro resulting in increased oxidant stress and decreased cGMP levels similar to what is observed in G6pd-deficient cells. Aldosterone decreases G6pd expression by protein kinase A activation to increase expression of Crem, which interferes with Creb binding to the G6pd promoter. In vivo, infusion of aldosterone decreases vascular G6pd expression and impairs vascular reactivity. These effects are abrogated by spironolactone or vascular gene transfer of G6pd. These studies demonstrate that aldosterone induces a G6pd-deficient phenotype to impair endothelial function; aldosterone antagonism or gene transfer of G6pd improves vascular reactivity by restoring G6pd activity. PMID:17273168

  9. A hemolysis trigger in glucose-6-phosphate dehydrogenase enzyme deficiency. Vicia sativa (Vetch).

    PubMed

    Bicakci, Zafer

    2009-02-01

    Glucose-6-phosphate dehydrogenase (G6PD) is an enzyme, playing an important role in the redox metabolism of all aerobic cells. It was reported that certain medications, fava beans, and infections can trigger acute hemolytic anemia in patients with G6PD deficiency. An 8-year-old male patient was admitted to the hospital with blood in the urine, headache, dizziness, fatigue, loss of appetite, and jaundice in the eyes, 24 hours after eating large amounts of fresh, vetch grains. Laboratory investigation revealed hemolytic anemia, hyperbilirubinemia, and G6PD deficiency. Approximately 0.5% of fava bean seeds have 2 pyrimidine beta-glycosides called, vicine and convicine. Vetch has 0.731% vicine, 0.081% convicine, and 0.530% beta cyanoalanine glycosides. The aim of this case report is to emphasize the importance of vetch seeds as a cause for hemolytic crisis in our country, where approximately one million tons of vetch is produced per year, especially in the agricultural regions.

  10. Expression and characterization of a cytosolic glucose 6 phosphate dehydrogenase isoform from barley (Hordeum vulgare) roots.

    PubMed

    Castiglia, Daniela; Cardi, Manuela; Landi, Simone; Cafasso, Donata; Esposito, Sergio

    2015-08-01

    In plant cells, glucose 6 phosphate dehydrogenase (G6PDH-EC 1.1.1.49) regulates the oxidative pentose phosphate pathway (OPPP), a metabolic route involved in the production of NADPH for various biosynthetic processes and stress response. In this study, we report the overexpression of a cytosolic G6PDH isoform from barley (Hordeum vulgare) roots in bacteria, and the biochemical characterization of the purified recombinant enzyme (HvCy-G6PDH). A full-length cDNA coding for a cytosolic isoform of G6PDH was isolated, and the sequence was cloned into pET3d vector; the protein was overexpressed in Escherichia coli BL21 (DE3) and purified by anion exchange and affinity chromatography. The kinetic properties were calculated: the recombinant HvCy-G6PDH showed KMs and KINADPH comparable to those observed for the enzyme purified from barley roots; moreover, the analysis of NADPH inhibition suggested a competitive mechanism. Therefore, this enzyme could be utilised for the structural and regulatory characterization of this isoform in higher plants.

  11. Enhanced expression of glucose-6-phosphate dehydrogenase in human cells sustaining oxidative stress.

    PubMed Central

    Ursini, M V; Parrella, A; Rosa, G; Salzano, S; Martini, G

    1997-01-01

    Recent reports have demonstrated that glucose-6-phosphate dehydrogenase (G6PD) activity in mammalian cells is necessary in order to ensure cell survival when damage is produced by reactive oxygen intermediates. In this paper we demonstrate that oxidative stress, caused by agents acting at different steps in the biochemical pathway controlling the intracellular redox status, determines the increase in G6PD-specific activity in human cell lines of different tissue origins. The intracellular level of G6PD-specific mRNA also increases, with kinetics compatible with the induction of new enzyme synthesis. We carried out experiments in which cells were exposed to oxidative stress in the presence of inhibitors of protein or RNA synthesis. These demonstrated that increased G6PD expression is mainly due to an increased rate of transcription, with a minor but significant contribution of regulatory mechanisms acting at post-transcriptional levels. These results provide new information on the defence systems that eukaryotic cells possess in order to prevent damage caused by potentially harmful oxygen derivatives. PMID:9169615

  12. Abscisic acid effects on activity and expression of barley (Hordeum vulgare) plastidial glucose-6-phosphate dehydrogenase

    PubMed Central

    Cardi, Manuela; Chibani, Kamel; Cafasso, Donata; Rouhier, Nicolas; Jacquot, Jean-Pierre; Esposito, Sergio

    2011-01-01

    Total glucose-6-phosphate dehydrogenase (G6PDH) activity, protein abundance, and transcript levels of G6PDH isoforms were measured in response to exogenous abscisic acid (ABA) supply to barley (Hordeum vulgare cv Nure) hydroponic culture. Total G6PDH activity increased by 50% in roots treated for 12 h with exogenous 0.1 mM ABA. In roots, a considerable increase (35%) in plastidial P2-G6PDH transcript levels was observed during the first 3 h of ABA treatment. Similar protein variations were observed in immunoblotting analyses. In leaves, a 2-fold increase in total G6PDH activity was observed after ABA treatment, probably related to an increase in the mRNA level (increased by 50%) and amount of protein (increased by 85%) of P2-G6PDH. Together these results suggest that the plastidial P2-isoform plays an important role in ABA-treated barley plants. PMID:21464159

  13. Detection of Occult Acute Kidney Injury in Glucose-6-Phosphate Dehydrogenase Deficiency Anemia

    PubMed Central

    Abdel Hakeem, Gehan Lotfy; Abdel Naeem, Emad Allam; Swelam, Salwa Hussein; El Morsi Aboul Fotoh, Laila; El Mazary, Abdel Azeem Mohamed; Abdel Fadil, Ashraf Mohamed; Abdel Hafez, Asmaa Hosny

    2016-01-01

    Background Glucose-6-phosphate dehydrogenase (G6PD) deficiency anemia is associated with intravascular hemolysis. The freely filtered hemoglobin can damage the kidney. We aimed to assess any subclinical renal injury in G6PD children. Methods Sixty children were included. Thirty G6PD deficiency anemia children were enrolled during the acute hemolytic crisis and after the hemolytic episode had elapsed. Another thirty healthy children were included as controls. Serum cystatin C, creatinine levels, and urinary albumin/creatinine (A/C) ratio were measured, and the glomerular filtration rate (GFR) was calculated. Results Significantly higher urinary A/C ratio (p=0.001,0.002 respectively) and lower GFR (p=0.001 for both) were found during hemolysis and after the hemolytic episode compared to the controls. Also, significant higher serum cystatin C (p=0.001), creatinine (p=0.05) and A/C (p= 0.001) ratio and insignificant lower GFR (p=0.3) during acute hemolytic crisis compared to the same children after the hemolytic episode subsided. Conclusions G6PD deficiency anemia is associated with a variable degree of acute renal injury during acute hemolytic episodes which may persist after elapsing of the hemolytic crises.

  14. Genetic heterogeneity of glucose-6-phosphate dehydrogenase deficiency in south-east Sicily.

    PubMed

    Cittadella, R; Civitelli, D; Manna, I; Azzia, N; Di Cataldo, A; Schilirò, G; Brancati, C

    1997-05-01

    In order to explore the nature of glucose-6-phosphate dehydrogenase (G6PD) deficiency in south-east Sicily, we have analysed the G6PD gene in 25 unrelated males with abnormal G6PD activity and/or electrophoretic mobility, by using the analysis of the appropriate PCR-amplified fragment of DNA and subsequent digestion by appropriate restriction-enzymes, looking for the presence of certain known G6PD mutations. We amplified the entire G6PD coding sequence into eight fragments, followed by single-strand conformation polymorphism (SSCP) analysis and sequencing of those individual fragments that were found to be abnormal by SSCP. Through these methods we found a total of twelve G6PD Mediterranean variants with the association of a silent mutation 1311 (also known as polymorphic site Bcl I), one G6PD Mediterranean without this association, four G6PD A-Val 68 and two G6PD Santamaria and five G6PD Chatham. In a subject with normal activity a mutation was found in exon 5, designated as G6PD Sao Borja. This is the first report on the molecular analysis of G6PD mutations in Sicily and we have obtained evidence for four distinct classes of variants.

  15. Nitrogen Assimilation, Abiotic Stress and Glucose 6-Phosphate Dehydrogenase: The Full Circle of Reductants

    PubMed Central

    Esposito, Sergio

    2016-01-01

    Glucose 6 phosphate dehydrogenase (G6PDH; EC 1.1.1.49) is well-known as the main regulatory enzyme of the oxidative pentose phosphate pathway (OPPP) in living organisms. Namely, in Planta, different G6PDH isoforms may occur, generally localized in cytosol and plastids/chloroplasts. These enzymes are differently regulated by distinct mechanisms, still far from being defined in detail. In the last decades, a pivotal function for plant G6PDHs during the assimilation of nitrogen, providing reductants for enzymes involved in nitrate reduction and ammonium assimilation, has been described. More recently, several studies have suggested a main role of G6PDH to counteract different stress conditions, among these salinity and drought, with the involvement of an ABA depending signal. In the last few years, this recognized vision has been greatly widened, due to studies clearly showing the non-conventional subcellular localization of the different G6PDHs, and the peculiar regulation of the different isoforms. The whole body of these considerations suggests a central question: how do the plant cells distribute the reductants coming from G6PDH and balance their equilibrium? This review explores the present knowledge about these mechanisms, in order to propose a scheme of distribution of reductants produced by G6PDH during nitrogen assimilation and stress. PMID:27187489

  16. A hemolysis trigger in glucose-6-phosphate dehydrogenase enzyme deficiency. Vicia sativa (Vetch).

    PubMed

    Bicakci, Zafer

    2009-02-01

    Glucose-6-phosphate dehydrogenase (G6PD) is an enzyme, playing an important role in the redox metabolism of all aerobic cells. It was reported that certain medications, fava beans, and infections can trigger acute hemolytic anemia in patients with G6PD deficiency. An 8-year-old male patient was admitted to the hospital with blood in the urine, headache, dizziness, fatigue, loss of appetite, and jaundice in the eyes, 24 hours after eating large amounts of fresh, vetch grains. Laboratory investigation revealed hemolytic anemia, hyperbilirubinemia, and G6PD deficiency. Approximately 0.5% of fava bean seeds have 2 pyrimidine beta-glycosides called, vicine and convicine. Vetch has 0.731% vicine, 0.081% convicine, and 0.530% beta cyanoalanine glycosides. The aim of this case report is to emphasize the importance of vetch seeds as a cause for hemolytic crisis in our country, where approximately one million tons of vetch is produced per year, especially in the agricultural regions. PMID:19198723

  17. Glucose-6-phosphate dehydrogenase deficiency A- variant in febrile patients in Haiti.

    PubMed

    Carter, Tamar E; Maloy, Halley; von Fricken, Michael; St Victor, Yves; Romain, Jean R; Okech, Bernard A; Mulligan, Connie J

    2014-08-01

    Haiti is one of two remaining malaria-endemic countries in the Caribbean. To decrease malaria transmission in Haiti, primaquine was recently added to the malaria treatment public health policy. One limitation of primaquine is that, at certain doses, primaquine can cause hemolytic anemia in individuals with glucose-6-phosphate dehydrogenase (G6PD) deficiency (G6PDd). In this study, we genotyped two mutations (A376G and G202A), which confer the most common G6PDd variant in West African populations, G6PDd A-. We estimated the frequency of G6PDd A- in a sample of febrile patients enrolled in an on-going malaria study who represent a potential target population for a primaquine mass drug administration. We found that 33 of 168 individuals carried the G6PDd A- allele (includes A- hemizygous males, A- homozygous or heterozygous females) and could experience toxicity if treated with primaquine. These data inform discussions on safe and effective primaquine dosing and future malaria elimination strategies for Haiti.

  18. Glucose-6-phosphate dehydrogenase is the target for the trypanocidal action of human steroids.

    PubMed

    Gupta, Shreedhara; Cordeiro, Artur T; Michels, Paul A M

    2011-04-01

    Steroids such as dehydroepiandrosterone (DHEA) and epiandrosterone (EA) exert multiple effects in mammals including the inhibition of glucose-6-phosphate dehydrogenase (G6PDH). Initially, the inhibition was considered specific for the mammalian enzyme. The beneficial effect of these steroids on infections by protists and nematodes was attributed to stimulation of the immune system. However, we showed previously that DHEA and EA also inhibit Trypanosoma brucei and T. cruzi G6PDH, with low micromolar K(i)' values, but not the enzyme from Leishmania species, and kill in vitro cultured trypanosomes. We report here that, contrary to wild-type trypanosomes, mutant bloodstream-form T. brucei cells expressing L. mexicana G6PDH are not susceptible to the steroids, proving that G6PDH is the in situ target. Moreover, bromo-derivatives of the steroids show 50-100 fold lower K(i)' values for the enzyme and display an increased potency to kill the parasites. Therefore, the compounds offer promise for use in development of parasite-selective drugs.

  19. Nitrogen Assimilation, Abiotic Stress and Glucose 6-Phosphate Dehydrogenase: The Full Circle of Reductants.

    PubMed

    Esposito, Sergio

    2016-01-01

    Glucose 6 phosphate dehydrogenase (G6PDH; EC 1.1.1.49) is well-known as the main regulatory enzyme of the oxidative pentose phosphate pathway (OPPP) in living organisms. Namely, in Planta, different G6PDH isoforms may occur, generally localized in cytosol and plastids/chloroplasts. These enzymes are differently regulated by distinct mechanisms, still far from being defined in detail. In the last decades, a pivotal function for plant G6PDHs during the assimilation of nitrogen, providing reductants for enzymes involved in nitrate reduction and ammonium assimilation, has been described. More recently, several studies have suggested a main role of G6PDH to counteract different stress conditions, among these salinity and drought, with the involvement of an ABA depending signal. In the last few years, this recognized vision has been greatly widened, due to studies clearly showing the non-conventional subcellular localization of the different G6PDHs, and the peculiar regulation of the different isoforms. The whole body of these considerations suggests a central question: how do the plant cells distribute the reductants coming from G6PDH and balance their equilibrium? This review explores the present knowledge about these mechanisms, in order to propose a scheme of distribution of reductants produced by G6PDH during nitrogen assimilation and stress. PMID:27187489

  20. Testis-specific expression of a functional retroposon encoding glucose-6-phosphate dehydrogenase in the mouse

    SciTech Connect

    Hendriksen, P.J.M. |; Hoogerbrugge, J.W.; Baarends, W.M.

    1997-05-01

    The X-chromosomal gene glucose-6-phosphate dehydrogenase (G6pd) is known to be expressed in most cell types of mammalian species. In the mouse, we have detected a novel gene, designated G6pd-2, encoding a G6PD isoenzyme. G6pd-2 does not contain introns and appears to represent a retroposed gene. This gene is uniquely transcribed in postmeiotic spermatogenic cells in which the X-encoded G6pd gene is not transcribed. Expression of the G6pd-2 sequence in a bacterial system showed that the encoded product is an active enzyme. Zymogramic analysis demonstrated that recombinant G6PD-2, but not recombinant G6PD-1 (the X-chromosome-encoded G6PD), formed tetramers under reducing conditions. Under the same conditions, G6PD tetramers were also found in extracts of spermatids and spermatozoa, indicating the presence of G6pd-2-encoded isoenzyme in these cell types. G6pd-2 is one of the very few known expressed retroposons encoding a functional protein, and the presence of this gene is probably related to X chromosome inactivation during spermatogenesis. 62 refs., 7 figs.

  1. [Glucose-6-phosphate dehydrogenase (G6PD) deficiency--a cause of anaemia in pregnant women].

    PubMed

    Kuliszkiewicz-Janus, Małgorzata; Zimny, Anna

    2003-11-01

    Glucose-6-phosphate dehydrogenase (G6PD) is one of the most important cytoprotective enzymes for oxidative stress. The WHO classification of G6PD deficiency, based on enzyme activity and clinical significance, distinguishes five variants. Chronic haemolytic process is rare and the main factors causing haemolysis are: infections, substances derived from plants, drugs with high oxidation-reduction potential, stress, ketoacidosis in diabetes and surgery operations. We report two cases of women belonging to the class 3 of the WHO classification in whom haemolysis occured during pregnancy. One of the patients developed two incidents of haemolytic anaemia. The cause of the first episode, nine months before pregnancy, was probably infection of the urinary tract caused by Escherichia coli, but the influence of the drugs also cannot be excluded. Because of the genetic background of this enzymopathy we also examined members of the patients, families but did not find any evidence of G6PD deficiency among them. The reported cases indicate that haemolytic anaemia caused by G6PD deficiency may occur during pregnancy what can lead to many not only haematological but also serious obstetrical complications such as infertility, fetus malformations and even its death. We also draw attention to several difficulties in diagnosing G6PD deficiency especially during haemolysis. PMID:16737003

  2. Glucose-6-Phosphate Dehydrogenase-Deficiency in Transfusion Medicine: The Unknown Risks

    PubMed Central

    Francis, Richard O.; Jhang, Jeffrey S.; Pham, Huy P.; Hod, Eldad A.; Zimring, James C.; Spitalnik, Steven L.

    2013-01-01

    The hallmark of glucose-6-phosphate dehydrogenase (G6PD) deficiency is red blood cell (RBC) destruction in response to oxidative stress. Patients requiring RBC transfusions may simultaneously receive oxidative medications or have concurrent infections, both of which can induce hemolysis in G6PD-deficient RBCs. Although it is not routine practice to screen healthy blood donors for G6PD deficiency, case reports identified transfusion of G6PD-deficient RBCs as causing hemolysis and other adverse events. In addition, some patient populations may be more at risk for complications associated with transfusions of G6PD-deficient RBCs because they receive RBCs from donors who are more likely to have G6PD deficiency. This review discusses G6PD deficiency, its importance in transfusion medicine, changes in the RBC antioxidant system (of which G6PD is essential) during refrigerated storage, and mechanisms of hemolysis. In addition, as yet unanswered questions that could be addressed by translational and clinical studies are identified and discussed. PMID:23815264

  3. Trehalose 6-phosphate regulates starch synthesis via posttranslational redox activation of ADP-glucose pyrophosphorylase.

    PubMed

    Kolbe, Anna; Tiessen, Axel; Schluepmann, Henriette; Paul, Matthew; Ulrich, Silke; Geigenberger, Peter

    2005-08-01

    Trehalose is the most widespread disaccharide in nature, occurring in bacteria, fungi, insects, and plants. Its precursor, trehalose 6-phosphate (T6P), is also indispensable for the regulation of sugar utilization and growth, but the sites of action are largely unresolved. Here we use genetic and biochemical approaches to investigate whether T6P acts to regulate starch synthesis in plastids of higher plants. Feeding of trehalose to Arabidopsis leaves led to stimulation of starch synthesis within 30 min, accompanied by activation of ADP-glucose pyrophosphorylase (AGPase) via posttranslational redox modification. The response resembled sucrose but not glucose feeding and depended on the expression of SNF1-related kinase. We also analyzed transgenic Arabidopsis plants with T6P levels increased by expression of T6P synthase or decreased by expression of T6P phosphatase (TPP) in the cytosol. Compared with wild type, leaves of T6P synthase-expressing plants had increased redox activation of AGPase and increased starch, whereas TPP-expressing plants showed the opposite. Moreover, TPP expression prevented the increase in AGPase activation in response to sucrose or trehalose feeding. Incubation of intact isolated chloroplasts with 100 muM T6P significantly and specifically increased reductive activation of AGPase within 15 min. Results provide evidence that T6P is synthesized in the cytosol and acts on plastidial metabolism by promoting thioredoxin-mediated redox transfer to AGPase in response to cytosolic sugar levels, thereby allowing starch synthesis to be regulated independently of light. The discovery informs about the evolution of plant metabolism and how chloroplasts of prokaryotic origin use an intermediate of the ancient trehalose pathway to report the metabolic status of the cytosol.

  4. Metabolic impact of an NADH-producing glucose-6-phosphate dehydrogenase in Escherichia coli.

    PubMed

    Olavarria, K; De Ingeniis, J; Zielinski, D C; Fuentealba, M; Muñoz, R; McCloskey, D; Feist, A M; Cabrera, R

    2014-12-01

    In Escherichia coli, the oxidative branch of the pentose phosphate pathway (oxPPP) is one of the major sources of NADPH when glucose is the sole carbon nutrient. However, unbalanced NADPH production causes growth impairment as observed in a strain lacking phosphoglucoisomerase (Δpgi). In this work, we studied the metabolic response of this bacterium to the replacement of its glucose-6-phosphate dehydrogenase (G6PDH) by an NADH-producing variant. The homologous enzyme from Leuconostoc mesenteroides was studied by molecular dynamics and site-directed mutagenesis to obtain the NAD-preferring LmG6PDH(R46E,Q47E). Through homologous recombination, the zwf loci (encoding G6PDH) in the chromosomes of WT and Δpgi E. coli strains were replaced by DNA encoding LmG6PDH(R46E,Q47E). Contrary to some predictions performed with flux balance analysis, the replacements caused a substantial effect on the growth rates, increasing 59 % in the Δpgi strain, while falling 44 % in the WT. Quantitative PCR (qPCR) analysis of the zwf locus showed that the expression level of the mutant enzyme was similar to the native enzyme and the expression of genes encoding key enzymes of the central pathways also showed moderate changes among the studied strains. The phenotypic and qPCR data were integrated into in silico modelling, showing an operative G6PDH flux contributing to the NADH pool. Our results indicated that, in vivo, the generation of NADH by G6PDH is beneficial or disadvantageous for growth depending on the operation of the upper Embden-Meyerhof pathway. Interestingly, a genomic database search suggested that in bacteria lacking phosphofructokinase, the G6PDHs tend to have similar preferences for NAD and NADP. The importance of the generation of NADPH in a pathway such as the oxPPP is discussed.

  5. What is the role of the second "structural" NADP+-binding site in human glucose 6-phosphate dehydrogenase?

    PubMed

    Wang, Xiao-Tao; Chan, Ting Fai; Lam, Veronica M S; Engel, Paul C

    2008-08-01

    Human glucose 6-phosphate dehydrogenase, purified after overexpression in E. coli, was shown to contain one molecule/subunit of acid-extractable "structural" NADP+ and no NADPH. This tightly bound NADP+ was reduced by G6P, presumably following migration to the catalytic site. Gel-filtration yielded apoenzyme, devoid of bound NADP+ but, surprisingly, still fully active. Mr of the main component of "stripped" enzyme by gel filtration was approximately 100,000, suggesting a dimeric apoenzyme (subunit Mr = 59,000). Holoenzyme also contained tetramer molecules and, at high protein concentration, a dynamic equilibrium gave an apparent intermediate Mr of 150 kDa. Fluorescence titration of the stripped enzyme gave the K d for structural NADP+ as 37 nM, 200-fold lower than for "catalytic" NADP+. Structural NADP+ quenches 91% of protein fluorescence. At 37 degrees C, stripped enzyme, much less stable than holoenzyme, inactivated irreversibly within 2 d. Inactivation at 4 degrees C was partially reversed at room temperature, especially with added NADP+. Apoenzyme was immediately active, without any visible lag, in rapid-reaction studies. Human G6PD thus forms active dimer without structural NADP+. Apparently, the true role of the second, tightly bound NADP+ is to secure long-term stability. This fits the clinical pattern, G6PD deficiency affecting the long-lived non-nucleate erythrocyte. The Kd values for two class I mutants, G488S and G488V, were 273 nM and 480 nM, respectively (seven- and 13-fold elevated), matching the structural prediction of weakened structural NADP+ binding, which would explain decreased stability and consequent disease. Preparation of native apoenzyme and measurement of Kd constant for structural NADP+ will now allow quantitative assessment of this defect in clinical G6PD mutations.

  6. The preparation of nylon-tube-supported hexokinase and glucose 6-phosphate dehydrogenase and the use of the co-immobilized enzymes in the automated determination of glucose.

    PubMed Central

    Morris, D L; Campbell, J; Hornby, W E

    1975-01-01

    Triethyloxonium tetrafluoroborate was used to O-alkylate nylon-tube thus producing the imidate salt of the nylon which was further made to react with 1,6-diaminohexane. 2. Hexokinase (EC 2.7.1.1) and glucose 6-phosphate dehydrogenase (EC 1.1.1.49) were immobilized on the amino-substituted nylon tube through glutaraldeyde and bisimidates. 3. The effect of varying the conditions of O-alkylation and the amount of enzyme immobilized on the activity of nylon tube-hexokinase derivatives was determined. 4. The effect of varying the amount of enzyme immobilized on the activity of nylon-tube-glucose 6-phosphate dehydrogenase derivatives was determined. 5. The thermal stability of nylon-tube-hexokinase and nylon-tube-glucose 6-phosphate dehydrogenase derivatives was studied. 6. Different ratios of hexokinase and glucose 6-phosphate dehydrogenase were co-immobilized on nylon tube, and the rate of conversion of glucose into 6-phosphogluconolactone was compared with the individual activities of the immobilized enzymes. 7. Hexokinase and glucose 6-phosphate dehydrogenase co-immobilized on nylon tube were used in the automated analysis of glucose. PMID:1167161

  7. Are free glucose and glucose-6-phosphate in milk indicators of specific physiological states in the cow?

    PubMed

    Larsen, T; Moyes, K M

    2015-01-01

    A total of 3200 milk samples from Holstein and Jersey cows were analysed for free glucose and glucose-6-phosphate (G6P) by an enzymatic-fluorometric method that requires no pre-treatment. The cows were primiparous as well as multiparous, and samples were taken throughout the entire lactation period. In addition, lactose, protein, fat, citrate and β-hydroxybutyrate were determined and comparisons between these variables were made. Data were analysed using GLM model for the effect of parity, breed, time from last milking and stage of lactation on variations in parameters in milk. Pearson's correlations were generated between milk variables. P<0.05 was considered significant. Concentration of free glucose and G6P were on average 331 and 81 μM, respectively. Time from last milking (stay in the gland cistern) did not increase the concentration of these monosaccharides, indicating that they are not hydrolysis product from lactose post secretion, but rather reflecting the energy status of the mammary epithelial cells pre-secretion. Wide variation in range of these metabolites, that is, from 90 to 630 μM and 5 to 324 μM, for glucose and G6P, respectively, was observed. During the first 21 weeks in milk, free glucose increased whereas G6P decreased. Concentration of free glucose in milk is greater for primiparous than multiparous cows and greater for Holstein than Jersey cows. Concentration of G6P was not affected by parity or breed. The use of free glucose and G6P as indicators of physiological conditions and risk of disease is warranted for use as potential biomarkers for in-line surveillance systems on-farm.

  8. Producing glucose 6-phosphate from cellulosic biomass: Structural insights into levoglucosan bioconversion

    SciTech Connect

    Bacik, John -Paul; Klesmith, Justin R.; Whitehead, Timothy A.; Jarboe, Laura R.; Unkefer, Clifford J.; Mark, Brian L.; Michalczyk, Ryszard

    2015-09-09

    The most abundant carbohydrate product of cellulosic biomass pyrolysis is the anhydrosugar levoglucosan (1,6-anhydro-β-d-glucopyranose), which can be converted to glucose 6-phosphate by levoglucosan kinase (LGK). In addition to the canonical kinase phosphotransfer reaction, the conversion requires cleavage of the 1,6-anhydro ring to allow ATP-dependent phosphorylation of the sugar O6 atom. Using x-ray crystallography, we show that LGK binds two magnesium ions in the active site that are additionally coordinated with the nucleotide and water molecules to result in ideal octahedral coordination. To further verify the metal binding sites, we co-crystallized LGK in the presence of manganese instead of magnesium and solved the structure de novo using the anomalous signal from four manganese atoms in the dimeric structure. The first metal is required for catalysis, whereas our work suggests that the second is either required or significantly promotes the catalytic rate. Although the enzyme binds its sugar substrate in a similar orientation to the structurally related 1,6-anhydro-N-acetylmuramic acid kinase (AnmK), it forms markedly fewer bonding interactions with the substrate. In this orientation, the sugar is in an optimal position to couple phosphorylation with ring cleavage. We also observed a second alternate binding orientation for levoglucosan, and in these structures, ADP was found to bind with lower affinity. These combined observations provide an explanation for the high Km of LGK for levoglucosan. Furthermore, greater knowledge of the factors that contribute to the catalytic efficiency of LGK can be used to improve applications of this enzyme for levoglucosan-derived biofuel production.

  9. Producing glucose 6-phosphate from cellulosic biomass: Structural insights into levoglucosan bioconversion

    DOE PAGES

    Bacik, John -Paul; Klesmith, Justin R.; Whitehead, Timothy A.; Jarboe, Laura R.; Unkefer, Clifford J.; Mark, Brian L.; Michalczyk, Ryszard

    2015-09-09

    The most abundant carbohydrate product of cellulosic biomass pyrolysis is the anhydrosugar levoglucosan (1,6-anhydro-β-d-glucopyranose), which can be converted to glucose 6-phosphate by levoglucosan kinase (LGK). In addition to the canonical kinase phosphotransfer reaction, the conversion requires cleavage of the 1,6-anhydro ring to allow ATP-dependent phosphorylation of the sugar O6 atom. Using x-ray crystallography, we show that LGK binds two magnesium ions in the active site that are additionally coordinated with the nucleotide and water molecules to result in ideal octahedral coordination. To further verify the metal binding sites, we co-crystallized LGK in the presence of manganese instead of magnesium andmore » solved the structure de novo using the anomalous signal from four manganese atoms in the dimeric structure. The first metal is required for catalysis, whereas our work suggests that the second is either required or significantly promotes the catalytic rate. Although the enzyme binds its sugar substrate in a similar orientation to the structurally related 1,6-anhydro-N-acetylmuramic acid kinase (AnmK), it forms markedly fewer bonding interactions with the substrate. In this orientation, the sugar is in an optimal position to couple phosphorylation with ring cleavage. We also observed a second alternate binding orientation for levoglucosan, and in these structures, ADP was found to bind with lower affinity. These combined observations provide an explanation for the high Km of LGK for levoglucosan. Furthermore, greater knowledge of the factors that contribute to the catalytic efficiency of LGK can be used to improve applications of this enzyme for levoglucosan-derived biofuel production.« less

  10. Binding Mode and Selectivity of Steroids towards Glucose-6-phosphate Dehydrogenase from the Pathogen Trypanosoma cruzi.

    PubMed

    Ortiz, Cecilia; Moraca, Francesca; Medeiros, Andrea; Botta, Maurizio; Hamilton, Niall; Comini, Marcelo A

    2016-01-01

    Glucose-6-phosphate dehydrogenase (G6PDH) plays a housekeeping role in cell metabolism by generating reducing power (NADPH) and fueling the production of nucleotide precursors (ribose-5-phosphate). Based on its indispensability for pathogenic parasites from the genus Trypanosoma, G6PDH is considered a drug target candidate. Several steroid-like scaffolds were previously reported to target the activity of G6PDH. Epiandrosterone (EA) is an uncompetitive inhibitor of trypanosomal G6PDH for which its binding site to the enzyme remains unknown. Molecular simulation studies with the structure of Trypanosoma cruzi G6PDH revealed that EA binds in a pocket close to the G6P binding-site and protrudes into the active site blocking the interaction between substrates and hence catalysis. Site directed mutagenesis revealed the important steroid-stabilizing effect of residues (L80, K83 and K84) located on helix α-1 of T. cruzi G6PDH. The higher affinity and potency of 16α-Br EA by T. cruzi G6PDH is explained by the formation of a halogen bond with the hydrogen from the terminal amide of the NADP+-nicotinamide. At variance with the human enzyme, the inclusion of a 21-hydroxypregnane-20-one moiety to a 3β-substituted steroid is detrimental for T. cruzi G6PDH inhibition. The species-specificity of certain steroid derivatives towards the parasite G6PDH and the corresponding biochemically validated binding models disclosed in this work may prove valuable for the development of selective inhibitors against the pathogen's enzyme. PMID:26999093

  11. Point-of-Care Quantitative Measure of Glucose-6-Phosphate Dehydrogenase Enzyme Deficiency

    PubMed Central

    Kaplan, Michael; Glader, Bertil; Cotten, Michael; Kleinert, Jairus; Pamula, Vamsee

    2015-01-01

    BACKGROUND AND OBJECTIVES: Widespread newborn screening on a point-of-care basis could prevent bilirubin neurotoxicity in newborns with glucose-6-phosphate dehydrogenase (G6PD) deficiency. We evaluated a quantitative G6PD assay on a digital microfluidic platform by comparing its performance with standard clinical methods. METHODS: G6PD activity was measured quantitatively by using digital microfluidic fluorescence and the gold standard fluorescence biochemical test on a convenience sample of 98 discarded blood samples. Twenty-four samples were designated as G6PD deficient. RESULTS: Mean ± SD G6PD activity for normal samples using the digital microfluidic method and the standard method, respectively, was 9.7 ± 2.8 and 11.1 ± 3.0 U/g hemoglobin (Hb), respectively; for G6PD-deficient samples, it was 0.8 ± 0.7 and 1.4 ± 0.9 U/g Hb. Bland-Altman analysis determined a mean difference of –0.96 ± 1.8 U/g Hb between the digital microfluidic fluorescence results and the standard biochemical test results. The lower and upper limits for the digital microfluidic platform were 4.5 to 19.5 U/g Hb for normal samples and 0.2 to 3.7 U/g Hb for G6PD-deficient samples. The lower and upper limits for the Stanford method were 5.5 to 20.7 U/g Hb for normal samples and 0.1 to 2.8 U/g Hb for G6PD-deficient samples. The measured activity discriminated between G6PD-deficient samples and normal samples with no overlap. CONCLUSIONS: Pending further validation, a digital microfluidics platform could be an accurate point-of-care screening tool for rapid newborn G6PD screening. PMID:26459646

  12. Importance of glucose-6-phosphate dehydrogenase (G6PDH) for vanillin tolerance in Saccharomyces cerevisiae.

    PubMed

    Nguyen, Trinh Thi My; Kitajima, Sakihito; Izawa, Shingo

    2014-09-01

    Vanillin is derived from lignocellulosic biomass and, as one of the major biomass conversion inhibitors, inhibits yeast growth and fermentation. Vanillin was recently shown to induce the mitochondrial fragmentation and formation of mRNP granules such as processing bodies and stress granules in Saccharomyces cerevisiae. Furfural, another major biomass conversion inhibitor, also induces oxidative stress and is reduced in an NAD(P)H-dependent manner to its less toxic alcohol derivative. Therefore, the pentose phosphate pathway (PPP), through which most NADPH is generated, plays a role in tolerance to furfural. Although vanillin also induces oxidative stress and is reduced to vanillyl alcohol in a NADPH-dependent manner, the relationship between vanillin and PPP has not yet been investigated. In the present study, we examined the importance of glucose-6-phosphate dehydrogenase (G6PDH), which catalyzes the rate-limiting NADPH-producing step in PPP, for yeast tolerance to vanillin. The growth of the null mutant of G6PDH gene (zwf1Δ) was delayed in the presence of vanillin, and vanillin was efficiently reduced in the culture of wild-type cells but not in the culture of zwf1Δ cells. Furthermore, zwf1Δ cells easily induced the activation of Yap1, an oxidative stress responsive transcription factor, mitochondrial fragmentation, and P-body formation with the vanillin treatment, which indicated that zwf1Δ cells were more susceptible to vanillin than wild type cells. These findings suggest the importance of G6PDH and PPP in the response of yeast to vanillin.

  13. Producing glucose 6-phosphate from cellulosic biomass: structural insights into levoglucosan bioconversion.

    PubMed

    Bacik, John-Paul; Klesmith, Justin R; Whitehead, Timothy A; Jarboe, Laura R; Unkefer, Clifford J; Mark, Brian L; Michalczyk, Ryszard

    2015-10-30

    The most abundant carbohydrate product of cellulosic biomass pyrolysis is the anhydrosugar levoglucosan (1,6-anhydro-β-d-glucopyranose), which can be converted to glucose 6-phosphate by levoglucosan kinase (LGK). In addition to the canonical kinase phosphotransfer reaction, the conversion requires cleavage of the 1,6-anhydro ring to allow ATP-dependent phosphorylation of the sugar O6 atom. Using x-ray crystallography, we show that LGK binds two magnesium ions in the active site that are additionally coordinated with the nucleotide and water molecules to result in ideal octahedral coordination. To further verify the metal binding sites, we co-crystallized LGK in the presence of manganese instead of magnesium and solved the structure de novo using the anomalous signal from four manganese atoms in the dimeric structure. The first metal is required for catalysis, whereas our work suggests that the second is either required or significantly promotes the catalytic rate. Although the enzyme binds its sugar substrate in a similar orientation to the structurally related 1,6-anhydro-N-acetylmuramic acid kinase (AnmK), it forms markedly fewer bonding interactions with the substrate. In this orientation, the sugar is in an optimal position to couple phosphorylation with ring cleavage. We also observed a second alternate binding orientation for levoglucosan, and in these structures, ADP was found to bind with lower affinity. These combined observations provide an explanation for the high Km of LGK for levoglucosan. Greater knowledge of the factors that contribute to the catalytic efficiency of LGK can be used to improve applications of this enzyme for levoglucosan-derived biofuel production. PMID:26354439

  14. Subcellular Characterization of Porcine Oocytes with Different Glucose-6-phosphate Dehydrogenase Activities

    PubMed Central

    Fu, Bo; Ren, Liang; Liu, Di; Ma, Jian-Zhang; An, Tie-Zhu; Yang, Xiu-Qin; Ma, Hong; Zhang, Dong-Jie; Guo, Zhen-Hua; Guo, Yun-Yun; Zhu, Meng; Bai, Jing

    2015-01-01

    The in vitro maturation (IVM) efficiency of porcine embryos is still low because of poor oocyte quality. Although brilliant cresyl blue positive (BCB+) oocytes with low glucose-6-phosphate dehydrogenase (G6PDH) activity have shown superior quality than BCB negative (−) oocytes with high G6PDH activity, the use of a BCB staining test before IVM is still controversial. This study aimed to shed more light on the subcellular characteristics of porcine oocytes after selection using BCB staining. We assessed germinal vesicle chromatin configuration, cortical granule (CG) migration, mitochondrial distribution, the levels of acetylated lysine 9 of histone H3 (AcH3K9) and nuclear apoptosis features to investigate the correlation between G6PDH activity and these developmentally related features. A pattern of chromatin surrounding the nucleoli was seen in 53.0% of BCB+ oocytes and 77.6% of BCB+ oocytes showed peripherally distributed CGs. After IVM, 48.7% of BCB+ oocytes had a diffused mitochondrial distribution pattern. However, there were no significant differences in the levels of AcH3K9 in the nuclei of blastocysts derived from BCB+ and BCB− oocytes; at the same time, we observed a similar incidence of apoptosis in the BCB+ and control groups. Although this study indicated that G6PDH activity in porcine oocytes was correlated with several subcellular characteristics such as germinal vesicle chromatin configuration, CG migration and mitochondrial distribution, other features such as AcH3K9 level and nuclear apoptotic features were not associated with G6PDH activity and did not validate the BCB staining test. In using this test for selecting porcine oocytes, subcellular characteristics such as the AcH3K9 level and apoptotic nuclear features should also be considered. Adding histone deacetylase inhibitors or apoptosis inhibitors into the culture medium used might improve the efficiency of IVM of BCB+ oocytes. PMID:26580437

  15. Producing glucose 6-phosphate from cellulosic biomass: structural insights into levoglucosan bioconversion.

    PubMed

    Bacik, John-Paul; Klesmith, Justin R; Whitehead, Timothy A; Jarboe, Laura R; Unkefer, Clifford J; Mark, Brian L; Michalczyk, Ryszard

    2015-10-30

    The most abundant carbohydrate product of cellulosic biomass pyrolysis is the anhydrosugar levoglucosan (1,6-anhydro-β-d-glucopyranose), which can be converted to glucose 6-phosphate by levoglucosan kinase (LGK). In addition to the canonical kinase phosphotransfer reaction, the conversion requires cleavage of the 1,6-anhydro ring to allow ATP-dependent phosphorylation of the sugar O6 atom. Using x-ray crystallography, we show that LGK binds two magnesium ions in the active site that are additionally coordinated with the nucleotide and water molecules to result in ideal octahedral coordination. To further verify the metal binding sites, we co-crystallized LGK in the presence of manganese instead of magnesium and solved the structure de novo using the anomalous signal from four manganese atoms in the dimeric structure. The first metal is required for catalysis, whereas our work suggests that the second is either required or significantly promotes the catalytic rate. Although the enzyme binds its sugar substrate in a similar orientation to the structurally related 1,6-anhydro-N-acetylmuramic acid kinase (AnmK), it forms markedly fewer bonding interactions with the substrate. In this orientation, the sugar is in an optimal position to couple phosphorylation with ring cleavage. We also observed a second alternate binding orientation for levoglucosan, and in these structures, ADP was found to bind with lower affinity. These combined observations provide an explanation for the high Km of LGK for levoglucosan. Greater knowledge of the factors that contribute to the catalytic efficiency of LGK can be used to improve applications of this enzyme for levoglucosan-derived biofuel production.

  16. Effect of red blood cell glucose-6-phosphate dehydrogenase deficiency on patients with dengue hemorrhagic fever.

    PubMed

    Tanphaichitr, Voravarn S; Chonlasin, Rachaneekorn; Suwantol, Lerlugsn; Pung-Amritt, Parichat; Tachavanich, Kalaya; Yogsan, Suthee; Viprakasit, Vip

    2002-08-01

    Eighty nine males aged 1-13 years diagnosed with dengue haemorrhagic fever (DHF) and admitted to the Department of Pediatrics Siriraj Hospital from March 1998 to April 2000 were included in this study. 17 cases (19.1%) had red blood cell glucose-6-phosphate dehydrogenase (G-6-PD) deficiency and 72 cases (80.9%) had normal G-6-PD enzyme activities. Most of the patients were classified as DHF grade II in severity. 3 of 17 G-6-PD deficient cases had serious complications and all of them had acute intravascular hemolysis requiring blood transfusions. One of these also had hematemesis, one had azothemia and the other one had renal failure and severe liver failure with hepatic encephalopathy. In the cases without obvious hemolytic or hepatic complications, G-6-PD deficient cases had mildly but significantly higher total birirubin and indirect bilirubin, as well as a lower hematocrit than those who had normal G-6-PD. Reticulocyte count was low during the acute phase, however, during recovery, the levels were significantly increased in both groups. In the non G-6-PD deficient group, G-6-PD enzyme levels were significantly decreased during the acute phase compared to the normal controls but rose significantly to normal levels during the recovery phase. There were no statistically significant differences in other laboratory data. All patients recovered fully from DHF. The prevalence of G-6-PD deficiency in male patients who had DHF in this study was 19.1 per cent which was higher than the prevalence in a previous study of 12 per cent in Bangkok. This may imply that G-6-PD deficient males suffer more from DHF compared to normal G-6-PD subjects.

  17. Regulation of a plant SNF1-related protein kinase by glucose-6-phosphate

    SciTech Connect

    Toroser, D.; Plaut, Z.; Huber, S.C.

    2000-05-01

    One of the major protein kinases (PK{sub III}) that phosphorylates serine-158 of spinach sucrose-phosphate synthase (SPS), which is responsible for light/dark modulation of activity, is known to be a member of the SNF1-related family of protein kinases. In the present study, the authors have developed a fluorescence-based continuous assay for measurement of PK{sub III} activity. Using the continuous assay, along with the fixed-time-point {sup 32}P-incorporation assay, they demonstrate that PK{sub III} activity is inhibited by glucose-6-phosphate (Glc-6-P). Relative inhibition by Glc-6-P was increased by decreasing pH from 8.5 to 5.5 and by reducing the concentration of Mg{sup 2+} in the assay from 10 to 2 nM. Under likely physiological conditions (PH 7.0 and 2 mM Mg{sup 2+}), 10 nM Glc-6-P inhibited kinase activity approximately 70%. Inhibition by Glc-6-P could not be ascribed to contaminants in the commercial preparations. Other metabolites inhibited PK{sub III} in the following order: Glc-6-P > mannose-6-P, fructose-1,6P{sub 2} > ribose-5-P, 3-PGA, fructose-6-P. Inorganic phosphate, Glc, and AMP were not inhibitory, and free Glc did not reverse the inhibition by Glc-6-P. Because SNF1-related protein kinases are thought to function broadly in the regulation of enzyme activity and gene expression, Glc-6-P inhibition of PK{sub III} activity potentially provides a mechanism for metabolic regulation of the reactions catalyzed by these important protein kinases.

  18. Lowering effect of firefly squid powder on triacylglycerol content and glucose-6-phosphate dehydrogenase activity in rat liver.

    PubMed

    Takeuchi, Hiroyuki; Morita, Ritsuko; Shirai, Yoko; Nakagawa, Yoshihisa; Terashima, Teruya; Ushikubo, Shun; Matsuo, Tatsuhiro

    2014-01-01

    Effects of dietary firefly squid on serum and liver lipid levels were investigated. Male Wistar rats were fed a diet containing 5% freeze-dried firefly squid or Japanese flying squid for 2 weeks. There was no significant difference in the liver triacylglycerol level between the control and Japanese flying squid groups, but the rats fed the firefly squid diet had a significantly lower liver triacylglycerol content than those fed the control diet. No significant difference was observed in serum triacylglycerol levels between the control and firefly squid groups. The rats fed the firefly squid had a significantly lower activity of liver glucose-6-phosphate dehydrogenase compared to the rats fed the control diet. There was no significant difference in liver fatty acid synthetase activity among the three groups. Hepatic gene expression and lipogenic enzyme activity were investigated; a DNA microarray showed that the significantly enriched gene ontology category of down-regulated genes in the firefly squid group was "lipid metabolic process". The firefly squid group had lower mRNA level of glucose-6-phosphate dehydrogenase compared to the controls. These results suggest that an intake of firefly squid decreases hepatic triacylglycerol in rats, and the reduction of mRNA level and enzyme activity of glucose-6-phosphate dehydrogenase might be related to the mechanisms.

  19. Purification and characterization of glucose 6-phosphate dehydrogenase from Lake Van fish (Chalcalburnus tarichii pallas, 1811) liver.

    PubMed

    Turkoglu, V; Altun, M; Ciftçi, M

    2006-09-01

    Glucose 6-phosphate dehydrogenase (D-glucose 6-phosphate: NADP+ oxidoreductase, EC 1.1.1.49; G6PD) was purified from Lake Van fish (Chalcalburnus tarichii pallas, 1811) liver, using a simple and rapid method, and some characteristics of the enzyme were investigated. The purification procedure was composed of two steps: homogenate preparation and 2', 5'-ADP Sepharose 4B affinity gel chromatography, which took 7-8 hours. Thanks to the two consecutive procedures, the enzyme, having specific activity of 38 EU/mg protein, was purified with a yield of 44.39% and 1310 fold. In order to control the enzyme purification SDS polyacrylamide gel electrophoresis (SDS-PAGE) was done. SDS polyacrylamide gel electrophoresis showed a single band for enzyme. Optimal pH, stable pH, optimal temperature, Km and, Vmax values for NADP+ and glucose 6-phosphate (G6P) were also determined for the enzyme. In addition, molecular weight and subunit molecular weights were found by sodium dodecyl sulfate polyacrilamide gel electrophoresis (SDS-PAGE) and gel filtration chromatography respectively.

  20. Glucose 6-phosphate dehydrogenase deficiency enhances germ cell apoptosis and causes defective embryogenesis in Caenorhabditis elegans

    PubMed Central

    Yang, H-C; Chen, T-L; Wu, Y-H; Cheng, K-P; Lin, Y-H; Cheng, M-L; Ho, H-Y; Lo, S J; Chiu, D T-Y

    2013-01-01

    Glucose 6-phosphate dehydrogenase (G6PD) deficiency, known as favism, is classically manifested by hemolytic anemia in human. More recently, it has been shown that mild G6PD deficiency moderately affects cardiac function, whereas severe G6PD deficiency leads to embryonic lethality in mice. How G6PD deficiency affects organisms has not been fully elucidated due to the lack of a suitable animal model. In this study, G6PD-deficient Caenorhabditis elegans was established by RNA interference (RNAi) knockdown to delineate the role of G6PD in animal physiology. Upon G6PD RNAi knockdown, G6PD activity was significantly hampered in C. elegans in parallel with increased oxidative stress and DNA oxidative damage. Phenotypically, G6PD-knockdown enhanced germ cell apoptosis (2-fold increase), reduced egg production (65% of mock), and hatching (10% of mock). To determine whether oxidative stress is associated with G6PD knockdown-induced reproduction defects, C. elegans was challenged with a short-term hydrogen peroxide (H2O2). The early phase egg production of both mock and G6PD-knockdown C. elegans were significantly affected by H2O2. However, H2O2-induced germ cell apoptosis was more dramatic in mock than that in G6PD-deficient C. elegans. To investigate the signaling pathways involved in defective oogenesis and embryogenesis caused by G6PD knockdown, mutants of p53 and mitogen-activated protein kinase (MAPK) pathways were examined. Despite the upregulation of CEP-1 (p53), cep-1 mutation did not affect egg production and hatching in G6PD-deficient C. elegans. Neither pmk-1 nor mek-1 mutation significantly affected egg production, whereas sek-1 mutation further decreased egg production in G6PD-deficient C. elegans. Intriguingly, loss of function of sek-1 or mek-1 dramatically rescued defective hatching (8.3- and 9.6-fold increase, respectively) induced by G6PD knockdown. Taken together, these findings show that G6PD knockdown reduces egg production and hatching in C. elegans

  1. Evaluation of Glucose-6-Phosphate Dehydrogenase stability in stored blood samples

    PubMed Central

    Jalil, Norunaluwar; Azma, Raja Zahratul; Mohamed, Emida; Ithnin, Azlin; Alauddin, Hafiza; Baya, Siti Noor; Othman, Ainoon

    2016-01-01

    Glucose-6-Phosphate Dehydrogenase (G6PD) deficiency is the commonest cause of neonatal jaundice in Malaysia. Recently, OSMMR2000-D G6PD Assay Kit has been introduced to quantitate the level of G6PD activity in newborns delivered in Universiti Kebangsaan Malaysia Medical Centre (UKMMC). As duration of sample storage prior to analysis is one of the matters of concern, this study was conducted to identify the stability of G6PD enzyme during storage. A total of 188 cord blood samples from normal term newborns delivered at UKMMC were selected for this study. The cord bloods samples were collected in ethylene-diamine-tetra-acetic acid (EDTA) tubes and refrigerated at 2-8 °C. In addition, 32 out of 188 cord blood samples were spotted on chromatography paper, air-dried and stored at room temperature. G6PD enzyme activities were measured daily for 7 days using the OSMMR2000-D G6PD Assay Kit on both the EDTA blood and dried blood samples. The mean value for G6PD activity was compared between days of analysis using Student Paired T-Test. In this study, 172 out of 188 cord blood samples showed normal enzyme levels while 16 had levels corresponding to severe enzyme deficiency. The daily mean G6PD activity for EDTA blood samples of newborns with normal G6PD activity showed a significant drop on the fourth day of storage (p < 0.005) while for samples with severely deficient G6PD activity, significant drop was seen on third day of storage (p = 0.002). Analysis of dried cord blood showed a significant reduction in enzyme activity as early as the second day of storage (p = 0.001). It was also noted that mean G6PD activity for spotted blood samples were lower compared to those in EDTA tubes for all days (p = 0.001). Thus, EDTA blood samples stored at 2-8 °C appeared to have better stability in terms of their G6PD enzyme level as compared to dried blood samples on filter paper, giving a storage time of up to 3 days. PMID:27103895

  2. Glucose-6-phosphate dehydrogenase polymorphisms and susceptibility to mild malaria in Dogon and Fulani, Mali

    PubMed Central

    2014-01-01

    Background Glucose-6-phosphate dehydrogenase (G6PD) deficiency is associated with protection from severe malaria, and potentially uncomplicated malaria phenotypes. It has been documented that G6PD deficiency in sub-Saharan Africa is due to the 202A/376G G6PD A-allele, and association studies have used genotyping as a convenient technique for epidemiological studies. However, recent studies have shown discrepancies in G6PD202/376 associations with severe malaria. There is evidence to suggest that other G6PD deficiency alleles may be common in some regions of West Africa, and that allelic heterogeneity could explain these discrepancies. Methods A cross-sectional epidemiological study of malaria susceptibility was conducted during 2006 and 2007 in the Sahel meso-endemic malaria zone of Mali. The study included Dogon (n = 375) and Fulani (n = 337) sympatric ethnic groups, where the latter group is characterized by lower susceptibility to Plasmodium falciparum malaria. Fifty-three G6PD polymorphisms, including 202/376, were genotyped across the 712 samples. Evidence of association of these G6PD polymorphisms and mild malaria was assessed in both ethnic groups using genotypic and haplotypic statistical tests. Results It was confirmed that the Fulani are less susceptible to malaria, and the 202A mutation is rare in this group (< 1% versus Dogon 7.9%). The Betica-Selma 968C/376G (~11% enzymatic activity) was more common in Fulani (6.1% vs Dogon 0.0%). There are differences in haplotype frequencies between Dogon and Fulani, and association analysis did not reveal strong evidence of protective G6PD genetic effects against uncomplicated malaria in both ethnic groups and gender. However, there was some evidence of increased risk of mild malaria in Dogon with the 202A mutation, attaining borderline statistical significance in females. The rs915942 polymorphism was found to be associated with asymptomatic malaria in Dogon females, and the rs61042368 polymorphism was

  3. Measurements of glucose phosphorylation with FDG and PET are not reduced by dephosphorylation of FDG-6-phosphate

    SciTech Connect

    Kuwabara, H.; Gjedde, A. )

    1991-04-01

    To improve the measurements of glucose metabolism in the human brain, we imposed biologic constraints on the deoxyglucose model with and without dephosphorylation of FDG-6-phosphate (the k4*- and k3*-models). The constraints included constant transport and phosphorylation ratios (tau and phi) and a common partition volume (K1/k2) for tracer ({sup 18}F)FDG and glucose. In the presence of significant dephosphorylation, the k3*-model yielded time-dependent estimates of the phosphorylation coefficient (k3*), while the K4*-model yielded time-independent estimates. However, the two models yielded practically identical measurements of regional cerebral glucose metabolism in PET studies of six normal volunteers when the phosphorylation affinity ratio (the k3*/k3 ratio of FDG and glucose) and tracer circulation time were 0.30 and 20 min for the k3*-model and 0.33 and 45 min for the k4*-model.

  4. High Frequency of Diabetes and Impaired Fasting Glucose in Patients with Glucose-6-Phosphate Dehydrogenase Deficiency in the Western Brazilian Amazon

    PubMed Central

    Santana, Marli S.; Monteiro, Wuelton M.; Costa, Mônica R. F.; Sampaio, Vanderson S.; Brito, Marcelo A. M.; Lacerda, Marcus V. G.; Alecrim, Maria G. C.

    2014-01-01

    Glucose-6-phosphate dehydrogenase (G6PD) deficiency is one of the most common human genetic abnormalities, and it has a significant prevalence in the male population (X chromosome linked). The purpose of this study was to estimate the frequency of impaired fasting glucose and diabetes among G6PD-deficient persons in Manaus, Brazil, an area in the Western Brazilian Amazon to which malaria is endemic. Glucose-6-phosphate dehydrogenase–deficient males had more impaired fasting glucose and diabetes. This feature could be used as a screening tool for G6PD-deficient persons who are unable to use primaquine for the radical cure of Plasmodium vivax malaria. PMID:24865682

  5. Purification and investigation of some kinetic properties of glucose-6-phosphate dehydrogenase from parsley (Petroselinum hortense) leaves.

    PubMed

    Coban, T Abdül Kadir; Ciftçi, Mehmet; Küfrevioğlu, O Irfan

    2002-05-01

    In this study, glucose-6-phosphate dehydrogenase (D-glucose-6-phosphate: NADP+ oxidoreductase, EC 1.1.1.49; G6PD) was purified from parsley (Petroselinum hortense) leaves, and analysis of the kinetic behavior and some properties of the enzyme were investigated. The purification consisted of three steps: preparation of homogenate, ammonium sulfate fractionation, and DEAE-Sephadex A50 ion exchange chromatography. The enzyme was obtained with a yield of 8.79% and had a specific activity of 2.146 U (mg protein)(-1). The overall purification was about 58-fold. Temperature of +4 degrees C was maintained during the purification process. Enzyme activity was spectrophotometrically measured according to the Beutler method, at 340 nm. In order to control the purification of enzyme, SDS-polyacrylamide gel electrophoresis was carried out in 4% and 10% acrylamide for stacking and running gel, respectively. SDS-polyacrylamide gel electrophoresis showed a single band for enzyme. The molecular weight was found to be 77.6 kDa by Sephadex G-150 gel filtration chromatography. A protein band corresponding to a molecular weight of 79.3 kDa was obtained on SDS-polyacrylamide gel electrophoresis. For the enzymes, the stable pH, optimum pH, and optimum temperature were found to be 6.0, 8.0, and 60 degrees C, respectively. Moreover, KM and Vmax values for NADP+ and G6-P at optimum pH and 25 degrees C were determined by means of Lineweaver-Burk graphs. Additionally, effects of streptomycin sulfate and tetracycline antibiotics were investigated for the enzyme activity of glucose-6-phosphate dehydrogenase in vitro.

  6. Mutational Analyses of Glucose Dehydrogenase and Glucose-6-Phosphate Dehydrogenase Genes in Pseudomonas fluorescens Reveal Their Effects on Growth and Alginate Production.

    PubMed

    Maleki, Susan; Mærk, Mali; Valla, Svein; Ertesvåg, Helga

    2015-05-15

    The biosynthesis of alginate has been studied extensively due to the importance of this polymer in medicine and industry. Alginate is synthesized from fructose-6-phosphate and thus competes with the central carbon metabolism for this metabolite. The alginate-producing bacterium Pseudomonas fluorescens relies on the Entner-Doudoroff and pentose phosphate pathways for glucose metabolism, and these pathways are also important for the metabolism of fructose and glycerol. In the present study, the impact of key carbohydrate metabolism enzymes on growth and alginate synthesis was investigated in P. fluorescens. Mutants defective in glucose-6-phosphate dehydrogenase isoenzymes (Zwf-1 and Zwf-2) or glucose dehydrogenase (Gcd) were evaluated using media containing glucose, fructose, or glycerol. Zwf-1 was shown to be the most important glucose-6-phosphate dehydrogenase for catabolism. Both Zwf enzymes preferred NADP as a coenzyme, although NAD was also accepted. Only Zwf-2 was active in the presence of 3 mM ATP, and then only with NADP as a coenzyme, indicating an anabolic role for this isoenzyme. Disruption of zwf-1 resulted in increased alginate production when glycerol was used as the carbon source, possibly due to decreased flux through the Entner-Doudoroff pathway rendering more fructose-6-phosphate available for alginate biosynthesis. In alginate-producing cells grown on glucose, disruption of gcd increased both cell numbers and alginate production levels, while this mutation had no positive effect on growth in a non-alginate-producing strain. A possible explanation is that alginate synthesis might function as a sink for surplus hexose phosphates that could otherwise be detrimental to the cell.

  7. Effects of some drugs on hepatic glucose 6-phosphate dehydrogenase activity in Lake Van fish (Chalcalburnus tarischii Pallas, 1811).

    PubMed

    Ciftci, Mehmet; Turkoglu, Vedat; Coban, T Abdulkadir

    2007-05-01

    Inhibitory effects of some drugs on hepatic glucose 6-phosphate dehydrogenase from Lake Van fish (chalcalburnus tarischii pallas, 1811) were investigated. For this purpose, initially liver glucose 6-phosphate dehydrogenase was purified 899-fold in a yield of 46.24% by using 2',5'-ADP Sepharose 4B affinity gel. In order to control the purification of enzyme was done SDS polyacrylamide gel electrophoresis. SDS polyacrylamide gel electrophoresis showed a single band for enzyme. A constant temperature (+4 degrees C) was maintained during the purification process. Enzyme activity was determined with the Beutler method by using a spectrophotometer at 340 nm. Vankomycine, sulfanylamide, sulfanylacetamide, nidazole, ciprofloxacin, amoxicillin and KMnO(4) were used as drugs. These drugs exhibited inhibitory effects on the enzyme. IC(50) values of vankomycine, sulfanylamide, sulfanylacetamide, nidazole, ciprofloxacin, amoxicillin and KMnO(4) were 1.88, 0.037, 0.032, 1.178, 2.26, 643.5 and 0.0002 mM, and the K(i) constants 1.18+/-0.148, 0.119+/-0.021, 0.075+/-0.015, 1.15+/-0.21, 7.69+/-0.67, 1007+/-69, and 0.001+/-0.00022 mM, respectively. While vankomycine and nidazole showed competitive inhibition, others displayed noncompetitive inhibition. K(i) constants and IC(50) values for drugs were determined by Lineweaver-Burk graphs and plotting activity percentage versus [I], respectively.

  8. [Frequency of glucose-6-phosphate dehydrogenase deficiency (A-376/202) in three Malian ethnic groups].

    PubMed

    Dolo, A; Maiga, B; Guindo, A; Diakité, S A S; Diakite, M; Tapily, A; Traoré, M; Sangaré, B; Arama, C; Daou, M; Doumbo, O

    2014-08-01

    Erythrocyte G6PD deficiency is the most common worldwide enzymopathy. The aim of this study was to determine erythrocyte G6PD deficiency in 3 ethnic groups of Mali and to investigate whether erythrocyte G6PD deficiency was associated to the observed protection against malaria seen in Fulani ethnic group. The study was conducted in two different areas of Mali: in the Sahel region of Mopti where Fulani and Dogon live as sympatric ethnic groups and in the Sudanese savannah area where lives mostly the Malinke ethnic group. The study was conducted in 2007 in Koro and in 2008 in Naguilabougou. It included a total 90 Dogon, 42 Fulani and 80 Malinke ethnic groups. Malaria was diagnosed using microscopic examination after Giemsa-staining of thick and thin blood smear. G6PD deficiency (A-(376/202)) samples were identified using RFLP (Restriction Fragment Length Polymorphism) assay and analysis of PCR-amplified DNA amplicon. G6PD deficiency (A-(376/202)) rate was 11.1%, 2.4%, and 13.3% in Dogon, Fulani, and Malinke ethnic group respectively. Heterozygous state for G6PD (A-(376/202)) was found in 7.8% in Dogon; 2.4% in Fulani and 9.3% in Malinke ethnic groups while hemizygous state was found at the frequency of 2.2% in Dogon and 4% in Malinke. No homozygous state was found in our study population.We conclude that G6PD deficiency is not differing significantly between the three ethnic groups, Fulani, Dogon and Malinke.

  9. Effect of feeding and of DDT on the activity of hepatic glucose 6- phosphate dehydrogenase in two salmonids

    USGS Publications Warehouse

    Buhler, Donald R.; Benville, P.

    1969-01-01

    The specific activity of liver glucose 6-phosphate dehydrogenase in yearling rainbow trout remained unchanged when the fish were starved for periods as long as 8 weeks and when starved animals were fed diets of various compositions. Injection of insulin concurrently with refeeding also failed to alter the specific activity of the enzyme in trout. The absence of a dietary or insulin influence on the teleost enzyme system is to be contrasted with studies in mammals in which the activity of hepatic glucose 6-P dehydrogenase was markedly stimulated after refeeding starved animals or injection of insulin.Ingestion of the pesticide DDT by juvenile coho salmon or adult rainbow trout also had no effect on the specific activity of liver glucose 6-P dehydrogenase and DDT failed to inhibit the rainbow trout enzyme in vitro. These results also differ considerably from those found in higher animals.These results suggest that the glucose 6-P dehydrogenase enzyme in teleosts may be under a different type of regulatory control from that found in mammals.

  10. Oxidant injury of caucasian glucose-6-phosphate dehydrogenase—deficient red blood cells by phagocytosing leukocytes during infection

    PubMed Central

    Baehner, Robert L.; Nathan, David G.; Castle, William B.

    1971-01-01

    Patients with glucose-6-phosphate dehydrogenase (G6PD) deficiency of red blood cells (RBC) may develop sudden hemolytic anemia during infection. Since phagocytizing polymorphonuclear leukocytes (PMN) are known to generate hydrogen peroxide, we explored the influence of this oxidant product of PMN on juxtaposed G6PD-deficient and normal RBC. The oxidant stress induced by phagocytosis depleted G6PD-deficient RBC of reduced glutathione (GSH) and this was associated with rapid removal of these cells from the circulation by the liver and spleen. No such effect was observed on normal RBC. Phagocytizing chronic granulomatous disease (CGD) PMN which lack hydrogen peroxide generation, failed to diminish GSH level in G6PD-deficient RBC. Thus, PMN can pose as a source of oxidant damage to G6PD-deficient RBC due to hydrogen peroxide generated during phagocytosis. PMID:5129301

  11. X-linked glucose-6-phosphate dehydrogenase (G6PD) and autosomal 6-phosphogluconate dehydrogenase (6PGD) polymorphisms in baboons

    SciTech Connect

    VandeBerg, J.L.; Aivaliotis, M.J.; Samollow, P.B. )

    1992-12-01

    Electrophoretic polymorphisms of glucose-6-phosphate dehydrogenase (G6PD) and 6-phosphogluconate dehydrogenase (6PGD) were examined in captive colonies of five subspecies of baboons (Papio hamadryas). Phenotype frequencies and family data verified the X-linked inheritance of the G6PD polymorphism. Insufficient family data were available to confirm autosomal inheritance of the 6PGD polymorphism, but the electrophoretic patterns of variant types (putative heterozygotes) suggested the codominant expression of alleles at an autosomal locus. Implications of the G6PD polymorphism are discussed with regard to its utility as a marker system for research on X-chromosome inactivation during baboon development and for studies of clonal cell proliferation and/or cell selection during the development of atherosclerotic lesions in the baboon model. 61 refs., 1 fig., 4 tabs.

  12. [Stability of glucose 6-phosphate dehydrogenase complexed with its substrate and/or cofactor in aqueous and micellar environment].

    PubMed

    Puchkaev, A V; Vlasov, A P; Metelitsa, D I

    2002-01-01

    Inactivation of glucose 6-phosphate dehydrogenase (G6PDH) complexed with its substrate, glucose 6-phosphate (GP), and/or cofactor, NADP+, has been studied within the range 20-40 degrees C in three media: (a) 0.04 M NaOH-glycine buffer (pH 9.1); (b) Aerosol OT (AOT) reversed micelles in octane; and (c) Triton X-100 micelles in octane supplemented with 10% hexanol. The enzyme inactivation was characterized quantitatively by first order rate constants, kin (s-1). In the case of G6PDH-NADP+ complexes, the values of kin were independent of the initial concentrations of G6PDH, either in aqueous medium or AOT micelles. The values of kin for the complex G6PDH-GP were inversely related to the initial concentration of the enzyme, in both aqueous and micellar media. When inactivation of both complexes were studied in AOT micelles, minimum values of kin corresponded to the degree of hydration W0 = 16.7; at W0 > 16.7 and W0 < 16.7, kin increased. Within the range 20-40 degrees C, the values of kin measured for both complexes in aqueous medium were significantly lower than those measured in AOT micelles. Temperature dependences of kin were characterized by inflections in Arrhenius plots, which corresponded, depending on the medium, to certain temperatures from 33.6 degrees C to 40 degrees C. In all media studied, NADP+ complexes of the enzyme exhibited higher stability than their GP counterparts. The parameters of G6PDH and G6PDH-NADP+ melting, measured by differential scanning microcalorimetry (maximum temperature and half-width of the transition, enthalpy of denaturation, and van't Hoff enthalpy), provided unequivocal evidence of the higher stability of the complex as compared to that of the enzyme. In addition, this approach demonstrated that G6PDH undergoes destabilization in AOT micelles.

  13. A gene on chromosome 11q23 coding for a putative glucose- 6-phosphate translocase is mutated in glycogen-storage disease types Ib and Ic.

    PubMed Central

    Veiga-da-Cunha, M; Gerin, I; Chen, Y T; de Barsy, T; de Lonlay, P; Dionisi-Vici, C; Fenske, C D; Lee, P J; Leonard, J V; Maire, I; McConkie-Rosell, A; Schweitzer, S; Vikkula, M; Van Schaftingen, E

    1998-01-01

    Glycogen-storage diseases type I (GSD type I) are due to a deficiency in glucose-6-phosphatase, an enzymatic system present in the endoplasmic reticulum that plays a crucial role in blood glucose homeostasis. Unlike GSD type Ia, types Ib and Ic are not due to mutations in the phosphohydrolase gene and are clinically characterized by the presence of associated neutropenia and neutrophil dysfunction. Biochemical evidence indicates the presence of a defect in glucose-6-phosphate (GSD type Ib) or inorganic phosphate (Pi) (GSD type Ic) transport in the microsomes. We have recently cloned a cDNA encoding a putative glucose-6-phosphate translocase. We have now localized the corresponding gene on chromosome 11q23, the region where GSD types Ib and Ic have been mapped. Using SSCP analysis and sequencing, we have screened this gene, for mutations in genomic DNA, from patients from 22 different families who have GSD types Ib and Ic. Of 20 mutations found, 11 result in truncated proteins that are probably nonfunctional. Most other mutations result in substitutions of conserved or semiconserved residues. The two most common mutations (Gly339Cys and 1211-1212 delCT) together constitute approximately 40% of the disease alleles. The fact that the same mutations are found in GSD types Ib and Ic could indicate either that Pi and glucose-6-phosphate are transported in microsomes by the same transporter or that the biochemical assays used to differentiate Pi and glucose-6-phosphate transport defects are not reliable. PMID:9758626

  14. Time course of radiolabeled 2-deoxy-D-glucose 6-phosphate turnover in cerebral cortex of goats

    SciTech Connect

    Pelligrino, D.A.; Miletich, D.J.; Albrecht, R.F.

    1987-02-01

    The vivo dephosphorylation rate of 2-deoxy-D-glucose 6-phosphate (DGP) in the cerebral cortex of goats injected intravenously with radiolabeled 2-deoxy-D-glucose (DG) was investigated. Serial rapidly frozen samples of parietal cortical gray tissue were obtained at regular intervals over time periods from 45 min to 3 h in awake goats or in paralyzed and artificially ventilated goats maintained under 70% N/sub 2/O or pentobarbital sodium anesthesia. The samples were analyzed for glucose content and separate DG and DGP activities. The rate parameters for phosphorylation (k/sup *//sub 4/) and dephosphorylation (k/sup *//sub 4/) were estimated in each animal. The glucose phosphorylation rate (PR) was calculated over the intervals 3-5 (or 6), 3-10, 3-20, 3-30, and 3-45 min, assuming k/sup *//sub 4/ = O. As the evaluation period was extended beyond 10 min, the calculated PR became increasingly less when compared with that calculated over the 3- to 5- (or 6) min interval (PR/sub i/). Furthermore, as metabolic activity decreased, the magnitude of the error increased such that at 45 min pentobarbital-anesthetize goats underestimated the PR/sub i/ by 46.5% compared with only 23.1 in N/sub 2/O-anesthetized goats. This was also reflected in the >twofold higher k/sup *//sub 4//k/sup *//sub 3/ ratio in the pentobarbital vs. N/sub 2/O-anesthetized group. It is concluded that when using the DG method in the goat, DGP dephosphorylation cannot be ignored when employing >10-min evaluation periods.

  15. Glucose-6-Phosphate-Dehydrogenase Is Also Increased in Erythrocytes from Adolescents with Down Syndrome

    ERIC Educational Resources Information Center

    Ordonez, Francisco J.; Rosety-Plaza, Manuel; Rosety-Rodriguez, Manuel

    2006-01-01

    For some time it has been claimed that trisomic cells are more sensitive to oxidative stress since there is an imbalance in hydrogen peroxide metabolism due to an increase in superoxide dismutase (SOD) catalytic activity. We designed the present study to assess activity levels of antioxidant enzymes [superoxide dismutase (SOD), glutathione…

  16. Glucose-6-phosphate dehydrogenase status and risk of hemolysis in Plasmodium falciparum-infected African children receiving single-dose primaquine.

    PubMed

    Eziefula, Alice C; Pett, Helmi; Grignard, Lynn; Opus, Salome; Kiggundu, Moses; Kamya, Moses R; Yeung, Shunmay; Staedke, Sarah G; Bousema, Teun; Drakeley, Chris

    2014-08-01

    Glucose-6-phosphate dehydrogenase (G6PD) enzyme function and genotype were determined in Ugandan children with uncomplicated falciparum malaria enrolled in a primaquine trial after exclusion of severe G6PD deficiency by fluorescent spot test. G6PD A- heterozygotes and hemizygotes/homozygotes experienced dose-dependent lower hemoglobin concentrations after treatment. No severe anemia was observed. PMID:24913169

  17. Clonal evolution following chemotherapy-induced stem cell depletion in cats heterozygous for glucose-6-phosphate dehydrogenase

    SciTech Connect

    Abkowitz, J.L.; Ott, R.M.; Holly, R.D.; Adamson, J.W.

    1988-06-01

    The number of hematopoietic stem cells necessary to support normal hematopoiesis is not known but may be small. If so, the depletion or damage of such cells could result in apparent clonal dominance. To test this hypothesis, dimethylbusulfan (2 to 4 mg/kg intravenously (IV) x 3) was given to cats heterozygous for the X-linked enzyme glucose-6-phosphate dehydrogenase (G-6-PD). These cats were the daughters of domestic X Geoffroy parents. After the initial drug-induced cytopenias (2 to 4 weeks), peripheral blood counts and the numbers of marrow progenitors detected in culture remained normal, although the percentages of erythroid burst-forming cells (BFU-E) and granulocyte/macrophage colony-forming cells (CFU-GM) in DNA synthesis increased, as determined by the tritiated thymidine suicide technique. In three of six cats treated, a dominance of Geoffroy-type G-6-PD emerged among the progenitor cells, granulocytes, and RBCs. These skewed ratios of domestic to Geoffroy-type G-6-PD have persisted greater than 3 years. No changes in cell cycle kinetics or G-6-PD phenotypes were noted in similar studies in six control cats. These data suggest that clonal evolution may reflect the depletion or damage of normal stem cells and not only the preferential growth and dominance of neoplastic cells.

  18. [Cloning and expression analysis of glucose-6-phosphate dehydrogenase 1 (G6PDH1) gene from Chimonanthus praecox].

    PubMed

    Wang, Xiao-hui; Liu, Xiao; Gao, Bo-wen; Zhang, Zhong-xiu; Shi, She-po; Tu, Peng-fei

    2015-11-01

    Glucose-6-phosphate dehydrogenase is main regulatory enzyme for pentose phosphate pathway. To amplify the core sequence of G6PDH gene from Chimonanthus praecox, the primers were synthesized, based on the conserved nucleotide sequence of other reported plant G6PDH genes. The specific primers were designed according to the major fragment. The full length cDNA of the G6PDH1 gene was isolated by the 3' and 5' rapid amplification of cDNA ends approach. Transcript levels of G6PDH1 isoform was measured by real-time quantitative RT-PCR in different tissues and in responds to cold treatment. The G6PDH1 subcellular localization, transmembrane domain, three-dimensional structure, and phylogenetic analysis were predicted by different software to analysis the bioinformatics of G6PDH1 protein. The G6PDH1 cDNA sequence was 2 011 bp in length and consisted of 1 551 bp Open Reading Frame (ORF) , encoding a protein of 516 amino acids. Expression analysis results in different tissues showed that G6PDH1 was primarily observed in flowers and roots, as opposed to the leaves and stems. Cold treatment experiments indicated that cold treatment caused a rapid increase in G6PDH1 expression in flowers within 12 h. The full-length cDNA of G6PDH1 and its expression analysis will play an important role for further study on cold stress responses in Ch. praecox. PMID:27071249

  19. Ultrasound-Guided Regional Anesthesia in a Glucose-6-Phosphate Dehydrogenase (G6PD)-Deficient Geriatric Trauma Patient

    PubMed Central

    Födinger, Agnes M.; Kammerlander, Christian; Luger, Thomas J.

    2012-01-01

    Objective: Glucose-6-phosphate dehydrogenase (G6PD) deficiency is a genetic enzymatic disorder causing hemolytic anemia. Exposure to drugs is considered to be the most common cause of acute hemolysis in patients with G6PD deficiency. Experience with regional anesthesia, in particular peripheral nerve blocks, is rarely described in patients with G6PD deficiency, but is of great clinical interest. For this reason, we now report on the successful management of ultrasound-guided axillary brachial plexus block in a patient with geriatric G6PD deficiency. Case report: A female, 75-year-old geriatric trauma patient with G6PD deficiency and a fracture of the left forearm, was scheduled for osteosynthesis of the left forearm. For surgery regional anesthesia with ultrasound-guided axillary brachial plexus block with 30 mL bupivacaine 0.5% was established. Surgical operation und postoperative course were uneventful and with no signs of hemolysis. Conclusion: Ultrasound-guided axillary brachial plexus block with bupivacaine was a safe and effective technique in this patient with G6PD deficiency. Peripheral nerve block is a major analgesic approach and of great value for anesthesiologists and surgeons, especially in our aging and multimorbid society. PMID:23569708

  20. Determination of the inhibitory effect of green tea extract on glucose-6-phosphate dehydrogenase based on multilayer capillary enzyme microreactor.

    PubMed

    Camara, Mohamed Amara; Tian, Miaomiao; Liu, Xiaoxia; Liu, Xin; Wang, Yujia; Yang, Jiqing; Yang, Li

    2016-08-01

    Natural herbal medicines are an important source of enzyme inhibitors for the discovery of new drugs. A number of natural extracts such as green tea have been used in prevention and treatment of diseases due to their low-cost, low toxicity and good performance. The present study reports an online assay of the activity and inhibition of the green tea extract of the Glucose 6-phosphate dehydrogenase (G6PDH) enzyme using multilayer capillary electrophoresis based immobilized enzyme microreactors (CE-IMERs). The multilayer CE-IMERs were produced with layer-by-layer electrostatic assembly, which can easily enhance the enzyme loading capacity of the microreactor. The activity of the G6PDH enzyme was determined and the enzyme inhibition by the inhibitors from green tea extract was investigated using online assay of the multilayer CE-IMERs. The Michaelis constant (Km ) of the enzyme, the IC50 and Ki values of the inhibitors were achieved and found to agree with those obtained using offline assays. The results show a competitive inhibition of green tea extract on the G6PDH enzyme. The present study provides an efficient and easy-to-operate approach for determining G6PDH enzyme reaction and the inhibition of green tea extract, which may be beneficial in research and the development of natural herbal medicines. Copyright © 2016 John Wiley & Sons, Ltd.

  1. Application of capillary enzyme micro-reactor in enzyme activity and inhibitors studies of glucose-6-phosphate dehydrogenase.

    PubMed

    Camara, Mohamed Amara; Tian, Miaomiao; Guo, Liping; Yang, Li

    2015-05-15

    In this study, we present an on-line measurement of enzyme activity and inhibition of Glucose-6-phosphate dehydrogenase (G6PDH) enzyme using capillary electrophoresis based immobilized enzyme micro-reactor (CE-based IMER). The IMER was prepared using a two-step protocol based on electrostatic assembly. The micro-reactor exhibited good stability and reproducibility for on-line assay of G6PDH enzyme. Both the activity as well as the inhibition of the G6PDH enzyme by six inhibitors, including three metals (Cu(2+), Pb(2+), Cd(2+)), vancomycin, urea and KMnO4, were investigated using on-line assay of the CE-based IMERs. The enzyme activity and inhibition kinetic constants were measured using the IMERs which were found to be consistent with those using traditional off-line enzyme assays. The kinetic mechanism of each inhibitor was also determined. The present study demonstrates the feasibility of using CE-based IMERs for rapid and efficient on-line assay of G6PDH, an important enzyme in the pentosephosphate pathway of human metabolism.

  2. Influence of the Inherited Glucose-6-phosphate Dehydrogenase Deficiency on the Appearance of Neonatal Hyperbilirubinemia in Southern Croatia

    PubMed Central

    Cherepnalkovski, Anet Papazovska; Marusic, Eugenija; Piperkova, Katica; Lozic, Bernarda; Skelin, Ana; Gruev, Todor; Krzelj, Vjekoslav

    2015-01-01

    Background: Neonatal hyperbilirubinemia is a common clinical manifestation of the inherited glucose-6-phosphate dehydrogenase (G6PD) deficiency. Aim of the study: The aim of this study was to investigate the influence of the inherited G6PD deficiency on the appearance of neonatal hyperbilirubinemia in southern Croatia. Methods: The fluorescent spot test (FST) was used in a retrospective study to screen blood samples of 513 male children who had neonatal hyperbilirubinemia, of unknown cause, higher than 240 μmol/L. Fluorescence readings were performed at the beginning and at the fifth and tenth minute of incubation and were classified into three groups bright fluorescence (BF), weak fluorescence (WF) and no fluorescence (NF). Normal samples show bright fluorescence. All NF and WF samples at the fifth minute were quantitatively measured using the spectrophotometric method. Results: Bright fluorescence was present in 461 patients (89.9%) at the fifth minute. The remaining 52 (10.1%) were quantitatively estimated using the spectrophotometric method. G6PD deficiency was observed in 38 patients (7.4%). Conclusions: Prevalence rate of G6PD deficiency among male newborns with hyperbilirubinemia in southern Croatia is significantly higher (p < 0.01) compared with the previously reported prevalence rate among male in general population of southern Croatia (0.75%). We recommend FST to be performed in hyperbilirubinemic newborns in southern Croatia. PMID:26635431

  3. Clinical mutants of human glucose 6-phosphate dehydrogenase: impairment of NADP(+) binding affects both folding and stability.

    PubMed

    Wang, Xiao-Tao; Engel, Paul C

    2009-08-01

    Human glucose 6-phosphate dehydrogenase (G6PD) has both the "catalytic" NADP(+) site and a "structural" NADP(+) site where a number of severe G6PD deficiency mutations are located. Two pairs of G6PD clinical mutants, G6PD(Wisconsin) (R393G) and G6PD(Nashville) (R393H), and G6PD(Fukaya) (G488S) and G6PD(Campinas) (G488V), in which the mutations are in the vicinity of the "structural" NADP(+) site, showed elevated K(d) values of the "structural" NADP(+), ranging from 53 nM to 500 nM compared with 37 nM for the wild-type enzyme. These recombinant enzymes were denatured by Gdn-HCl and refolded by rapid dilution in the presence of l-Arg, NADP(+) and DTT at 25 degrees C. The refolding yields of the mutants exhibited strong NADP(+)-dependence and ranged from 1.5% to 59.4% with 1000 microM NADP(+), in all cases lower than the figure of 72% for the wild-type enzyme. These mutant enzymes also displayed decreased thermostability and high susceptibility to chymotrypsin digestion, in good agreement with their corresponding melting temperatures in CD experiments. Taken together, the results support the view that impaired binding of "structural" NADP(+) can hinder folding as well as cause instability of these clinical mutant enzymes in the fully folded state.

  4. Elevation of Glucose 6-Phosphate Dehydrogenase Activity Induced by Amplified Insulin Response in Low Glutathione Levels in Rat Liver

    PubMed Central

    Taniguchi, Misako; Mori, Nobuko; Iramina, Chizuru

    2016-01-01

    Weanling male Wistar rats were fed on a 10% soybean protein isolate (SPI) diet for 3 weeks with or without supplementing 0.3% sulfur-containing amino acids (SAA; methionine or cystine) to examine relationship between glutathione (GSH) levels and activities of NADPH-producing enzymes, glucose 6-phosphate dehydrogenase (G6PD) and malic enzyme (ME), in the liver. Of rats on the 10% SPI diet, GSH levels were lower and the enzyme activities were higher than of those fed on an SAA-supplemented diet. Despite the lower GSH level, γ-glutamylcysteine synthetase (γ-GCS) activity was higher in the 10% SPI group than other groups. Examination of mRNAs of G6PD and ME suggested that the GSH-suppressing effect on enzyme induction occurred prior to and/or at transcriptional levels. Gel electrophoresis of G6PD indicated that low GSH status caused a decrease in reduced form and an increase in oxidized form of the enzyme, suggesting an accelerated turnover rate of the enzyme. In primary cultured hepatocytes, insulin response to induce G6PD activity was augmented in low GSH levels manipulated in the presence of buthionine sulfoximine. These findings indicated that elevation of the G6PD activity in low GSH levels was caused by amplified insulin response for expression of the enzyme and accelerated turnover rate of the enzyme molecule. PMID:27597985

  5. Elevation of Glucose 6-Phosphate Dehydrogenase Activity Induced by Amplified Insulin Response in Low Glutathione Levels in Rat Liver

    PubMed Central

    Taniguchi, Misako; Mori, Nobuko; Iramina, Chizuru

    2016-01-01

    Weanling male Wistar rats were fed on a 10% soybean protein isolate (SPI) diet for 3 weeks with or without supplementing 0.3% sulfur-containing amino acids (SAA; methionine or cystine) to examine relationship between glutathione (GSH) levels and activities of NADPH-producing enzymes, glucose 6-phosphate dehydrogenase (G6PD) and malic enzyme (ME), in the liver. Of rats on the 10% SPI diet, GSH levels were lower and the enzyme activities were higher than of those fed on an SAA-supplemented diet. Despite the lower GSH level, γ-glutamylcysteine synthetase (γ-GCS) activity was higher in the 10% SPI group than other groups. Examination of mRNAs of G6PD and ME suggested that the GSH-suppressing effect on enzyme induction occurred prior to and/or at transcriptional levels. Gel electrophoresis of G6PD indicated that low GSH status caused a decrease in reduced form and an increase in oxidized form of the enzyme, suggesting an accelerated turnover rate of the enzyme. In primary cultured hepatocytes, insulin response to induce G6PD activity was augmented in low GSH levels manipulated in the presence of buthionine sulfoximine. These findings indicated that elevation of the G6PD activity in low GSH levels was caused by amplified insulin response for expression of the enzyme and accelerated turnover rate of the enzyme molecule.

  6. Functional and Biochemical Characterization of Three Recombinant Human Glucose-6-Phosphate Dehydrogenase Mutants: Zacatecas, Vanua-Lava and Viangchan.

    PubMed

    Gómez-Manzo, Saúl; Marcial-Quino, Jaime; Vanoye-Carlo, America; Serrano-Posada, Hugo; González-Valdez, Abigail; Martínez-Rosas, Víctor; Hernández-Ochoa, Beatriz; Sierra-Palacios, Edgar; Castillo-Rodríguez, Rosa Angélica; Cuevas-Cruz, Miguel; Rodríguez-Bustamante, Eduardo; Arreguin-Espinosa, Roberto

    2016-05-21

    Glucose-6-phosphate dehydrogenase (G6PD) deficiency in humans causes severe disease, varying from mostly asymptomatic individuals to patients showing neonatal jaundice, acute hemolysis episodes or chronic nonspherocytic hemolytic anemia. In order to understand the effect of the mutations in G6PD gene function and its relation with G6PD deficiency severity, we report the construction, cloning and expression as well as the detailed kinetic and stability characterization of three purified clinical variants of G6PD that present in the Mexican population: G6PD Zacatecas (Class I), Vanua-Lava (Class II) and Viangchan (Class II). For all the G6PD mutants, we obtained low purification yield and altered kinetic parameters compared with Wild Type (WT). Our results show that the mutations, regardless of the distance from the active site where they are located, affect the catalytic properties and structural parameters and that these changes could be associated with the clinical presentation of the deficiency. Specifically, the structural characterization of the G6PD Zacatecas mutant suggests that the R257L mutation have a strong effect on the global stability of G6PD favoring an unstable active site. Using computational analysis, we offer a molecular explanation of the effects of these mutations on the active site.

  7. Genetic heterogeneity of glucose-6-phosphate dehydrogenase deficiency revealed by single-strand conformation and sequence analysis

    SciTech Connect

    Calabro, V.; Mason, P.J.; Luzzatto, L. ); Filosa, S.; Martini, G. ); Civitelli, D.; Cittadella, R.; Brancati, C. )

    1993-03-01

    The authors have carried out a systematic study of the molecular basis of glucose-6-phosphate dehydrogenase (G6PD) deficiency on a sample of 53 male subjects from Calabria, in southern Italy. Their sequential approach consisted of the following steps: (1) Partial biochemical characterization was used to pinpoint candidate known variants. The identity of these was then varified by restriction-enzyme or allele-specific oligonucleotide hybridization analysis of the appropriate PCR-amplified fragment. (2) On samples for which there was no obvious candidate mutation, they proceeded to amplify the entire coding region in eight fragments, followed by single-strand conformation polymorphism (SSCP) analysis of each fragment. (3) The next step was M13 phage cloning and sequencing of those individual fragments that were found to be abnormal by SSCP. Through this approach they have identified the molecular lesion in 51 of the 53 samples. In these they found a total of nine different G6PD-deficient variants, five of which (G6PD Mediterranean, G6PD A[sup [minus

  8. Functional and Biochemical Characterization of Three Recombinant Human Glucose-6-Phosphate Dehydrogenase Mutants: Zacatecas, Vanua-Lava and Viangchan.

    PubMed

    Gómez-Manzo, Saúl; Marcial-Quino, Jaime; Vanoye-Carlo, America; Serrano-Posada, Hugo; González-Valdez, Abigail; Martínez-Rosas, Víctor; Hernández-Ochoa, Beatriz; Sierra-Palacios, Edgar; Castillo-Rodríguez, Rosa Angélica; Cuevas-Cruz, Miguel; Rodríguez-Bustamante, Eduardo; Arreguin-Espinosa, Roberto

    2016-01-01

    Glucose-6-phosphate dehydrogenase (G6PD) deficiency in humans causes severe disease, varying from mostly asymptomatic individuals to patients showing neonatal jaundice, acute hemolysis episodes or chronic nonspherocytic hemolytic anemia. In order to understand the effect of the mutations in G6PD gene function and its relation with G6PD deficiency severity, we report the construction, cloning and expression as well as the detailed kinetic and stability characterization of three purified clinical variants of G6PD that present in the Mexican population: G6PD Zacatecas (Class I), Vanua-Lava (Class II) and Viangchan (Class II). For all the G6PD mutants, we obtained low purification yield and altered kinetic parameters compared with Wild Type (WT). Our results show that the mutations, regardless of the distance from the active site where they are located, affect the catalytic properties and structural parameters and that these changes could be associated with the clinical presentation of the deficiency. Specifically, the structural characterization of the G6PD Zacatecas mutant suggests that the R257L mutation have a strong effect on the global stability of G6PD favoring an unstable active site. Using computational analysis, we offer a molecular explanation of the effects of these mutations on the active site. PMID:27213370

  9. Elevation of Glucose 6-Phosphate Dehydrogenase Activity Induced by Amplified Insulin Response in Low Glutathione Levels in Rat Liver.

    PubMed

    Taniguchi, Misako; Mori, Nobuko; Iramina, Chizuru; Yasutake, Akira

    2016-01-01

    Weanling male Wistar rats were fed on a 10% soybean protein isolate (SPI) diet for 3 weeks with or without supplementing 0.3% sulfur-containing amino acids (SAA; methionine or cystine) to examine relationship between glutathione (GSH) levels and activities of NADPH-producing enzymes, glucose 6-phosphate dehydrogenase (G6PD) and malic enzyme (ME), in the liver. Of rats on the 10% SPI diet, GSH levels were lower and the enzyme activities were higher than of those fed on an SAA-supplemented diet. Despite the lower GSH level, γ-glutamylcysteine synthetase (γ-GCS) activity was higher in the 10% SPI group than other groups. Examination of mRNAs of G6PD and ME suggested that the GSH-suppressing effect on enzyme induction occurred prior to and/or at transcriptional levels. Gel electrophoresis of G6PD indicated that low GSH status caused a decrease in reduced form and an increase in oxidized form of the enzyme, suggesting an accelerated turnover rate of the enzyme. In primary cultured hepatocytes, insulin response to induce G6PD activity was augmented in low GSH levels manipulated in the presence of buthionine sulfoximine. These findings indicated that elevation of the G6PD activity in low GSH levels was caused by amplified insulin response for expression of the enzyme and accelerated turnover rate of the enzyme molecule. PMID:27597985

  10. Overexpression, purification and enzymatic characterization of a recombinant plastidial glucose-6-phosphate dehydrogenase from barley (Hordeum vulgare cv. Nure) roots.

    PubMed

    Cardi, Manuela; Chibani, Kamel; Castiglia, Daniela; Cafasso, Donata; Pizzo, Elio; Rouhier, Nicolas; Jacquot, Jean-Pierre; Esposito, Sergio

    2013-12-01

    In plant cells, the plastidial glucose 6-phosphate dehydrogenase (P2-G6PDH, EC 1.1.1.49) represents one of the most important sources of NADPH. However, previous studies revealed that both native and recombinant purified P2-G6PDHs show a great instability and a rapid loss of catalytic activity. Therefore it has been difficult to describe accurately the catalytic and physico-chemical properties of these isoforms. The plastidial G6PDH encoding sequence from barley roots (Hordeum vulgare cv. Nure), devoid of a long plastidial transit peptide, was expressed as recombinant protein in Escherichia coli, either untagged or with an N-terminal his-tag. After purification from both the soluble fraction and inclusion bodies, we have explored its kinetic parameters, as well as its sensitivity to reduction. The obtained results are consistent with values determined for other P2-G6PDHs previously purified from barley roots and from other land plants. Overall, these data shed light on the catalytic mechanism of plant P2-G6PDH, summarized in a proposed model in which the sequential mechanism is very similar to the mammalian cytosolic G6PDH. This study provides a rational basis to consider the recombinant barley root P2-G6PDH as a good model for further kinetic and structural studies.

  11. Inhibition of Glucose-6-Phosphate Dehydrogenase Could Enhance 1,4-Benzoquinone-Induced Oxidative Damage in K562 Cells

    PubMed Central

    Cao, Meng; Yang, Wenwen; Sun, Fengmei; Xu, Cheng

    2016-01-01

    Benzene is a chemical contaminant widespread in industrial and living environments. The oxidative metabolites of benzene induce toxicity involving oxidative damage. Protecting cells and cell membranes from oxidative damage, glucose-6-phosphate dehydrogenase (G6PD) maintains the reduced state of glutathione (GSH). This study aims to investigate whether the downregulation of G6PD in K562 cell line can influence the oxidative toxicity induced by 1,4-benzoquinone (BQ). G6PD was inhibited in K562 cell line transfected with the specific siRNA of G6PD gene. An empty vector was transfected in the control group. Results revealed that G6PD was significantly upregulated in the control cells and in the cells with inhibited G6PD after they were exposed to BQ. The NADPH/NADP and GSH/GSSG ratio were significantly lower in the cells with inhibited G6PD than in the control cells at the same BQ concentration. The relative reactive oxygen species (ROS) level and DNA oxidative damage were significantly increased in the cell line with inhibited G6PD. The apoptotic rate and G2 phase arrest were also significantly higher in the cells with inhibited G6PD and exposed to BQ than in the control cells. Our results suggested that G6PD inhibition could reduce GSH activity and alleviate oxidative damage. G6PD deficiency is also a possible susceptible risk factor of benzene exposure.

  12. Inhibition of Glucose-6-Phosphate Dehydrogenase Could Enhance 1,4-Benzoquinone-Induced Oxidative Damage in K562 Cells

    PubMed Central

    Cao, Meng; Yang, Wenwen; Sun, Fengmei; Xu, Cheng

    2016-01-01

    Benzene is a chemical contaminant widespread in industrial and living environments. The oxidative metabolites of benzene induce toxicity involving oxidative damage. Protecting cells and cell membranes from oxidative damage, glucose-6-phosphate dehydrogenase (G6PD) maintains the reduced state of glutathione (GSH). This study aims to investigate whether the downregulation of G6PD in K562 cell line can influence the oxidative toxicity induced by 1,4-benzoquinone (BQ). G6PD was inhibited in K562 cell line transfected with the specific siRNA of G6PD gene. An empty vector was transfected in the control group. Results revealed that G6PD was significantly upregulated in the control cells and in the cells with inhibited G6PD after they were exposed to BQ. The NADPH/NADP and GSH/GSSG ratio were significantly lower in the cells with inhibited G6PD than in the control cells at the same BQ concentration. The relative reactive oxygen species (ROS) level and DNA oxidative damage were significantly increased in the cell line with inhibited G6PD. The apoptotic rate and G2 phase arrest were also significantly higher in the cells with inhibited G6PD and exposed to BQ than in the control cells. Our results suggested that G6PD inhibition could reduce GSH activity and alleviate oxidative damage. G6PD deficiency is also a possible susceptible risk factor of benzene exposure. PMID:27656260

  13. On-plate enzyme and inhibition assay of glucose-6-phosphate dehydrogenase using thin-layer chromatography.

    PubMed

    Tian, Miaomiao; Mohamed, Amara Camara; Wang, Shengtian; Yang, Li

    2015-08-01

    We performed on-plate enzyme and inhibition assays of glucose 6-phosphate dehydrogenase using thin-layer chromatography. The assays were accomplished based on different retardation factors of the substrates, enzyme, and products. All the necessary steps were integrated on-plate in one developing process, including substrate/enzyme mixing, reaction starting, and quenching as well as product separation. In order to quantitatively measure the enzyme reaction, the developed plate was then densitometrically evaluated to determine the peak area of the product. Rapid and high-throughput assays were achieved by loading different substrate spots and/or enzyme (and inhibition) spots in different tracks on the plate. The on-plate enzyme assay could be finished in a developing time of only 4 min, with good track-to-track and plate-to-plate repeatability. Moreover, we determined the Km values of the enzyme reaction and Ki values of the inhibition (Pb(2+) Cd(2+) and Cu(2+) as inhibitors), as well as the corresponding kinetics using the on-plate assay. Taken together, our method expanded the application of thin-layer chromatography in enzyme assays, and it could be potentially used in research fields for rapid and quantitative measurement of enzyme activity and inhibition.

  14. Functional and Biochemical Characterization of Three Recombinant Human Glucose-6-Phosphate Dehydrogenase Mutants: Zacatecas, Vanua-Lava and Viangchan

    PubMed Central

    Gómez-Manzo, Saúl; Marcial-Quino, Jaime; Vanoye-Carlo, America; Serrano-Posada, Hugo; González-Valdez, Abigail; Martínez-Rosas, Víctor; Hernández-Ochoa, Beatriz; Sierra-Palacios, Edgar; Castillo-Rodríguez, Rosa Angélica; Cuevas-Cruz, Miguel; Rodríguez-Bustamante, Eduardo; Arreguin-Espinosa, Roberto

    2016-01-01

    Glucose-6-phosphate dehydrogenase (G6PD) deficiency in humans causes severe disease, varying from mostly asymptomatic individuals to patients showing neonatal jaundice, acute hemolysis episodes or chronic nonspherocytic hemolytic anemia. In order to understand the effect of the mutations in G6PD gene function and its relation with G6PD deficiency severity, we report the construction, cloning and expression as well as the detailed kinetic and stability characterization of three purified clinical variants of G6PD that present in the Mexican population: G6PD Zacatecas (Class I), Vanua-Lava (Class II) and Viangchan (Class II). For all the G6PD mutants, we obtained low purification yield and altered kinetic parameters compared with Wild Type (WT). Our results show that the mutations, regardless of the distance from the active site where they are located, affect the catalytic properties and structural parameters and that these changes could be associated with the clinical presentation of the deficiency. Specifically, the structural characterization of the G6PD Zacatecas mutant suggests that the R257L mutation have a strong effect on the global stability of G6PD favoring an unstable active site. Using computational analysis, we offer a molecular explanation of the effects of these mutations on the active site. PMID:27213370

  15. Genetic heterogeneity of glucose-6-phosphate dehydrogenase deficiency revealed by single-strand conformation and sequence analysis.

    PubMed Central

    Calabrò, V; Mason, P J; Filosa, S; Civitelli, D; Cittadella, R; Tagarelli, A; Martini, G; Brancati, C; Luzzatto, L

    1993-01-01

    We have carried out a systematic study of the molecular basis of glucose-6-phosphate dehydrogenase (G6PD) deficiency on a sample of 53 male subjects from Calabria, in southern Italy. Our sequential approach consisted of the following steps: (1) Partial biochemical characterization was used to pinpoint candidate known variants. The identity of these was then verified by restriction-enzyme or allele-specific oligonucleotide hybridization analysis of the appropriate PCR-amplified fragment. (2) On samples for which there was no obvious candidate mutation, we proceeded to amplify the entire coding region in eight fragments, followed by single-strand conformation polymorphism (SSCP) analysis of each fragment. (3) The next step was M13 phage cloning and sequencing of those individual fragments that were found to be abnormal by SSCP. Through this approach we have identified the molecular lesion in 51 of the 53 samples. In these we found a total of nine different G6PD-deficient variants, five of which (G6PD Mediterranean, G6PD A-, G6PD Coimbra, G6PD Seattle, and G6PD Montalbano) were already known, whereas four are new (G6PD Cassano, G6PD Cosenza, G6PD Sibari, and G6PD Maewo). G6PD Mediterranean is the commonest variant, followed by G6PD Seattle. At least seven of the variants are present, at polymorphic frequencies, in the Calabria region, and some have a nonrandom distribution within the region. This study shows that the genetic heterogeneity of G6PD deficiency in Calabria, when analyzed at the DNA level, is even greater than had been anticipated from biochemical characterization. The sequential approach that we have followed is fast and efficient and could be applied to other populations. Images Figure 1 Figure 2 Figure 3 Figure 4 PMID:8447319

  16. Origin and spread of the glucose-6-phosphate dehydrogenase variant (G6PD-Mediterranean) in the Middle East.

    PubMed Central

    Kurdi-Haidar, B; Mason, P J; Berrebi, A; Ankra-Badu, G; al-Ali, A; Oppenheim, A; Luzzatto, L

    1990-01-01

    A common glucose-6-phosphate dehydrogenase (G6PD) variant characterized by severe enzyme deficiency and B-like electrophoretic mobility is called "G6PD-Mediterranean" because it is found in different populations around the Mediterranean Sea. Sequence analysis of Italian subjects has revealed that the molecular basis of G6PD-Mediterranean is a single C-T transition at nucleotide position 563, causing a serine phenylalanine replacement at amino acid position 188. Most G6PD-Mediterranean subjects also have a silent C-T transition (without amino acid replacement) at nucleotide position 1311. Twenty-one unrelated individuals from Saudi Arabia, Iraq, Iran, Jordan, Lebanon, and Israel with both severe G6PD deficiency and B-like electrophoretic mobility were tested for both mutations by using amplification followed by digestion with appropriate restriction enzymes. All but one had the 563 mutation, and, of these, all but one had the 1311 mutation. Another 24 unrelated Middle Eastern individuals with normal G6PD activity or not known to be G6PD deficient were similarly tested. Four had the silent mutation at position 1311 in the absence of the deficiency mutation at position 563. We conclude that (1) the large majority of Middle Eastern subjects with the G6PD-Mediterranean phenotype have the same mutation found in Italy, (2) the silent mutation is an independent polymorphism in the Middle East, with a frequency of about .13, and (3) the mutation leading to the G6PD-Mediterranean deficiency has probably arisen on a chromosome that already carried the silent mutation. Images Figure 2 Figure 3 PMID:1978555

  17. Identification and Characterization of the Glucose-6-Phosphate Dehydrogenase Gene Family in the Para Rubber Tree, Hevea brasiliensis

    PubMed Central

    Long, Xiangyu; He, Bin; Fang, Yongjun; Tang, Chaorong

    2016-01-01

    As a key enzyme in the pentose phosphate pathway (PPP), glucose-6-phosphate dehydrogenase (G6PDH) provides nicotinamide adenine dinucleotide phosphate (NADPH) and intermediary metabolites for rubber biosynthesis, and plays an important role in plant development and stress responses. In this study, four Hevea brasiliensis (Para rubber tree) G6PDH genes (HbG6PDH1 to 4) were identified and cloned using a genome-wide scanning approach. All four HbG6PDH genes encode functional G6PDH enzymes as shown by heterologous expression in E. coli. Phylogeny analysis and subcellular localization prediction show that HbG6PDH3 is a cytosolic isoform, while the other three genes (HbG6PDH1, 2 and 4) are plastidic isoforms. The subcellular locations of HbG6PDH3 and 4, two latex-abundant isoforms were further verified by transient expression in rice protoplasts. Enzyme activity assay and expression analysis showed HbG6PDH3 and 4 were implicated in PPP during latex regeneration, and to influence rubber production positively in rubber tree. The cytosolic HbG6PDH3 is a predominant isoform in latex, implying a principal role for this isoform in controlling carbon flow and NADPH production in the PPP during latex regeneration. The expression pattern of plastidic HbG6PDH4 correlates well with the degree of tapping panel dryness, a physiological disorder that stops the flow of latex from affected rubber trees. In addition, the four HbG6PDHs responded to temperature and drought stresses in root, bark, and leaves, implicating their roles in maintaining redox balance and defending against oxidative stress. PMID:26941770

  18. Autosomal Factors with Correlated Effects on the Activities of the Glucose 6-Phosphate and 6-Phosphogluconate Dehydrogenases in DROSOPHILA MELANOGASTER

    PubMed Central

    Laurie-Ahlberg, C. C.; Williamson, J. H.; Cochrane, B. J.; Wilton, A. N.; Chasalow, F. I.

    1981-01-01

    Isogenic lines, in which chromosomes sampled from natural populations of D. melanogaster are substituted into a common genetic background, were used to detect and partially characterize autosomal factors that affect the activities of the two pentose phosphate pathway enzymes, glucose 6-phosphate dehydrogenase (G6PD) and 6-phosphogluconate dehydrogenase (6PGD). The chromosome 3 effects on G6PD and 6PGD are clearly correlated; the chromosome 2 effects, which are not so great, also appear to be correlated, but the evidence in this case is not so strong. Examination of activity variation of ten other enzymes revealed that G6PD and 6PGD are not the only pair of enzymes showing a high positive correlation, but it is among the highest in both sets of lines. In addition, there was some evidence that the factor(s) affecting G6PD and 6PGD may also affect two other metabolically related enzymes, transaldolase and phosphoglucose isomerase.—Rocket immunoelectrophoresis was used to estimate specific CRM levels for three of the enzymes studied: G6PD, 6PGD and ME. This experiment shows that a large part of the activity variation is accounted for by variation in CRM level (especially for chromosome 3 lines), but there remains a significant fraction of the genetic component of activity variation that is not explained by CRM level.—These results suggest that the autosomal factors are modifiers involved in regulation of the expression of the X-linked structural genes for G6PD and 6PGD, but a role in determining part of the enzymes' primary structure cannot be excluded with the present evidence. PMID:6804300

  19. Prevalence of glucose-6-phosphate dehydrogenase deficiency and diagnostic challenges in 1500 immigrants in Denmark examined for haemoglobinopathies.

    PubMed

    Warny, Marie; Klausen, Tobias Wirenfeldt; Petersen, Jesper; Birgens, Henrik

    2015-09-01

    Similar to the thalassaemia syndromes, glucose-6-phosphate dehydrogenase (G6PD) deficiency is highly prevalent in areas historically exposed to malaria. In the present study, we used quantitative and molecular methods to determine the prevalence of G6PD deficiency in a population of 1508 immigrants in Denmark. We found the allele frequency to be between 2.4 and 2.9% in the female immigrants. Furthermore, the mutation pattern in the studied population showed a high prevalence of the G6PD A-(202A) variant in African and African-American immigrants, a high prevalence of the G6PD Mediterranean variant in Mediterranean European and Western Asian immigrants, and substantial heterogeneity in the variants found in the Eastern Asian/Pacific immigrants. Inasmuch as many of the patients included in this investigation had various thalassaemic syndromes, we were able to evaluate the effects of the interaction between a low mean corpuscular haemoglobin (MCH) value and G6PD activity, particularly in heterozygous females. The activity level was markedly influenced by the MCH value in females with normal G6PD activity, but not in heterozygous and homozygous females. Comparison of patients with normal G6PD activity and heterozygous females indicated considerable overlap in activity levels. To help separating heterozygous females from females with wild-type genes, a DNA analysis is necessary when the female activity level is between 4.0 and 4.9 U/g hgb corresponding to 50-60% of the median activity of unaffected males.

  20. Aspirin inhibits glucose-6-phosphate dehydrogenase activity in HCT 116 cells through acetylation: Identification of aspirin-acetylated sites

    PubMed Central

    Ai, Guoqiang; Dachineni, Rakesh; Kumar, D. Ramesh; Alfonso, Lloyd F.; Marimuthu, Srinivasan; Bhat, G. Jayarama

    2016-01-01

    Glucose-6-phosphate dehydrogenase (G6PD) catalyzes the first reaction in the pentose phosphate pathway, and generates ribose sugars, which are required for nucleic acid synthesis, and nicotinamide adenine dinucleotide phosphate (NADPH), which is important for neutralization of oxidative stress. The expression of G6PD is elevated in several types of tumor, including colon, breast and lung cancer, and has been implicated in cancer cell growth. Our previous study demonstrated that exposure of HCT 116 human colorectal cancer cells to aspirin caused acetylation of G6PD, and this was associated with a decrease in its enzyme activity. In the present study, this observation was expanded to HT-29 colorectal cancer cells, in order to compare aspirin-mediated acetylation of G6PD and its activity between HCT 116 and HT-29 cells. In addition, the present study aimed to determine the acetylation targets of aspirin on recombinant G6PD to provide an insight into the mechanisms of inhibition. The results demonstrated that the extent of G6PD acetylation was significantly higher in HCT 116 cells compared with in HT-29 cells; accordingly, a greater reduction in G6PD enzyme activity was observed in the HCT 116 cells. Mass spectrometry analysis of aspirin-acetylated G6PD (isoform a) revealed that aspirin acetylated a total of 14 lysine residues, which were dispersed throughout the length of the G6PD protein. One of the important amino acid targets of aspirin included lysine 235 (K235, in isoform a) and this corresponds to K205 in isoform b, which has previously been identified as being important for catalysis. Acetylation of G6PD at several sites, including K235 (K205 in isoform b), may mediate inhibition of G6PD activity, which may contribute to the ability of aspirin to exert anticancer effects through decreased synthesis of ribose sugars and NADPH. PMID:27356773

  1. A Population Survey of the Glucose-6-Phosphate Dehydrogenase (G6PD) 563C>T (Mediterranean) Mutation in Afghanistan

    PubMed Central

    Jamornthanyawat, Natsuda; Awab, Ghulam R.; Tanomsing, Naowarat; Pukrittayakamee, Sasithon; Yamin, Fazel; Dondorp, Arjen M.; Day, Nicholas P. J.; White, Nicholas J.; Woodrow, Charles J.; Imwong, Mallika

    2014-01-01

    Glucose-6-phosphate dehydrogenase (G6PD) deficiency is a common inherited enzyme defect and an important problem in areas with Plasmodium vivax infection because of the risk of haemolysis following administration of primaquine to treat the liver forms of the parasite. We undertook a genotypic survey of 713 male individuals across nine provinces of Afghanistan in which malaria is found, four in the north and five in the east. RFLP typing at nucleotide position 563 detected 40 individuals with the Mediterranean mutation 563C>T, an overall prevalence of 5.6%. This varied according to self-reported ethnicity, with prevalence in the Pashtun/Pashai group of 33/369 (8.9%) compared to 7/344 individuals in the rest of the population (2.0%; p<0.001, Chi-squared test). Multivariate analysis of ethnicity and geographical location indicated an adjusted odds ratio of 3.50 (95% CI 1.36–9.02) for the Pashtun/Pashai group, while location showed only a trend towards higher prevalence in eastern provinces (adjusted odds ratio = 1.73, 0.73–4.13). Testing of known polymorphic markers (1311C>T in exon 11, and C93T in intron XI) in a subset of 82 individuals wild-type at C563 revealed a mixture of 3 haplotypes in the background population and was consistent with data from the 1000 Genomes Project and published studies. By comparison individuals with G6PD deficiency showed a highly skewed haplotype distribution, with 95% showing the CT haplotype, a finding consistent with relatively recent appearance and positive selection of the Mediterranean variant in Afghanistan. Overall, the data confirm that the Mediterranean variant of G6PD is common in many ethnic groups in Afghanistan, indicating that screening for G6PD deficiency is required in all individuals before radical treatment of P. vivax with primaquine. PMID:24586352

  2. Glucose-6-Phosphate Dehydrogenase and NADPH Redox Regulates Cardiac Myocyte L-Type Calcium Channel Activity and Myocardial Contractile Function

    PubMed Central

    Rawat, Dhwajbahadur K.; Hecker, Peter; Watanabe, Makino; Chettimada, Sukrutha; Levy, Richard J.; Okada, Takao; Edwards, John G.; Gupte, Sachin A.

    2012-01-01

    We recently demonstrated that a 17-ketosteroid, epiandrosterone, attenuates L-type Ca2+ currents (ICa-L) in cardiac myocytes and inhibits myocardial contractility. Because 17-ketosteroids are known to inhibit glucose-6-phosphate dehydrogenase (G6PD), the rate-limiting enzyme in the pentose phosphate pathway, and to reduce intracellular NADPH levels, we hypothesized that inhibition of G6PD could be a novel signaling mechanism which inhibit ICa-L and, therefore, cardiac contractile function. We tested this idea by examining myocardial function in isolated hearts and Ca2+ channel activity in isolated cardiac myocytes. Myocardial function was tested in Langendorff perfused hearts and ICa-L were recorded in the whole-cell patch configuration by applying double pulses from a holding potential of −80 mV and then normalized to the peak amplitudes of control currents. 6-Aminonicotinamide, a competitive inhibitor of G6PD, increased pCO2 and decreased pH. Additionally, 6-aminonicotinamide inhibited G6PD activity, reduced NADPH levels, attenuated peak ICa-L amplitudes, and decreased left ventricular developed pressure and ±dp/dt. Finally, dialyzing NADPH into cells from the patch pipette solution attenuated the suppression of ICa-L by 6-aminonicotinamide. Likewise, in G6PD-deficient mice, G6PD insufficiency in the heart decreased GSH-to-GSSG ratio, superoxide, cholesterol and acetyl CoA. In these mice, M-mode echocardiographic findings showed increased diastolic volume and end-diastolic diameter without changes in the fraction shortening. Taken together, these findings suggest that inhibiting G6PD activity and reducing NADPH levels alters metabolism and leads to inhibition of L-type Ca2+ channel activity. Notably, this pathway may be involved in modulating myocardial contractility under physiological and pathophysiological conditions during which the pentose phosphate pathway-derived NADPH redox is modulated (e.g., ischemia-reperfusion and heart failure). PMID:23071515

  3. Prevalence of glucose-6-phosphate dehydrogenase (G6PD) deficiency in the Ouest and Sud-Est departments of Haiti.

    PubMed

    von Fricken, Michael E; Weppelmann, Thomas A; Eaton, Will T; Alam, Meer T; Carter, Tamar E; Schick, Laura; Masse, Roseline; Romain, Jean R; Okech, Bernard A

    2014-07-01

    Malaria remains a significant public health issue in Haiti, with chloroquine (CQ) used almost exclusively for the treatment of uncomplicated infections. Recently, single dose primaquine (PQ) was added to the Haitian national malaria treatment policy, despite a lack of information on the prevalence of glucose-6-phosphate dehydrogenase (G6PD) deficiency within the population. G6PD deficient individuals who take PQ are at risk of developing drug induced hemolysis (DIH). In this first study to examine G6PD deficiency rates in Haiti, 22.8% (range 14.9%-24.7%) of participants were found to be G6PD deficient (class I, II, or III) with 2.0% (16/800) of participants having severe deficiency (class I and II). Differences in deficiency were observed by gender, with males having a much higher prevalence of severe deficiency (4.3% vs. 0.4%) compared to females. Male participants were 1.6 times more likely to be classified as deficient and 10.6 times more likely to be classified as severely deficient compared to females, as expected. Finally, 10.6% (85/800) of the participants were considered to be at risk for DIH. Males also had much higher rates than females (19.3% vs. 4.6%) with 4.9 times greater likelihood (p value 0.000) of having an activity level that could lead to DIH. These findings provide useful information to policymakers and clinicians who are responsible for the implementation of PQ to control and manage malaria in Haiti.

  4. The oxidative pentose phosphate pathway in the haloarchaeon Haloferax volcanii involves a novel type of glucose-6-phosphate dehydrogenase--The archaeal Zwischenferment.

    PubMed

    Pickl, Andreas; Schönheit, Peter

    2015-04-28

    The oxidative pentose phosphate pathway (OPPP), catalyzing the oxidation of glucose-6-phosphate to ribulose-5-phosphate is ubiquitous in eukarya and bacteria but has not yet been reported in archaea. In haloarchaea a putative 6-phosphogluconate dehydrogenase (6PGDH) is annotated, whereas a gene coding for glucose-6-phosphate dehydrogenase (Glc6PDH) could not be identified. Here we report the purification and characterization of a novel type of Glc6PDH in Haloferax volcanii that is not related to bacterial and eukaryal Glc6PDHs and the encoding gene is designated as azf (archaeal zwischenferment). Further, recombinant H. volcanii 6PGDH was characterized. Deletion mutant analyses indicate that both, Glc6PDH and 6PGDH, are functionally involved in pentose phosphate formation in vivo. This is the first report on the operation of the OPPP in the domain of archaea.

  5. Data on how several physiological parameters of stored red blood cells are similar in glucose 6-phosphate dehydrogenase deficient and sufficient donors.

    PubMed

    Tzounakas, Vassilis L; Kriebardis, Anastasios G; Georgatzakou, Hara T; Foudoulaki-Paparizos, Leontini E; Dzieciatkowska, Monika; Wither, Matthew J; Nemkov, Travis; Hansen, Kirk C; Papassideri, Issidora S; D'Alessandro, Angelo; Antonelou, Marianna H

    2016-09-01

    This article contains data on the variation in several physiological parameters of red blood cells (RBCs) donated by eligible glucose-6-phosphate dehydrogenase (G6PD) deficient donors during storage in standard blood bank conditions compared to control, G6PD sufficient (G6PD(+)) cells. Intracellular reactive oxygen species (ROS) generation, cell fragility and membrane exovesiculation were measured in RBCs throughout the storage period, with or without stimulation by oxidants, supplementation of N-acetylcysteine and energy depletion, following incubation of stored cells for 24 h at 37 °C. Apart from cell characteristics, the total or uric acid-dependent antioxidant capacity of the supernatant in addition to extracellular potassium concentration was determined in RBC units. Finally, procoagulant activity and protein carbonylation levels were measured in the microparticles population. Further information can be found in "Glucose 6-phosphate dehydrogenase deficient subjects may be better "storers" than donors of red blood cells" [1]. PMID:27437434

  6. Acute viral hepatitis E presenting with haemolytic anaemia and acute renal failure in a patient with glucose-6-phosphate dehydrogenase deficiency.

    PubMed

    Tomar, Laxmikant Ramkumarsingh; Aggarwal, Amitesh; Jain, Piyush; Rajpal, Surender; Agarwal, Mukul P

    2015-10-01

    The association of acute hepatitis E viral (HEV) infection with glucose-6-phosphate dehydrogenase (G6PD) deficiency leading to extensive intravascular haemolysis is a very rare clinical entity. Here we discuss such a patient, who presented with acute HEV illness, developed severe intravascular haemolysis and unusually high levels of bilirubin, complicated by acute renal failure (ARF), and was later on found to have a deficiency of G6PD. The patient recovered completely with haemodialysis and supportive management. PMID:25500531

  7. Determinants of Cofactor Specificity for the Glucose-6-Phosphate Dehydrogenase from Escherichia coli: Simulation, Kinetics and Evolutionary Studies

    PubMed Central

    Fuentealba, Matias; Muñoz, Rodrigo; Maturana, Pablo; Krapp, Adriana; Cabrera, Ricardo

    2016-01-01

    Glucose 6-Phosphate Dehydrogenases (G6PDHs) from different sources show varying specificities towards NAD+ and NADP+ as cofactors. However, it is not known to what extent structural determinants of cofactor preference are conserved in the G6PDH family. In this work, molecular simulations, kinetic characterization of site-directed mutants and phylogenetic analyses were used to study the structural basis for the strong preference towards NADP+ shown by the G6PDH from Escherichia coli. Molecular Dynamics trajectories of homology models showed a highly favorable binding energy for residues K18 and R50 when interacting with the 2'-phosphate of NADP+, but the same residues formed no observable interactions in the case of NAD+. Alanine mutants of both residues were kinetically characterized and analyzed with respect to the binding energy of the transition state, according to the kcat/KM value determined for each cofactor. Whereas both residues contribute to the binding energy of NADP+, only R50 makes a contribution (about -1 kcal/mol) to NAD+ binding. In the absence of both positive charges the enzyme was unable to discriminate NADP+ from NAD+. Although kinetic data is sparse, the observed distribution of cofactor preferences within the phylogenetic tree is sufficient to rule out the possibility that the known NADP+-specific G6PDHs form a monophyletic group. While the β1-α1 loop shows no strict conservation of K18, (rather, S and T seem to be more frequent), in the case of the β2-α2 loop, different degrees of conservation are observed for R50. Noteworthy is the fact that a K18T mutant is indistinguishable from K18A in terms of cofactor preference. We conclude that the structural determinants for the strict discrimination against NAD+ in the case of the NADP+-specific enzymes have evolved independently through different means during the evolution of the G6PDH family. We further suggest that other regions in the cofactor binding pocket, besides the β1-α1 and β2-α2

  8. Prevalence and molecular characterization of Glucose-6-Phosphate dehydrogenase deficient variants among the Kurdish population of Northern Iraq

    PubMed Central

    2010-01-01

    Background Glucose-6-Phosphate dehydrogenase (G6PD) is a key enzyme of the pentose monophosphate pathway, and its deficiency is the most common inherited enzymopathy worldwide. G6PD deficiency is common among Iraqis, including those of the Kurdish ethnic group, however no study of significance has ever addressed the molecular basis of this disorder in this population. The aim of this study is to determine the prevalence of this enzymopathy and its molecular basis among Iraqi Kurds. Methods A total of 580 healthy male Kurdish Iraqis randomly selected from a main regional premarital screening center in Northern Iraq were screened for G6PD deficiency using methemoglobin reduction test. The results were confirmed by quantitative enzyme assay for the cases that showed G6PD deficiency. DNA analysis was performed on 115 G6PD deficient subjects, 50 from the premarital screening group and 65 unrelated Kurdish male patients with documented acute hemolytic episodes due to G6PD deficiency. Analysis was performed using polymerase chain reaction/restriction fragment length polymorphism for five deficient molecular variants, namely G6PD Mediterranean (563 C→T), G6PD Chatham (1003 G→A), G6PD A- (202 G→A), G6PD Aures (143 T→C) and G6PD Cosenza (1376 G→C), as well as the silent 1311 (C→T) mutation. Results Among 580 random Iraqi male Kurds, 63 (10.9%) had documented G6PD deficiency. Molecular studies performed on a total of 115 G6PD deficient males revealed that 101 (87.8%) had the G6PD Mediterranean variant and 10 (8.7%) had the G6PD Chatham variant. No cases of G6PD A-, G6PD Aures or G6PD Cosenza were identified, leaving 4 cases (3.5%) uncharacterized. Further molecular screening revealed that the silent mutation 1311 was present in 93/95 of the Mediterranean and 1/10 of the Chatham cases. Conclusions The current study revealed a high prevalence of G6PD deficiency among Iraqi Kurdish population of Northern Iraq with most cases being due to the G6PD Mediterranean and

  9. Glucose-6-phosphate dehydrogenase regulation in the hepatopancreas of the anoxia-tolerant marine mollusc, Littorina littorea.

    PubMed

    Lama, Judeh L; Bell, Ryan A V; Storey, Kenneth B

    2013-01-01

    Glucose-6-phosphate dehydrogenase (G6PDH) gates flux through the pentose phosphate pathway and is key to cellular antioxidant defense due to its role in producing NADPH. Good antioxidant defenses are crucial for anoxia-tolerant organisms that experience wide variations in oxygen availability. The marine mollusc, Littorina littorea, is an intertidal snail that experiences daily bouts of anoxia/hypoxia with the tide cycle and shows multiple metabolic and enzymatic adaptations that support anaerobiosis. This study investigated the kinetic, physical and regulatory properties of G6PDH from hepatopancreas of L. littorea to determine if the enzyme is differentially regulated in response to anoxia, thereby providing altered pentose phosphate pathway functionality under oxygen stress conditions. Several kinetic properties of G6PDH differed significantly between aerobic and 24 h anoxic conditions; compared with the aerobic state, anoxic G6PDH (assayed at pH 8) showed a 38% decrease in K m G6P and enhanced inhibition by urea, whereas in pH 6 assays K m NADP and maximal activity changed significantly between the two states. The mechanism underlying anoxia-responsive changes in enzyme properties proved to be a change in the phosphorylation state of G6PDH. This was documented with immunoblotting using an anti-phosphoserine antibody, in vitro incubations that stimulated endogenous protein kinases versus protein phosphatases and significantly changed K m G6P, and phosphorylation of the enzyme with (32)P-ATP. All these data indicated that the aerobic and anoxic forms of G6PDH were the high and low phosphate forms, respectively, and that phosphorylation state was modulated in response to selected endogenous protein kinases (PKA or PKG) and protein phosphatases (PP1 or PP2C). Anoxia-induced changes in the phosphorylation state of G6PDH may facilitate sustained or increased production of NADPH to enhance antioxidant defense during long term anaerobiosis and/or during the transition

  10. Glucose-6-phosphate dehydrogenase deficiency among children attending the Emergency Paediatric Unit of Usmanu Danfodiyo University Teaching Hospital, Sokoto, Nigeria

    PubMed Central

    Isaac, IZ; Mainasara, AS; Erhabor, Osaro; Omojuyigbe, ST; Dallatu, MK; Bilbis, LS; Adias, TC

    2013-01-01

    Glucose-6-phosphate dehydrogenase (G6PD) deficiency is one of the most common human enzyme deficiencies in the world. It is particularly common in populations living in malaria-endemic areas, affecting more than 400 million people worldwide. This present study was conducted with the aim of determining the prevalence of G6PD deficiency among children visiting the Emergency Paediatric Unit of Usmanu Danfodiyo University Teaching Hospital for pediatric-related care. The study included 118 children, made up of 77 (65.3%) males and 41 (34.7%) females aged ≤5 years with mean age of 3.26 ± 1.90 years. Randox G6PD quantitative in vitro test screening was used for the diagnosis of G6PD deficiency. Of the 118 children tested, 17 (14.4%) were G6PD-deficient. Prevalence of G6PD deficiency was concentrated predominantly among male children (22.1%). Male sex was significantly correlated with G6PD deficiency among the children studied (r = 7.85, P = 0.01). The highest prevalence occurred among children in the 2- to 5-year age-group. Of the 17 G6PD-deficient children, twelve (70.2%) were moderately deficient, while five (29.4%) were severely deficient. Blood film from G6PD-deficient children indicated the following morphological changes; Heinz bodies, schistocytes, target cells, nucleated red cells, spherocytes, and polychromasia. This present study has shown a high prevalence of G6PD deficiency among children residing in Sokoto in the northwestern geopolitical zone of Nigeria. The study indicated a male sex bias in the prevalence of G6PD deficiency among the children studied. There is a need for the routine screening of children for G6PD deficiency in our environment, to allow for evidence-based management of these children and to ensure the avoidance of food, drugs, and infective agents that can potentially predispose these children to oxidative stress as well as diseases that deplete micronutrients that protect against oxidative stress. There is need to build capacity in our

  11. Determinants of Cofactor Specificity for the Glucose-6-Phosphate Dehydrogenase from Escherichia coli: Simulation, Kinetics and Evolutionary Studies.

    PubMed

    Fuentealba, Matias; Muñoz, Rodrigo; Maturana, Pablo; Krapp, Adriana; Cabrera, Ricardo

    2016-01-01

    Glucose 6-Phosphate Dehydrogenases (G6PDHs) from different sources show varying specificities towards NAD+ and NADP+ as cofactors. However, it is not known to what extent structural determinants of cofactor preference are conserved in the G6PDH family. In this work, molecular simulations, kinetic characterization of site-directed mutants and phylogenetic analyses were used to study the structural basis for the strong preference towards NADP+ shown by the G6PDH from Escherichia coli. Molecular Dynamics trajectories of homology models showed a highly favorable binding energy for residues K18 and R50 when interacting with the 2'-phosphate of NADP+, but the same residues formed no observable interactions in the case of NAD+. Alanine mutants of both residues were kinetically characterized and analyzed with respect to the binding energy of the transition state, according to the kcat/KM value determined for each cofactor. Whereas both residues contribute to the binding energy of NADP+, only R50 makes a contribution (about -1 kcal/mol) to NAD+ binding. In the absence of both positive charges the enzyme was unable to discriminate NADP+ from NAD+. Although kinetic data is sparse, the observed distribution of cofactor preferences within the phylogenetic tree is sufficient to rule out the possibility that the known NADP+-specific G6PDHs form a monophyletic group. While the β1-α1 loop shows no strict conservation of K18, (rather, S and T seem to be more frequent), in the case of the β2-α2 loop, different degrees of conservation are observed for R50. Noteworthy is the fact that a K18T mutant is indistinguishable from K18A in terms of cofactor preference. We conclude that the structural determinants for the strict discrimination against NAD+ in the case of the NADP+-specific enzymes have evolved independently through different means during the evolution of the G6PDH family. We further suggest that other regions in the cofactor binding pocket, besides the β1-α1 and β2-α2

  12. Expression, crystallization and preliminary X-ray crystallographic analysis of glucose-6-phosphate dehydrogenase from the human pathogen Trypanosoma cruzi in complex with substrate

    PubMed Central

    Ortíz, Cecilia; Larrieux, Nicole; Medeiros, Andrea; Botti, Horacio; Comini, Marcelo; Buschiazzo, Alejandro

    2011-01-01

    An N-terminally truncated version of the enzyme glucose-6-phosphate dehydrogenase from Trypanosoma cruzi lacking the first 37 residues was crystallized both in its apo form and in a binary complex with glucose 6-­phosphate. The crystals both belonged to space group P21 and diffracted to 2.85 and 3.35 Å resolution, respectively. Self-rotation function maps were consistent with point group 222. The structure was solved by molecular replacement, confirming a tetrameric quaternary structure. PMID:22102256

  13. Influence of glucose solution on the erythrocyte scattering properties

    NASA Astrophysics Data System (ADS)

    Naumenko, Elena K.

    2007-02-01

    The scattering characteristics of erythrocytes (the coefficients of extinction, scattering, absorption and indicatrixes) were calculated with using the theory Mie for spherical homogeneous spherical particles and the theory for two-layered spherical concentric particles. Transmission spectrums were measured with the spectrophotometer Cary500 in the wavelength range 460-860 n m. Specimens of liquid for imbedding of erythrocytes were preparing by mixing blood plasma a nd 50-% glucose solution with the different concentrations. The volume concentrations (hematocrit) of red blood cells (RBC) were maintained to have the same values in all specimens by adding equal volume of whole blood to immersion liquid of equal volumes. It has been shown that, contrary to theretical prediction, transmission is decreasing for all wavelengths with the addition of glucose solution in interval glucose volume concentrations 0.05 - 0.35-0.4. The subsequent increase of the glucose concentration leads to increasing of spectral transmission as a result of erythrocyte hemolysis.

  14. Effect of high glucose concentrations on human erythrocytes in vitro

    PubMed Central

    Viskupicova, Jana; Blaskovic, Dusan; Galiniak, Sabina; Soszyński, Mirosław; Bartosz, Grzegorz; Horakova, Lubica; Sadowska-Bartosz, Izabela

    2015-01-01

    Exposure to high glucose concentrations in vitro is often employed as a model for understanding erythrocyte modifications in diabetes. However, effects of such experiments may be affected by glucose consumption during prolonged incubation and changes of cellular parameters conditioned by impaired energy balance. The aim of this study was to compare alterations in various red cell parameters in this type of experiment to differentiate between those affected by glycoxidation and those affected by energy imbalance. Erythrocytes were incubated with 5, 45 or 100 mM glucose for up to 72 h. High glucose concentrations intensified lipid peroxidation and loss of activities of erythrocyte enzymes (glutathione S-transferase and glutathione reductase). On the other hand, hemolysis, eryptosis, calcium accumulation, loss of glutathione and increase in the GSSG/GSH ratio were attenuated by high glucose apparently due to maintenance of energy supply to the cells. Loss of plasma membrane Ca2+-ATPase activity and decrease in superoxide production were not affected by glucose concentration, being seemingly determined by processes independent of both glycoxidation and energy depletion. These results point to the necessity of careful interpretation of data obtained in experiments, in which erythrocytes are subject to treatment with high glucose concentrations in vitro. PMID:26141922

  15. Development and altered gravity dependent changes in glucose-6-phosphate dehydrogenase activity in the brain of the cichlid fish Oreochromis mossambicus.

    PubMed

    Slenzka, K; Appel, R; Rahmann, H

    1995-06-01

    Glucose-6-phosphate dehydrogenase activity was studied in the brain of the cichlid fish Oreochromis mossambicus during early ontogenetic development. In general a slight but continuous decrease in enzyme activity was found (9.5 +/- 0.5 nmol substrate cleaved per mg protein and per min at developmental stage 13 [= 1 day post hatch at 28 degrees C] to a value of 7.9 +/- 0.6 in adult brain). In order to investigate the possible influence of altered gravity during early ontogenetic brain development, fish larvae were exposed to an increased acceleration of three times earth gravity (3 g) or to functional weightlessness in a fast-rotating clinostat for 7 days. A significant increase of brain G6PDH activity of approx. 15% was found after exposure to hyper gravity, whereas a significant decrease of the enzyme activity, approximately 10%, was detected following functional weightlessness in respect to the corresponding 1 g controls. Analyses concerning the regain of normal control enzyme activity of the larvae revealed dramatic fluctuations within the first 5 h after exposure to an increased acceleration of 3 g. Thereafter, between day 1 and day 3 after exposure, brain glucose-6-phosphate dehydrogenase decreased slowly. At day 3 after exposure no further differences of the hyper-g larvae compared to the controls were found. Only slight changes in total brain glucose-6-phosphate dehydrogenase activity occur during ontogenetic development of cichlid fish. This suggests that a more or less constant enzyme activity is important during brain development, but is reacting very sensitively to changes in the environmental factor gravity.

  16. Unsuspected glucose-6-phosphate dehydrogenase deficiency presenting as symptomatic methemoglobinemia with severe hemolysis after fava bean ingestion in a 6-year-old boy.

    PubMed

    Odièvre, Marie-Hélène; Danékova, Névéna; Mesples, Bettina; Chemouny, Myriam; Couque, Nathalie; Parez, Nathalie; Ducrocq, Rolande; Elion, Jacques

    2011-05-01

    We report the occurrence of symptomatic methemoglobinemia in a previously healthy boy, who presented with severe acute hemolysis after fava bean ingestion. The methemoglobinemia revealed a previously unrecognized glucose-6-phosphate dehydrogenase (G6PD) deficiency. We discuss the pathophysiology of severe methemoglobinemia when associated with acute hemolysis, favism, and the common African G6PD A-variant [G6PD, VAL68MET, ASN126ASP]. In conclusion, screening for G6PD deficiency must be considered in symptomatic methemoglobinemia, especially in young boys, when associated with intravascular hemolysis.

  17. Unsuspected glucose-6-phosphate dehydrogenase deficiency presenting as symptomatic methemoglobinemia with severe hemolysis after fava bean ingestion in a 6-year-old boy.

    PubMed

    Odièvre, Marie-Hélène; Danékova, Névéna; Mesples, Bettina; Chemouny, Myriam; Couque, Nathalie; Parez, Nathalie; Ducrocq, Rolande; Elion, Jacques

    2011-05-01

    We report the occurrence of symptomatic methemoglobinemia in a previously healthy boy, who presented with severe acute hemolysis after fava bean ingestion. The methemoglobinemia revealed a previously unrecognized glucose-6-phosphate dehydrogenase (G6PD) deficiency. We discuss the pathophysiology of severe methemoglobinemia when associated with acute hemolysis, favism, and the common African G6PD A-variant [G6PD, VAL68MET, ASN126ASP]. In conclusion, screening for G6PD deficiency must be considered in symptomatic methemoglobinemia, especially in young boys, when associated with intravascular hemolysis. PMID:21479984

  18. pH-induced bistable dynamic behaviour in the reaction catalysed by glucose-6-phosphate dehydrogenase and conformational hysteresis of the enzyme.

    PubMed Central

    Aon, M A; Cortassa, S; Hervagault, J F; Thomas, D

    1989-01-01

    1. Bistable (multiple stationary states) dynamic behaviour in the activity of glucose-6-phosphate dehydrogenase that was subjected to successive pH change was demonstrated in an open continuously stirred tank reactor. Although the enzyme under study did not exhibit an autocatalytic effect and was homogeneously distributed, bistability was shown to occur. 2. The successive pH changes of the enzyme solution corresponded to a pH transition (8.3 in equilibrium 2), i.e. an acidification (forward direction) and an alkalinization (reverse direction). By use of intrinsic protein fluorescence methods, a glucose-6-phosphate dehydrogenase conformational hysteresis was shown to exist concomitant with the pH transition before and after enzyme injection into the reactor. 3. The results obtained suggest that the enzyme behaves, conformationally, as a memory device that stores information about its pH history (i.e. the enzyme records information in its structure about the environment to which it was previously exposed) and transduces it in a non-linear dynamic fashion, producing the bistable behaviour observed in the open reactor. PMID:2590166

  19. Deletion of the Glucose-6-Phosphate Dehydrogenase Gene KlZWF1 Affects both Fermentative and Respiratory Metabolism in Kluyveromyces lactis▿

    PubMed Central

    Saliola, Michele; Scappucci, Gina; De Maria, Ilaria; Lodi, Tiziana; Mancini, Patrizia; Falcone, Claudio

    2007-01-01

    In Kluyveromyces lactis, the pentose phosphate pathway is an alternative route for the dissimilation of glucose. The first enzyme of the pathway is the glucose-6-phosphate dehydrogenase (G6PDH), encoded by KlZWF1. We isolated this gene and examined its role. Like ZWF1 of Saccharomyces cerevisiae, KlZWF1 was constitutively expressed, and its deletion led to increased sensitivity to hydrogen peroxide on glucose, but unlike the case for S. cerevisiae, the Klzwf1Δ strain had a reduced biomass yield on fermentative carbon sources as well as on lactate and glycerol. In addition, the reduced yield on glucose was associated with low ethanol production and decreased oxygen consumption, indicating that this gene is required for both fermentation and respiration. On ethanol, however, the mutant showed an increased biomass yield. Moreover, on this substrate, wild-type cells showed an additional band of activity that might correspond to a dimeric form of G6PDH. The partial dimerization of the G6PDH tetramer on ethanol suggested the production of an NADPH excess that was negative for biomass yield. PMID:17085636

  20. Molecular characterization of the first two enzymes of the pentose-phosphate pathway of Trypanosoma brucei. Glucose-6-phosphate dehydrogenase and 6-phosphogluconolactonase.

    PubMed

    Duffieux, F; Van Roy, J; Michels, P A; Opperdoes, F R

    2000-09-01

    Trypanosomatids are parasitic protists that have part of their glycolytic pathway sequestered inside peroxisome-like organelles: the glycosomes. So far, at least one enzyme of the pentose-phosphate pathway has been found to be associated partially with glycosomes. Here, we describe how two genes from Trypanosoma brucei, coding for the first two enzymes of the pentose-phosphate pathway, i.e. glucose-6-phosphate dehydrogenase and 6-phosphogluconolactonase, were identified by in silico screening of trypanosome genome project data bases. These genes were cloned and sequenced. Analysis of the lactonase sequence revealed that it contained a C-terminal peroxisome targeting signal in agreement with its subcellular localization in the bloodstream form trypanosome (15% glycosomal and 85% cytosolic). However, the dehydrogenase sequence did not reveal any targeting signal, despite its localization inside glycosomes. The corresponding enzymes have been overexpressed in Escherichia coli and purified, and their biochemical characteristics have been determined.

  1. Marked differences in drug-induced methemoglobinemia in sheep are not due to RBC glucose-6-phosphate dehydrogenase, reduced glutathione, or methemoglobin reductase activity

    SciTech Connect

    Martin, D.G.; Guertler, A.T.; Lagutchik, M.S.; Woodard, C.L.; Leonard, D.A.

    1993-05-13

    Benzocaine is a commonly used topical anesthetic that is structurally similar to current candidates for cyanide prophylaxis. Benzocaine induces profound methemoglobinemia in some sheep but not others. After topical benzocaine administration certain sheep respond to form MHb (elevated MHb 16-50% after a 56-280 mg dose, a 2-10 second spray with benzocine), while other phenotypically similar sheep fail to significantly form MHb (less than a 2% increase from baseline). Deficiencies in Glucose-6-phosphate dehydrogenase (G-6-PD), reduced glutathione (GSH), and MHb reductase increase the susceptibility to methemoglobinemia in man and animals. Sheep are used as a model for G-6-PD deficiency in man, and differences in this enzyme level could cause the variable response seen in these sheep. Similarly, differences in GSH and MHb reductase could be responsible for the observed differences in MHb formation.

  2. Molecular Epidemiological Survey of Glucose-6-Phosphate Dehydrogenase Deficiency and Thalassemia in Uygur and Kazak Ethnic Groups in Xinjiang, Northwest China.

    PubMed

    Han, Luhao; Su, Hai; Wu, Hao; Jiang, Weiying; Chen, Suqin

    2016-06-01

    Glucose-6-phosphate dehydrogenase (G6PD) deficiency and thalassemia occur frequently in tropical and subtropical regions, while the prevalence of relationship between the two diseases in Xinjiang has not been reported. We aimed to determine the prevalence of these diseases and clarify the relationship between genotypes and phenotypes of the two diseases in the Uygur and Kazak ethnic groups in Xinjiang. We measured G6PD activity by G6PD:6PGD (glucose acid-6-phosphate dehydrogenase) ratio, identified the gene variants of G6PD and α- and β-globin genes by polymerase chain reaction (PCR)-DNA sequencing and gap-PCR and compared these variants in different ethnic groups in Xinjiang with those adjacent to it. Of the 149 subjects with molecular analysis of G6PD deficiency conducted, a higher prevalence of the combined mutations c.1311C > T/IVSXI + 93T > C and IVSXI + 93T > C, both with normal enzymatic activities, were observed in the Uygur and Kazak subjects. A case of rare mutation HBB: c.135delC [codon 44 (-C) in the heterozygous state], a heterozygous case of HBB: c.68A > G [Hb G-Taipei or β22(B4)Glu→Gly] and several common single nucleotide polymorphisms (SNPs) were found on the β-globin gene. In conclusion, G6PD deficiency with pathogenic mutations and three common α-thalassemia (α-thal) [- -(SEA), -α(3.7) (rightward), -α(4.2) (leftward)] deletions and point mutations of the α-globin gene were not detected in the present study. The average incidence of β-thalassemia (β-thal) in Uygurs was 1.45% (2/138) in Xinjiang. The polymorphisms of G6PD and β-globin genes might be useful genetic markers to trace the origin and migration of the Uygur and Kazak in Xinjiang. PMID:26950205

  3. Enhanced production of epsilon-caprolactone by overexpression of NADPH-regenerating glucose 6-phosphate dehydrogenase in recombinant Escherichia coli harboring cyclohexanone monooxygenase gene.

    PubMed

    Lee, Won-Heong; Park, Jin-Byung; Park, Kyungmoon; Kim, Myoung-Dong; Seo, Jin-Ho

    2007-08-01

    Whole-cell conversion of cyclohexanone to epsilon-caprolactone was attempted by recombinant Escherichia coli BL21(DE3) expressing cyclohexanone monooxygenase (CHMO) of Acinetobacter calcoaceticus NCIMB 9871. High concentrations of cyclohexanone and epsilon-caprolactone reduced CHMO-mediated bioconversion of cyclohexanone to epsilon-caprolactone in the resting recombinant E. coli cells. Metabolically active cells were employed by adopting a fed-batch culture to improve the production of epsilon-caprolactone from cyclohexanone. A glucose-limited fed-batch Baeyer-Villiger oxidation where a cyclohexanone level was maintained less than 6 g/l resulted in a maximum epsilon-caprolactone concentration of 11.0 g/l. The maximum epsilon-caprolactone concentration was improved further to 15.3 g/l by coexpression of glucose-6-phosphate dehydrogenase, an NADPH-generating enzyme encoded by the zwf gene which corresponded to a 39% enhancement in epsilon-caprolactone concentration compared with the control experiment performed under the same conditions.

  4. Metabolomic profile of glycolysis and the pentose phosphate pathway identifies the central role of glucose-6-phosphate dehydrogenase in clear cell-renal cell carcinoma.

    PubMed

    Lucarelli, Giuseppe; Galleggiante, Vanessa; Rutigliano, Monica; Sanguedolce, Francesca; Cagiano, Simona; Bufo, Pantaleo; Lastilla, Gaetano; Maiorano, Eugenio; Ribatti, Domenico; Giglio, Andrea; Serino, Grazia; Vavallo, Antonio; Bettocchi, Carlo; Selvaggi, Francesco Paolo; Battaglia, Michele; Ditonno, Pasquale

    2015-05-30

    The analysis of cancer metabolome has shown that proliferating tumor cells require a large quantities of different nutrients in order to support their high rate of proliferation. In this study we analyzed the metabolic profile of glycolysis and the pentose phosphate pathway (PPP) in human clear cell-renal cell carcinoma (ccRCC) and evaluate the role of these pathways in sustaining cell proliferation, maintenance of NADPH levels, and production of reactive oxygen species (ROS). Metabolomic analysis showed a clear signature of increased glucose uptake and utilization in ccRCC tumor samples. Elevated levels of glucose-6-phosphate dehydrogenase (G6PDH) in association with higher levels of PPP-derived metabolites, suggested a prominent role of this pathway in RCC-associated metabolic alterations. G6PDH inhibition, caused a significant decrease in cancer cell survival, a decrease in NADPH levels, and an increased production of ROS, suggesting that the PPP plays an important role in the regulation of ccRCC redox homeostasis. Patients with high levels of glycolytic enzymes had reduced progression-free and cancer-specific survivals as compared to subjects with low levels. Our data suggest that oncogenic signaling pathways may promote ccRCC through rerouting the sugar metabolism. Blocking the flux through this pathway may serve as a novel therapeutic target. PMID:25945836

  5. Metabolomic profile of glycolysis and the pentose phosphate pathway identifies the central role of glucose-6-phosphate dehydrogenase in clear cell-renal cell carcinoma.

    PubMed

    Lucarelli, Giuseppe; Galleggiante, Vanessa; Rutigliano, Monica; Sanguedolce, Francesca; Cagiano, Simona; Bufo, Pantaleo; Lastilla, Gaetano; Maiorano, Eugenio; Ribatti, Domenico; Giglio, Andrea; Serino, Grazia; Vavallo, Antonio; Bettocchi, Carlo; Selvaggi, Francesco Paolo; Battaglia, Michele; Ditonno, Pasquale

    2015-05-30

    The analysis of cancer metabolome has shown that proliferating tumor cells require a large quantities of different nutrients in order to support their high rate of proliferation. In this study we analyzed the metabolic profile of glycolysis and the pentose phosphate pathway (PPP) in human clear cell-renal cell carcinoma (ccRCC) and evaluate the role of these pathways in sustaining cell proliferation, maintenance of NADPH levels, and production of reactive oxygen species (ROS). Metabolomic analysis showed a clear signature of increased glucose uptake and utilization in ccRCC tumor samples. Elevated levels of glucose-6-phosphate dehydrogenase (G6PDH) in association with higher levels of PPP-derived metabolites, suggested a prominent role of this pathway in RCC-associated metabolic alterations. G6PDH inhibition, caused a significant decrease in cancer cell survival, a decrease in NADPH levels, and an increased production of ROS, suggesting that the PPP plays an important role in the regulation of ccRCC redox homeostasis. Patients with high levels of glycolytic enzymes had reduced progression-free and cancer-specific survivals as compared to subjects with low levels. Our data suggest that oncogenic signaling pathways may promote ccRCC through rerouting the sugar metabolism. Blocking the flux through this pathway may serve as a novel therapeutic target.

  6. Molecular Characterization of Glucose-6-phosphate Dehydrogenase Deficiency in Families from the Republic of Macedonia and Genotype-phenotype Correlation

    PubMed Central

    Cherepnalkovski, Anet Papazovska; Zemunik, Tatijana; Glamocanin, Sofijanka; Piperkova, Katica; Gunjaca, Ivana; Kocheva, Svetlana; Jovanova, Biljana Coneska; Krzelj, Vjekoslav

    2015-01-01

    Introduction: Glucose-6-phospahte dehydrogenase deficiency (G6PD) is one of the most common inherited disorders affecting around 400 million people worldwide. Molecular analysis of the G6PD gene identified more than 140 distinct mutations, the majority being single base missense mutations. G6PD Mediterranean is the most common variant found in populations of the Mediterranean area. Aim: The aim of our study was to perform molecular characterization of G6PD deficiency in families from the Republic of Macedonia and correlate the findings to disease phenotype. Patients and methods: Six patients and seven other family members were selected for genetic characterization, the selection procedure involved clinical evaluation and G6PD quantitative testing. All patients were first screened for the Mediterranean mutation, and subsequently for the Seattle mutation. Mutations were detected using PCR amplification and appropriate restriction endonuclease cleavage. Results: Four hemizygote and 3 heterozygous carriers for G6PD Mediterranean were detected. All G6PD deficient patients from this group showed clinical picture of hemolysis, and in 66.6% neonatal jaundice was confirmed based on history data. To our knowledge, this is the first study concerned with molecular aspects of the G6PD deficiency in R. Macedonia. Conclusion: This study represents a step towards a more comprehensive genetic evaluation in our population and better understanding of the health issues involved. PMID:26622077

  7. Polymorphic sites in the African population detected by sequence analysis of the glucose-6-phosphate dehydrogenase gene outline the evolution of the variants A and A-.

    PubMed Central

    Vulliamy, T J; Othman, A; Town, M; Nathwani, A; Falusi, A G; Mason, P J; Luzzatto, L

    1991-01-01

    The human X chromosome-linked gene encoding glucose-6-phosphate dehydrogenase (G6PD; EC 1.1.1.49) is known to be highly polymorphic from the biochemical characterization of enzyme variants. The variant A (with enzyme activity in the normal range) and the variant A- (associated with enzyme deficiency) each have a frequency of about 0.2 in several African populations. Two restriction fragment length polymorphisms have also been found in people of African descent, but not in other populations, whereas a silent mutation has been shown to be polymorphic in Mediterranean, Middle Eastern, African, and Indian populations. We report now on two additional polymorphisms that we have detected by sequence analysis, one in intron 7 and one in intron 8. The analysis of 54 African male subjects for the seven polymorphic sites, clustered within 3 kilobases of the G6PD gene, has revealed only 7 of the 128 possible haplotypes, indicating marked linkage disequilibrium. These data have enabled us to suggest an evolutionary pathway for the different mutations, with only a single ambiguity. The mutation underlying the A variant is the most ancient and the mutation underlying the A- variant is the most recent. Since it seems reasonable that the A- allele is subject to positive selection by malaria, whereas the other alleles are neutral, G6PD may lend itself to the analysis of the role of random genetic drift and selection in determining allele frequencies within a single genetic locus in human populations. Images PMID:1924316

  8. A New Glucose-6-Phosphate Dehydrogenase Variant, G6PD Orissa (44 Ala→Gly), is the Major Polymorphic Variant in Tribal Populations in India

    PubMed Central

    Kaeda, J. S.; Chhotray, G. P.; Ranjit, M. R.; Bautista, J. M.; Reddy, P. H.; Stevens, D.; Naidu, J. M.; Britt, R. P.; Vulliamy, T. J.; Luzzatto, L.; Mason, P. J.

    1995-01-01

    Deficiency of glucose-6-phosphate dehydrogenase (G6PD) is usually found at high frequencies in areas of the world where malaria has been endemic. The frequency and genetic basis of G6PD deficiency have been studied in Africa, around the Mediterranean, and in the Far East, but little such information is available about the situation in India. To determine the extent of heterogeneity of G6PD, we have studied several different Indian populations by screening for G6PD deficiency, followed by molecular analysis of deficient alleles. The frequency of G6PD deficiency varies between 3% and 15% in different tribal and urban groups. Remarkably, a previously unreported deficient variant, G6PD Orissa (44 Ala→Gly), is responsible for most of the G6PD deficiency in tribal Indian populations but is not found in urban populations, where most of the G6PD deficiency is due to the G6PD Mediterranean (188 Ser→Phe) variant. The K of G6PD Orissa is fivefold higher than that of the normal enzyme. This may be due to the fact that the alanine residue that is replaced by glycine is part of a putative coenzyme-binding site. ImagesFigure 2 PMID:8533762

  9. Glucose-6-phosphate dehydrogenase plays a central role in the response of tomato (Solanum lycopersicum) plants to short and long-term drought.

    PubMed

    Landi, Simone; Nurcato, Roberta; De Lillo, Alessia; Lentini, Marco; Grillo, Stefania; Esposito, Sergio

    2016-08-01

    The present study was undertaken to investigate the expression, occurrence and activity of glucose 6 phosphate dehydrogenase (G6PDH - EC 1.1.1.49), the key-enzyme of the Oxidative Pentose Phosphate Pathway (OPPP), in tomato plants (Solanum lycopersicum cv. Red Setter) exposed to short- and long-term drought stress. For the first time, drought effects have been evaluated in plants under different growth conditions: in hydroponic laboratory system, and in greenhouse pots under controlled conditions; and in open field, in order to evaluate drought response in a representative agricultural environment. Interestingly, changes observed appear strictly associated to the induction of well known stress response mechanisms, such as the increase of proline synthesis, accumulation of chaperone Hsp70, and ascorbate peroxidase. Results show significant increase in total activity of G6PDH, and specifically in expression and occurrence of cytosolic isoform (cy-G6PDH) in plants grown in any cultivation system upon drought. Intriguingly, the results clearly suggest that abscissic acid (ABA) pathway and signaling cascade (protein phosphatase 2C PP2C) could be strictly related to increased G6PDH expression, occurrence and activities. We hypothesized for G6PDH a specific role as one of the main reductants' suppliers to counteract the effects of drought stress, in the light of converging evidences given by young and adult tomato plants under stress of different duration and intensity. PMID:27085599

  10. DNA damage and apoptosis in mononuclear cells from glucose-6-phosphate dehydrogenase-deficient patients (G6PD Aachen variant) after UV irradiation.

    PubMed

    Efferth, T; Fabry, U; Osieka, R

    2001-03-01

    Patients affected with X chromosome-linked, hereditary glucose-6-phosphate dehydrogenase (G6PD) deficiency suffer from life-threatening hemolytic crises after intake of certain drugs or foods. G6PD deficiency is associated with low levels of reduced glutathione. We analyzed mononuclear white blood cells (MNC) of three males suffering from the German G6PD Aachen variant, four heterozygote females of this family, one G6PD-deficient male from another family coming from Iran, and six healthy male volunteers with respect to their DNA damage in two different genes (G6PD and T-cell receptor-delta) and their propensity to enter apoptosis after UV illumination (0.08-5.28 J/cm2). As determined by PCR stop assays, there was more UV-induced DNA damage in MNC of G6PD-deficient male patients than in those of healthy subjects. MNC of G6PD-deficient patients showed a higher rate of apoptosis after UV irradiation than MNC of healthy donors. MNC of heterozygote females showed intermediate rates of DNA damage and apoptosis. It is concluded that increased DNA damage may be a result of deficient detoxification of reactive oxygen species by glutathione and may ultimately account for the higher rate of apoptosis in G6PD-deficient MNC.

  11. Overcompensation in response to herbivory in Arabidopsis thaliana: the role of glucose-6-phosphate dehydrogenase and the oxidative pentose-phosphate pathway.

    PubMed

    Siddappaji, Madhura H; Scholes, Daniel R; Bohn, Martin; Paige, Ken N

    2013-10-01

    That some plants benefit from being eaten is counterintuitive, yet there is now considerable evidence demonstrating enhanced fitness following herbivory (i.e., plants can overcompensate). Although there is evidence that genetic variation for compensation exists, little is known about the genetic mechanisms leading to enhanced growth and reproduction following herbivory. We took advantage of the compensatory variation in recombinant inbred lines of Arabidopsis thaliana, combined with microarray and QTL analyses to assess the molecular basis of overcompensation. We found three QTL explaining 11.4, 10.1, and 26.7% of the variation in fitness compensation, respectively, and 109 differentially expressed genes between clipped and unclipped plants of the overcompensating ecotype Columbia. From the QTL/microarray screen we uncovered one gene that plays a significant role in overcompensation: glucose-6-phosphate-1-dehydrogenase (G6PDH1). Knockout studies of Transfer-DNA (T-DNA) insertion lines and complementation studies of G6PDH1 verify its role in compensation. G6PDH1 is a key enzyme in the oxidative pentose-phosphate pathway that plays a central role in plant metabolism. We propose that plants capable of overcompensating reprogram their transcriptional activity by up-regulating defensive genes and genes involved in energy metabolism and by increasing DNA content (via endoreduplication) with the increase in DNA content feeding back on pathways involved in defense and metabolism through increased gene expression.

  12. Mutations of Glucose-6-Phosphate Dehydrogenase Durham, Santa-Maria and A+ Variants Are Associated with Loss Functional and Structural Stability of the Protein

    PubMed Central

    Gómez-Manzo, Saúl; Marcial-Quino, Jaime; Vanoye-Carlo, America; Enríquez-Flores, Sergio; De la Mora-De la Mora, Ignacio; González-Valdez, Abigail; García-Torres, Itzhel; Martínez-Rosas, Víctor; Sierra-Palacios, Edgar; Lazcano-Pérez, Fernando; Rodríguez-Bustamante, Eduardo; Arreguin-Espinosa, Roberto

    2015-01-01

    Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common enzymopathy in the world. More than 160 mutations causing the disease have been identified, but only 10% of these variants have been studied at biochemical and biophysical levels. In this study we report on the functional and structural characterization of three naturally occurring variants corresponding to different classes of disease severity: Class I G6PD Durham, Class II G6PD Santa Maria, and Class III G6PD A+. The results showed that the G6PD Durham (severe deficiency), and the G6PD Santa Maria and A+ (less severe deficiency) (Class I, II and III, respectively) affect the catalytic efficiency of these enzymes, are more sensitive to temperature denaturing, and affect the stability of the overall protein when compared to the wild type WT-G6PD. In the variants, the exposure of more and buried hydrophobic pockets was induced and monitored with 8-Anilinonaphthalene-1-sulfonic acid (ANS) fluorescence, directly affecting the compaction of structure at different levels and probably reducing the stability of the protein. The degree of functional and structural perturbation by each variant correlates with the clinical severity reported in different patients. PMID:26633385

  13. Increased red cell calcium, decreased calcium adenosine triphosphatase, and altered membrane proteins during fava bean hemolysis in glucose-6-phosphate dehydrogenase-deficient (Mediterranean variant) individuals.

    PubMed

    Turrini, F; Naitana, A; Mannuzzu, L; Pescarmona, G; Arese, P

    1985-08-01

    RBCs from four glucose-6-phosphate dehydrogenase (G6PD)-deficient (Mediterranean variant) subjects were studied during fava bean hemolysis. In the density-fractionated RBC calcium level, Ca2+-ATPase activity, reduced glutathione level, and ghost protein pattern were studied. In the bottom fraction, containing most heavily damaged RBCs, calcium level ranged from 143 to 244 mumol/L RBCs (healthy G6PD-deficient controls: 17 +/- 5 mumol/L RBCs). The Ca2+-ATPase activity ranged from 0.87 to 1.84 mumol ATP consumed/g Hb/min (healthy G6PD-deficient controls: 2.27 +/- 0.4). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of ghosts showed: (1) the presence of high mol wt aggregates (in three cases they were reduced by dithioerythritol; in one case, only partial reduction was possible); (2) the presence of multiple, scattered new bands; and (3) the reduction of band 3. Oxidant-mediated damage to active calcium extrusion, hypothetically associated with increased calcium permeability, may explain the large increase in calcium levels. They, in turn, could activate calcium-dependent protease activity, giving rise to the profound changes in the ghost protein pattern.

  14. Increased activity of 6-phosphogluconate dehydrogenase and glucose-6-phosphate dehydrogenase in purified cell suspensions and single cells from the uterine cervix in cervical intraepithelial neoplasia.

    PubMed Central

    Jonas, S. K.; Benedetto, C.; Flatman, A.; Hammond, R. H.; Micheletti, L.; Riley, C.; Riley, P. A.; Spargo, D. J.; Zonca, M.; Slater, T. F.

    1992-01-01

    The activities of 6-phosphogluconate dehydrogenase and glucose-6-phosphate dehydrogenase have been measured in squamous epithelial cells of the uterine cervix from normal patients and cases of cervical intraepithelial neoplasia (CIN). A biochemical cycling method, which uses only simple equipment and is suited to routine use and to automation, was applied to cells separated by gradient centrifugation. In addition, cells were examined cytochemically, and the intensity of staining in the cytoplasm of single whole cells was measured using computerised microcytospectrophotometry. Twenty per cent of cells in samples from normal patients (n=61) showed staining intensities above an extinction of 0.15 at 540 nm, compared to 71% of cases of CIN 1 (n=14), 91% of cases of CIN 2 (n=11) and 67% of cases of CIN 3 (n=15). The cytochemical data do not allow definitive distinctions to be made between different grades of CIN whereas the biochemical assay applied to cell lysates shows convincing differences between normal samples and cases of CIN. There are no false negatives for CIN 3 (n=14) and CIN 2 (n=10) and 11% false negatives for CIN 1 (n=9) and 14% of false positives for normal cases (n=21). The results of this preliminary study with reference to automation are discussed [corrected]. Images Figure 1 PMID:1637668

  15. A new glucose-6-phosphate dehydrogenase variant, G6PD Orissa (44 Ala{yields}Gly), is the major polymorphic variant in tribal populations in India

    SciTech Connect

    Kaeda, J.S.; Bautista, J.M.; Stevens, D.

    1995-12-01

    Deficiency of glucose-6-phosphate dehydrogenase (G6PD) is usually found at high frequencies in areas of the world where malaria has been epidemic. The frequency and genetic basis of G6PD deficiency have been studied in Africa, around the Mediterranean, and in the Far East, but little such information is available about the situation in India. To determine the extent of heterogeneity of G6PD, we have studied several different Indian populations by screening for G6PD deficiency, followed by molecular analysis of deficient alleles. The frequency of G6PD deficiency varies between 3% and 15% in different tribal and urban groups. Remarkably, a previously unreported deficient variant, G6PD Orissa (44 Ala{yields}Gly), is responsible for most of the G6PD deficiency in tribal Indian populations but is not found in urban populations, where most of the G6PD deficiency is due to the G6PD Mediterranean (188 Ser{yields}Phe) variant. The K{sup NADP}{sub m} of G6PD Orissa is fivefold higher than that of the normal enzyme. This may be due to the fact that the alanine residue that is replaced by glycine is part of a putative coenzyme-binding site. 37 refs., 2 figs., 3 tabs.

  16. Avoiding Buffer Interference in ITC Experiments: A Case Study from the Analysis of Entropy-Driven Reactions of Glucose-6-Phosphate Dehydrogenase.

    PubMed

    Bianconi, M Lucia

    2016-01-01

    Isothermal titration calorimetry (ITC) is a label-free technique that allows the direct determination of the heat absorbed or released in a reaction. Frequently used to determining binding parameters in biomolecular interactions, it is very useful to address enzyme-catalyzed reactions as both kinetic and thermodynamic parameters can be obtained. Since calorimetry measures the total heat effects of a reaction, it is important to consider the contribution of the heat of protonation/deprotonation that is possibly taking place. Here, we show a case study of the reaction catalyzed by the glucose-6-phosphate dehydrogenase (G6PD) from Leuconostoc mesenteroides. This enzyme is able to use either NAD(+) or NADP(+) as a cofactor. The reactions were done in five buffers of different enthalpy of protonation. Depending on the buffer used, the observed calorimetric enthalpy (ΔH(cal)) of the reaction varied from -22.93 kJ/mol (Tris) to 19.37 kJ/mol (phosphate) for the NADP(+)-linked reaction, and -11.67 kJ/mol (Tris) to 7.32 kcal/mol or 30.63 kJ/mol (phosphate) for the NAD(+) reaction. We will use this system as an example of how to extract proton-independent reaction enthalpies from kinetic data to ensure that the reported accurately represent the intrinsic heat of reaction.

  17. Hereditary nonspherocytic hemolytic anemia caused by red cell glucose-6-phosphate isomerase (GPI) deficiency in two Portuguese patients: Clinical features and molecular study.

    PubMed

    Manco, Licínio; Bento, Celeste; Victor, Bruno L; Pereira, Janet; Relvas, Luís; Brito, Rui M; Seabra, Carlos; Maia, Tabita M; Ribeiro, M Letícia

    2016-09-01

    Glucose-6-phosphate isomerase (GPI) deficiency cause hereditary nonspherocytic hemolytic anemia (HNSHA) of variable severity in individuals homozygous or compound heterozygous for mutations in GPI gene. This work presents clinical features and genotypic results of two patients of Portuguese origin with GPI deficiency. The patients suffer from a mild hemolytic anemia (Hb levels ranging from 10 to 12.7g/mL) associated with macrocytosis, reticulocytosis, hyperbilirubinemia, hyperferritinemia and slight splenomegaly. Genomic DNA sequencing revealed in one patient homozygosity for a new missense mutation in exon 3, c.260G>C (p.Gly87Ala), and in the second patient compound heterozygosity for the same missense mutation (p.Gly87Ala), along with a frameshift mutation resulting from a single nucleotide deletion in exon 14, c.1238delA (p.Gln413Arg fs*24). Mutation p.Gln413Arg fs*24 is the first frameshift null mutation to be described in GPI deficiency. Molecular modeling suggests that the structural change induced by the p.Gly87Ala pathogenic variant has direct impact in the structural arrangement of the region close to the active site of the enzyme. PMID:27519939

  18. Rapid and Reliable Detection of Glucose-6-Phosphate Dehydrogenase (G6PD) Gene Mutations in Han Chinese Using High-Resolution Melting Analysis

    PubMed Central

    Yan, Jing-bin; Xu, Hong-ping; Xiong, Can; Ren, Zhao-rui; Tian, Guo-li; Zeng, Fanyi; Huang, Shu-zhen

    2010-01-01

    Glucose-6-phosphate dehydrogenase (G6PD) deficiency, an X-linked inherited disease, is one of the most common enzymopathies and affects over 400 million people worldwide. In China at least 21 distinct point mutations have been identified so far. In this study high-resolution melting (HRM) analysis was used to screen for G6PD mutations in 260 unrelated Han Chinese individuals, and the rapidity and reliability of this method was investigated. The mutants were readily differentiated by using HRM analysis, which produced distinct melting curves for each tested mutation. Interestingly, G1388A and G1376T, the two most common variants accounting for 50% to 60% of G6PD deficiency mutations in the Chinese population, could be differentiated in a single reaction. Further, two G6PD mutations not previously reported in the Chinese population were identified in this study. One of these mutations, designated “G6PD Jiangxi G1340T,” involved a G1340T substitution in exon 11, predicting a Gly447Val change in the protein. The other mutation involved a C406T substitution in exon 5. The frequencies of the common polymorphism site C1311T/IVS (intervening sequence) XI t93c between patients with G6PD and healthy volunteers were not significantly different. Thus, HRM analysis will be a useful alternative for screening G6PD mutations. PMID:20203002

  19. Rapid and reliable detection of glucose-6-phosphate dehydrogenase (G6PD) gene mutations in Han Chinese using high-resolution melting analysis.

    PubMed

    Yan, Jing-bin; Xu, Hong-ping; Xiong, Can; Ren, Zhao-rui; Tian, Guo-li; Zeng, Fanyi; Huang, Shu-zhen

    2010-05-01

    Glucose-6-phosphate dehydrogenase (G6PD) deficiency, an X-linked inherited disease, is one of the most common enzymopathies and affects over 400 million people worldwide. In China at least 21 distinct point mutations have been identified so far. In this study high-resolution melting (HRM) analysis was used to screen for G6PD mutations in 260 unrelated Han Chinese individuals, and the rapidity and reliability of this method was investigated. The mutants were readily differentiated by using HRM analysis, which produced distinct melting curves for each tested mutation. Interestingly, G1388A and G1376T, the two most common variants accounting for 50% to 60% of G6PD deficiency mutations in the Chinese population, could be differentiated in a single reaction. Further, two G6PD mutations not previously reported in the Chinese population were identified in this study. One of these mutations, designated "G6PD Jiangxi G1340T," involved a G1340T substitution in exon 11, predicting a Gly447Val change in the protein. The other mutation involved a C406T substitution in exon 5. The frequencies of the common polymorphism site C1311T/IVS (intervening sequence) XI t93c between patients with G6PD and healthy volunteers were not significantly different. Thus, HRM analysis will be a useful alternative for screening G6PD mutations. PMID:20203002

  20. Mutations of Glucose-6-Phosphate Dehydrogenase Durham, Santa-Maria and A+ Variants Are Associated with Loss Functional and Structural Stability of the Protein.

    PubMed

    Gómez-Manzo, Saúl; Marcial-Quino, Jaime; Vanoye-Carlo, America; Enríquez-Flores, Sergio; De la Mora-De la Mora, Ignacio; González-Valdez, Abigail; García-Torres, Itzhel; Martínez-Rosas, Víctor; Sierra-Palacios, Edgar; Lazcano-Pérez, Fernando; Rodríguez-Bustamante, Eduardo; Arreguin-Espinosa, Roberto

    2015-12-02

    Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common enzymopathy in the world. More than 160 mutations causing the disease have been identified, but only 10% of these variants have been studied at biochemical and biophysical levels. In this study we report on the functional and structural characterization of three naturally occurring variants corresponding to different classes of disease severity: Class I G6PD Durham, Class II G6PD Santa Maria, and Class III G6PD A+. The results showed that the G6PD Durham (severe deficiency), and the G6PD Santa Maria and A+ (less severe deficiency) (Class I, II and III, respectively) affect the catalytic efficiency of these enzymes, are more sensitive to temperature denaturing, and affect the stability of the overall protein when compared to the wild type WT-G6PD. In the variants, the exposure of more and buried hydrophobic pockets was induced and monitored with 8-Anilinonaphthalene-1-sulfonic acid (ANS) fluorescence, directly affecting the compaction of structure at different levels and probably reducing the stability of the protein. The degree of functional and structural perturbation by each variant correlates with the clinical severity reported in different patients.

  1. Prevalence of thalassaemia, iron-deficiency anaemia and glucose-6-phosphate dehydrogenase deficiency among Arab migrating nomad children, southern Islamic Republic of Iran.

    PubMed

    Pasalar, M; Mehrabani, D; Afrasiabi, A; Mehravar, Z; Reyhani, I; Hamidi, R; Karimi, M

    2014-12-17

    This study investigated the prevalence of iron-deficiency anaemia, glucose-6-phosphate dehydrogenase (G6PD) deficiency and β-thalassaemia trait among Arab migrating nomad children in southern Islamic Republic of Iran. Blood samples were analysed from 134 schoolchildren aged < 18 years (51 males, 83 females). Low serum ferritin (< 12 ng/dL) was present in 17.9% of children (21.7% in females and 11.8% in males). Low haemoglobin (Hb) correlated significantly with a low serum ferritin. Only 1 child had G6PD deficiency. A total of 9.7% of children had HbA2 ≥ 3.5 g/dL, indicating β-thalassaemia trait (10.8% in females and 7.8% in males). Mean serum iron, serum ferritin and total iron binding capacity were similar in males and females. Serum ferritin index was as accurate as Hb index in the diagnosis of iron-deficiency anaemia. A high prevalence of β-thalassaemia trait was the major potential risk factor in this population.

  2. Definitive localization of intracellular proteins: Novel approach using CRISPR-Cas9 genome editing, with glucose 6-phosphate dehydrogenase as a model.

    PubMed

    Spencer, Netanya Y; Yan, Ziying; Cong, Le; Zhang, Yulong; Engelhardt, John F; Stanton, Robert C

    2016-02-01

    Studies to determine subcellular localization and translocation of proteins are important because subcellular localization of proteins affects every aspect of cellular function. Such studies frequently utilize mutagenesis to alter amino acid sequences hypothesized to constitute subcellular localization signals. These studies often utilize fluorescent protein tags to facilitate live cell imaging. These methods are excellent for studies of monomeric proteins, but for multimeric proteins, they are unable to rule out artifacts from native protein subunits already present in the cells. That is, native monomers might direct the localization of fluorescent proteins with their localization signals obliterated. We have developed a method for ruling out such artifacts, and we use glucose 6-phosphate dehydrogenase (G6PD) as a model to demonstrate the method's utility. Because G6PD is capable of homodimerization, we employed a novel approach to remove interference from native G6PD. We produced a G6PD knockout somatic (hepatic) cell line using CRISPR-Cas9 mediated genome engineering. Transfection of G6PD knockout cells with G6PD fluorescent mutant proteins demonstrated that the major subcellular localization sequences of G6PD are within the N-terminal portion of the protein. This approach sets a new gold standard for similar studies of subcellular localization signals in all homodimerization-capable proteins.

  3. NADP+ and NAD+ binding to the dual coenzyme specific enzyme Leuconostoc mesenteroides glucose 6-phosphate dehydrogenase: different interdomain hinge angles are seen in different binary and ternary complexes.

    PubMed

    Naylor, C E; Gover, S; Basak, A K; Cosgrove, M S; Levy, H R; Adams, M J

    2001-05-01

    The reduced coenzymes NADH and NADPH only differ by one phosphate, but in the cell NADH provides reducing power for catabolism while NADPH is utilized in biosynthetic pathways. Enzymes almost invariably discriminate between the coenzymes, but glucose 6-phosphate dehydrogenase (G6PD) from Leuconostoc mesenteroides is rare in being functionally dual specific. In order to elucidate the coenzyme selectivity, the structures of NADP(+)- and NAD(+)-complexed L. mesenteroides G6PD have been determined including data to 2.2 and 2.5 A resolution, respectively, and compared with unliganded G6PD crystallized in the same space groups. Coenzyme binding is also compared with that in a ternary complex of a mutant in which Asp177 in the active site has been mutated to asparagine. There are no gross structural differences between the complexes. In both binary complexes, the enzyme interdomain hinge angle has opened. NADP(+) binds to the furthest open form; of the residues within the coenzyme domain, only Arg46 moves, interacting with the 2'-phosphate and adenine. NAD(+) is less well defined in the binding site; smaller hinge opening is seen but larger local changes: Arg46 is displaced, Thr14 bonds the 3'-hydroxyl and Gln47 bonds the 2'-hydroxyl. In the ternary complex, the hinge angle has closed; only the adenine nucleotide is ordered in the binding site. Arg46 again provides most binding interactions.

  4. Rapid kinetics of liver microsomal glucose-6-phosphatase. Evidence for tight-coupling between glucose-6-phosphate transport and phosphohydrolase activity

    SciTech Connect

    Berteloot, A.; Vidal, H.; van de Werve, G. )

    1991-03-25

    Rapid kinetics of both glucose-6-P uptake and hydrolysis in fasted rat liver microsomes were investigated with a recently developed fast-sampling, rapid-filtration apparatus. Experiments were confronted with both the substrate transport and conformational models currently proposed for the glucose-6-phosphatase system. Accumulation in microsomes of 14C products from (U-14C)glucose-6-P followed biexponential kinetics. From the inside to outside product concentrations, it could be inferred that mostly glucose should accumulate inside the vesicles. While biexponential kinetics are compatible with the mathematical predictions of a simplified substrate transport model, the latter fails in explaining the burst in total glucose production over a similar time scale to that used for the uptake measurements. Since the initial rate of the burst phase in untreated microsomes exactly matched the steady-state rate of glucose production in detergent-treated vesicles, it can be definitely concluded that the substrate transport model does not describe adequately our results. While the conformational model accounts for both the burst of glucose production and the kinetics of glucose accumulation into the vesicles, it cannot explain the burst in 32Pi production from (32P)glucose-6-P measured under the same conditions. Since the amplitude of the observed bursts is not compatible with a presteady state in enzyme activity, we propose that a hysteretic transition best explains our results in both untreated and permeabilized microsomes, thus providing a new rationale to understand the molecular mechanism of the glucose-6-phosphatase system.

  5. Aggregation ability of erythrocytes of patients with coronary heart disease depending on different glucose concentration

    NASA Astrophysics Data System (ADS)

    Malinova, Lidia I.; Simonenko, Georgy V.; Kirichuk, Vyacheslav F.; Denisova, Tatyana P.; Tuchin, Valery V.

    2002-07-01

    The aggregation ability of erythrocytes of patients with coronary heart disease comparing to practically healthy persons and patients with coronary heart disease combined with non insulin dependent diabetes mellitus depending on different glucose concentration in unguentums of blood incubates with the help of computer microphotometer - visual analyzer was studied. Two-phase behavior of erythrocytes size changing of practically healthy persons depending on glucose concentration in an incubation medium and instability erythrocyte systems of a whole blood to the influence of high glucose concentration were revealed. Influence of high glucose concentration on aggregation ability of erythrocytes of patients with coronary heart disease and its combination with non insulin dependent diabetes mellitus was revealed.

  6. Glucose-6-phosphate dehydrogenase deficiency

    MedlinePlus

    G6PD deficiency; Hemolytic anemia due to G6PD deficiency; Anemia - hemolytic due to G6PD deficiency ... Saunders; 2016:chap 161. Janz TG, Hamilton GC. Anemia, polycythemia, and white blood cell disorders. In: Marx ...

  7. Sequence Analysis and Molecular Characterization of Clonorchis sinensis Hexokinase, an Unusual Trimeric 50-kDa Glucose-6-Phosphate-Sensitive Allosteric Enzyme

    PubMed Central

    Chen, Tingjin; Ning, Dan; Sun, Hengchang; Li, Ran; Shang, Mei; Li, Xuerong; Wang, Xiaoyun; Chen, Wenjun; Liang, Chi; Li, Wenfang; Mao, Qiang; Li, Ye; Deng, Chuanhuan; Wang, Lexun; Wu, Zhongdao; Huang, Yan; Xu, Jin; Yu, Xinbing

    2014-01-01

    Clonorchiasis, which is induced by the infection of Clonorchis sinensis (C. sinensis), is highly associated with cholangiocarcinoma. Because the available examination, treatment and interrupting transmission provide limited opportunities to prevent infection, it is urgent to develop integrated strategies to prevent and control clonorchiasis. Glycolytic enzymes are crucial molecules for trematode survival and have been targeted for drug development. Hexokinase of C. sinensis (CsHK), the first key regulatory enzyme of the glycolytic pathway, was characterized in this study. The calculated molecular mass (Mr) of CsHK was 50.0 kDa. The obtained recombinant CsHK (rCsHK) was a homotrimer with an Mr of approximately 164 kDa, as determined using native PAGE and gel filtration. The highest activity was obtained with 50 mM glycine-NaOH at pH 10 and 100 mM Tris-HCl at pH 8.5 and 10. The kinetics of rCsHK has a moderate thermal stability. Compared to that of the corresponding negative control, the enzymatic activity was significantly inhibited by praziquantel (PZQ) and anti-rCsHK serum. rCsHK was homotropically and allosterically activated by its substrates, including glucose, mannose, fructose, and ATP. ADP exhibited mixed allosteric effect on rCsHK with respect to ATP, while inorganic pyrophosphate (PPi) displayed net allosteric activation with various allosteric systems. Fructose behaved as a dose-dependent V activator with the substrate glucose. Glucose-6-phosphate (G6P) displayed net allosteric inhibition on rCsHK with respect to ATP or glucose with various allosteric systems in a dose-independent manner. There were differences in both mRNA and protein levels of CsHK among the life stages of adult worm, metacercaria, excysted metacercaria and egg of C. sinensis, suggesting different energy requirements during different development stages. Our study furthers the understanding of the biological functions of CsHK and supports the need to screen for small molecule inhibitors

  8. Dengue Virus Type 2 (DENV2)-Induced Oxidative Responses in Monocytes from Glucose-6-Phosphate Dehydrogenase (G6PD)-Deficient and G6PD Normal Subjects

    PubMed Central

    Al-alimi, Abdullah Ahmed; Ali, Syed A.; Al-Hassan, Faisal Muti; Idris, Fauziah Mohd; Teow, Sin-Yeang; Mohd Yusoff, Narazah

    2014-01-01

    Background Dengue virus is endemic in peninsular Malaysia. The clinical manifestations vary depending on the incubation period of the virus as well as the immunity of the patients. Glucose-6-phosphate dehydrogenase (G6PD) deficiency is prevalent in Malaysia where the incidence is 3.2%. It has been noted that some G6PD-deficient individuals suffer from more severe clinical presentation of dengue infection. In this study, we aim to investigate the oxidative responses of DENV2-infected monocytes from G6PD-deficient individuals. Methodology Monocytes from G6PD-deficient individuals were infected with DENV2 and infection rate, levels of oxidative species, nitric oxide (NO), superoxide anions (O2−), and oxidative stress were determined and compared with normal controls. Principal Findings Monocytes from G6PD-deficient individuals exhibited significantly higher infection rates compared to normal controls. In an effort to explain the reason for this enhanced susceptibility, we investigated the production of NO and O2− in the monocytes of individuals with G6PD deficiency compared with normal controls. We found that levels of NO and O2− were significantly lower in the DENV-infected monocytes from G6PD-deficient individuals compared with normal controls. Furthermore, the overall oxidative stress in DENV-infected monocytes from G6PD-deficient individuals was significantly higher when compared to normal controls. Correlation studies between DENV-infected cells and oxidative state of monocytes further confirmed these findings. Conclusions/Significance Altered redox state of DENV-infected monocytes from G6PD-deficient individuals appears to augment viral replication in these cells. DENV-infected G6PD-deficient individuals may contain higher viral titers, which may be significant in enhanced virus transmission. Furthermore, granulocyte dysfunction and higher viral loads in G6PD-deificient individuals may result in severe form of dengue infection. PMID:24625456

  9. Involvement of ABA- and H2O2-dependent cytosolic glucose-6-phosphate dehydrogenase in maintaining redox homeostasis in soybean roots under drought stress.

    PubMed

    Wang, Huahua; Yang, Lidan; Li, Yan; Hou, Junjie; Huang, Junjun; Liang, Weihong

    2016-10-01

    The roles of abscisic acid (ABA) and hydrogen peroxide (H2O2) in inducing glucose-6-phosphate dehydrogenase (G6PDH, EC 1.1.1.49) activity and the possible roles of G6PDH in regulating ascorbate-glutathione (AsA-GSH) cycle were investigated in soybean (Glycine max L.) roots under drought stress. Drought caused a marked increase of the total and cytosolic G6PDH activities and triggered a rapid ABA and H2O2 accumulation in soybean roots. Exogenous ABA or H2O2 treatment elevated the total and cytosolic G6PDH activities, whereas suppressing ABA or H2O2 production inhibited the drought-induced increase in total and cytosolic G6PDH activities, suggesting that ABA and H2O2 are required for drought-induced increase of total G6PDH activity, namely cytosolic G6PDH activity. Furthermore, ABA induced H2O2 production by stimulating NADPH oxidase activity under drought stress. Moreover, drought significantly increased the contents of AsA and GSH and the activities of key enzymes in AsA-GSH cycle, while application of G6PDH inhibitor to seedlings significantly reduced the above effect induced by drought. Taken together, these results indicate that H2O2 acting as a downstream signaling molecule of ABA mediates drought-induced increase in cytosolic G6PDH activity, and that enhanced cytosolic G6PDH activity maintains cellular redox homeostasis by regulating AsA-GSH cycle in soybean roots. PMID:27285781

  10. Glucose-6-Phosphate Dehydrogenase Enhances Antiviral Response through Downregulation of NADPH Sensor HSCARG and Upregulation of NF-κB Signaling

    PubMed Central

    Wu, Yi-Hsuan; Chiu, Daniel Tsun-Yee; Lin, Hsin-Ru; Tang, Hsiang-Yu; Cheng, Mei-Ling; Ho, Hung-Yao

    2015-01-01

    Glucose-6-phosphate dehydrogenase (G6PD)-deficient cells are highly susceptible to viral infection. This study examined the mechanism underlying this phenomenon by measuring the expression of antiviral genes—tumor necrosis factor alpha (TNF-α) and GTPase myxovirus resistance 1 (MX1)—in G6PD-knockdown cells upon human coronavirus 229E (HCoV-229E) and enterovirus 71 (EV71) infection. Molecular analysis revealed that the promoter activities of TNF-α and MX1 were downregulated in G6PD-knockdown cells, and that the IκB degradation and DNA binding activity of NF-κB were decreased. The HSCARG protein, a nicotinamide adenine dinucleotide phosphate (NADPH) sensor and negative regulator of NF-κB, was upregulated in G6PD-knockdown cells with decreased NADPH/NADP+ ratio. Treatment of G6PD-knockdown cells with siRNA against HSCARG enhanced the DNA binding activity of NF-κB and the expression of TNF-α and MX1, but suppressed the expression of viral genes; however, the overexpression of HSCARG inhibited the antiviral response. Exogenous G6PD or IDH1 expression inhibited the expression of HSCARG, resulting in increased expression of TNF-α and MX1 and reduced viral gene expression upon virus infection. Our findings suggest that the increased susceptibility of the G6PD-knockdown cells to viral infection was due to impaired NF-κB signaling and antiviral response mediated by HSCARG. PMID:26694452

  11. Involvement of ABA- and H2O2-dependent cytosolic glucose-6-phosphate dehydrogenase in maintaining redox homeostasis in soybean roots under drought stress.

    PubMed

    Wang, Huahua; Yang, Lidan; Li, Yan; Hou, Junjie; Huang, Junjun; Liang, Weihong

    2016-10-01

    The roles of abscisic acid (ABA) and hydrogen peroxide (H2O2) in inducing glucose-6-phosphate dehydrogenase (G6PDH, EC 1.1.1.49) activity and the possible roles of G6PDH in regulating ascorbate-glutathione (AsA-GSH) cycle were investigated in soybean (Glycine max L.) roots under drought stress. Drought caused a marked increase of the total and cytosolic G6PDH activities and triggered a rapid ABA and H2O2 accumulation in soybean roots. Exogenous ABA or H2O2 treatment elevated the total and cytosolic G6PDH activities, whereas suppressing ABA or H2O2 production inhibited the drought-induced increase in total and cytosolic G6PDH activities, suggesting that ABA and H2O2 are required for drought-induced increase of total G6PDH activity, namely cytosolic G6PDH activity. Furthermore, ABA induced H2O2 production by stimulating NADPH oxidase activity under drought stress. Moreover, drought significantly increased the contents of AsA and GSH and the activities of key enzymes in AsA-GSH cycle, while application of G6PDH inhibitor to seedlings significantly reduced the above effect induced by drought. Taken together, these results indicate that H2O2 acting as a downstream signaling molecule of ABA mediates drought-induced increase in cytosolic G6PDH activity, and that enhanced cytosolic G6PDH activity maintains cellular redox homeostasis by regulating AsA-GSH cycle in soybean roots.

  12. Population screening for glucose-6-phosphate dehydrogenase deficiencies in Isabel Province, Solomon Islands, using a modified enzyme assay on filter paper dried bloodspots

    PubMed Central

    2010-01-01

    Background Glucose-6-phosphate dehydrogenase deficiency poses a significant impediment to primaquine use for the elimination of liver stage infection with Plasmodium vivax and for gametocyte clearance, because of the risk of life-threatening haemolytic anaemia that can occur in G6PD deficient patients. Although a range of methods for screening G6PD deficiency have been described, almost all require skilled personnel, expensive laboratory equipment, freshly collected blood, and are time consuming; factors that render them unsuitable for mass-screening purposes. Methods A published WST8/1-methoxy PMS method was adapted to assay G6PD activity in a 96-well format using dried blood spots, and used it to undertake population screening within a malaria survey undertaken in Isabel Province, Solomon Islands. The assay results were compared to a biochemical test and a recently marketed rapid diagnostic test. Results Comparative testing with biochemical and rapid diagnostic test indicated that results obtained by filter paper assay were accurate providing that blood spots were assayed within 5 days when stored at ambient temperature and 10 days when stored at 4 degrees. Screening of 8541 people from 41 villages in Isabel Province, Solomon Islands revealed the prevalence of G6PD deficiency as defined by enzyme activity < 30% of normal control was 20.3% and a prevalence of severe deficiency that would predispose to primaquine-induced hemolysis (WHO Class I-II) of 6.9%. Conclusions The assay enabled simple and quick semi-quantitative population screening in a malaria-endemic region. The study indicated a high prevalence of G6PD deficiency in Isabel Province and highlights the critical need to consider G6PD deficiency in the context of P. vivax malaria elimination strategies in Solomon Islands, particularly in light of the potential role of primaquine mass drug administration. PMID:20684792

  13. Glucose-6-phosphate dehydrogenase and alternative oxidase are involved in the cross tolerance of highland barley to salt stress and UV-B radiation.

    PubMed

    Zhao, Chengzhou; Wang, Xiaomin; Wang, Xiaoyu; Wu, Kunlun; Li, Ping; Chang, Ning; Wang, Jianfeng; Wang, Feng; Li, Jiaolong; Bi, Yurong

    2015-06-01

    In this study, a new mechanism involving glucose-6-phosphate dehydrogenase (G6PDH) and alternative pathways (AP) in salt pretreatment-induced tolerance of highland barley to UV-B radiation was investigated. When highland barley was exposed to UV-B radiation, the G6PDH activity decreased but the AP capacity increased. In contrast, under UV-B+NaCl treatment, the G6PDH activity was restored to the control level and the maximal AP capacity and antioxidant enzyme activities were reached. Glucosamine (Glucm, an inhibitor of G6PDH) obviously inhibited the G6PDH activity in highland barley under UV-B + NaCl treatment and a similar pattern was observed in reduced glutathione (GSH) and ascorbic acid (Asc) contents. Similarly, salicylhydroxamic acid (SHAM, an inhibitor of AOX) significantly reduced the AP capacity in highland barley under UV-B + NaCl treatment. The UV-B-induced hydrogen peroxide (H2O2) accumulation was also followed. Further studies indicated that non-functioning of G6PDH or AP under UV-B+NaCl + Glucm or UV-B + NaCl + SHAM treatment also caused damages in photosynthesis and stomatal movement. Western blot analysis confirmed that the alternative oxidase (AOX) and G6PDH were dependent each other in cross tolerance to UV-B and salt. The inhibition of AP or G6PDH activity resulted in a significant accumulation or reduction of NADPH content, respectively, under UV-B+NaCl treatment in highland barley leaves. Taken together, our results indicate that AP and G6PDH mutually regulate and maintain photosynthesis and stomata movement in the cross adaptation of highland barley seedlings to UV-B and salt by modulating redox homeostasis and NADPH content.

  14. Starvation actively inhibits splicing of glucose-6-phosphate dehydrogenase mRNA via a bifunctional ESE/ESS element bound by hnRNP K.

    PubMed

    Cyphert, T J; Suchanek, A L; Griffith, B N; Salati, L M

    2013-09-01

    Regulated expression of glucose-6-phosphate dehydrogenase (G6PD) is due to changes in the rate of pre-mRNA splicing and not changes in its transcription. Starvation alters pre-mRNA splicing by decreasing the rate of intron removal, leading to intron retention and a decrease in the accumulation of mature mRNA. A regulatory element within exon 12 of G6PD pre-mRNA controls splicing efficiency. Starvation caused an increase in the expression of heterogeneous nuclear ribonucleoprotein (hnRNP) K protein and this increase coincided with the increase in the binding of hnRNP K to the regulatory element and a decrease in the expression of G6PD mRNA. HnRNP K bound to two C-rich motifs forming an ESS within exon 12. Overexpression of hnRNP K decreased the splicing and expression of G6PD mRNA, while siRNA-mediated depletion of hnRNP K caused an increase in the splicing and expression of G6PD mRNA. Binding of hnRNP K to the regulatory element was enhanced in vivo by starvation coinciding with a decrease in G6PD mRNA. HnRNP K binding to the C-rich motifs blocked binding of serine-arginine rich, splicing factor 3 (SRSF3), a splicing enhancer. Thus hnRNP K is a nutrient regulated splicing factor responsible for the inhibition of the splicing of G6PD during starvation.

  15. Elevated glucose-6-phosphate dehydrogenase expression in the cervical cancer cases is associated with the cancerigenic event of high-risk human papillomaviruses.

    PubMed

    Hu, Tao; Li, Ya-Shan; Chen, Bo; Chang, Ye-Fei; Liu, Guang-Cai; Hong, Ying; Chen, Hong-Lan; Xiyang, Yan-Bin

    2015-10-01

    The most important etiologic agent in the pathogenesis of cervical cancers (CCs) is human papillomavirus (HPV), while the mechanisms underlying are still not well known. Glucose-6-phosphate dehydrogenase (G6PD) is reported to elevate in various tumor cells. However, no available references elucidated the correlation between the levels of G6PD and HPV-infected CC until now. In the present study, we explored the possible role of G6PD in the pathology of CC induced by HPV infection. Totally 48 patients with HPV + CC and another 63 healthy women enrolled in the clinical were employed in the present study. Overall, prevalence of cervical infection with high-risk-HPV (HR-HPV) type examined was HPV-16, followed by HPV-18. The expressions of G6PD in CC samples were also detected by immunohistochemistry (IHC), qRT-PCR, and Western blot. Regression analysis showed elevated G6PD level was positively correlated with the CC development in 30-40 aged patients with HR-HPV-16/18 infection. The HPV16 + Siha, HPV18 + Hela, and HPV-C33A cell lines were employed and transfected with G6PD deficient vectors developed in vitro. MTT and flow cytometry were also employed to determine the survival and apoptosis of CC cells after G6PD expressional inhibition. Our data revealed that G6PD down-regulation induced poor proliferation and more apoptosis of HPV18 + Hela cells, when compared with that of HPV16 + Siha and HPV-C33A cells. These findings suggest that G6PD expressions in the HR-HPV + human CC tissues and cell lines play an important role in tumor growth and proliferation.

  16. Elevated glucose-6-phosphate dehydrogenase expression in the cervical cancer cases is associated with the cancerigenic event of high-risk human papillomaviruses

    PubMed Central

    Hu, Tao; Li, Ya-Shan; Chen, Bo; Chang, Ye-Fei; Liu, Guang-Cai; Hong, Ying; Chen, Hong-Lan

    2015-01-01

    The most important etiologic agent in the pathogenesis of cervical cancers (CCs) is human papillomavirus (HPV), while the mechanisms underlying are still not well known. Glucose-6-phosphate dehydrogenase (G6PD) is reported to elevate in various tumor cells. However, no available references elucidated the correlation between the levels of G6PD and HPV-infected CC until now. In the present study, we explored the possible role of G6PD in the pathology of CC induced by HPV infection. Totally 48 patients with HPV + CC and another 63 healthy women enrolled in the clinical were employed in the present study. Overall, prevalence of cervical infection with high-risk-HPV (HR-HPV) type examined was HPV-16, followed by HPV-18. The expressions of G6PD in CC samples were also detected by immunohistochemistry (IHC), qRT-PCR, and Western blot. Regression analysis showed elevated G6PD level was positively correlated with the CC development in 30–40 aged patients with HR-HPV-16/18 infection. The HPV16 + Siha, HPV18 + Hela, and HPV-C33A cell lines were employed and transfected with G6PD deficient vectors developed in vitro. MTT and flow cytometry were also employed to determine the survival and apoptosis of CC cells after G6PD expressional inhibition. Our data revealed that G6PD down-regulation induced poor proliferation and more apoptosis of HPV18 + Hela cells, when compared with that of HPV16 + Siha and HPV-C33A cells. These findings suggest that G6PD expressions in the HR-HPV + human CC tissues and cell lines play an important role in tumor growth and proliferation. PMID:25616277

  17. Review of key knowledge gaps in glucose-6-phosphate dehydrogenase deficiency detection with regard to the safe clinical deployment of 8-aminoquinoline treatment regimens: a workshop report.

    PubMed

    von Seidlein, Lorenz; Auburn, Sarah; Espino, Fe; Shanks, Dennis; Cheng, Qin; McCarthy, James; Baird, Kevin; Moyes, Catherine; Howes, Rosalind; Ménard, Didier; Bancone, Germana; Winasti-Satyahraha, Ari; Vestergaard, Lasse S; Green, Justin; Domingo, Gonzalo; Yeung, Shunmay; Price, Ric

    2013-01-01

    The diagnosis and management of glucose-6-phosphate dehydrogenase (G6PD) deficiency is a crucial aspect in the current phases of malaria control and elimination, which will require the wider use of 8-aminoquinolines for both reducing Plasmodium falciparum transmission and achieving the radical cure of Plasmodium vivax. 8-aminoquinolines, such as primaquine, can induce severe haemolysis in G6PD-deficient individuals, potentially creating significant morbidity and undermining confidence in 8-aminoquinoline prescription. On the other hand, erring on the side of safety and excluding large numbers of people with unconfirmed G6PD deficiency from treatment with 8-aminoquinolines will diminish the impact of these drugs. Estimating the remaining G6PD enzyme activity is the most direct, accessible, and reliable assessment of the phenotype and remains the gold standard for the diagnosis of patients who could be harmed by the administration of primaquine. Genotyping seems an unambiguous technique, but its use is limited by cost and the large range of recognized G6PD genotypes. A number of enzyme activity assays diagnose G6PD deficiency, but they require a cold chain, specialized equipment, and laboratory skills. These assays are impractical for care delivery where most patients with malaria live. Improvements to the diagnosis of G6PD deficiency are required for the broader and safer use of 8-aminoquinolines to kill hypnozoites, while lower doses of primaquine may be safely used to kill gametocytes without testing. The discussions and conclusions of a workshop conducted in Incheon, Korea in May 2012 to review key knowledge gaps in G6PD deficiency are reported here.

  18. Red cell glucose 6-phosphate dehydrogenase deficiency in the northern region of Turkey: is G6PD deficiency exclusively a male disease?

    PubMed

    Albayrak, Canan; Albayrak, Davut

    2015-03-01

    Glucose-6-phosphate dehydrogenase (G6PD) deficiency is an X-linked recessive genetic defect that can cause hemolytic crisis. However, this disease affects both males and females. In Turkey, the frequency of this enzyme deficiency was reported to vary, from 0.25 to 18%, by the geographical area. Its prevalence in the northern Black Sea region of Turkey is unknown. The aims of this study were to assess the prevalence of G6PD deficiency in the northern region Turkey in children and adults with hyperbilirubinemia and hemolytic anemia. This report included a total of 976 G6PD enzyme results that were analyzed between May 2005 and January 2014. G6PD deficiency was detected in 5.0% of all patients. G6PD deficiency was significantly less frequent in females (1.9%, 6/323) than in males (6.6%, 43/653). G6PD deficiency was detected in 3.7% of infants with hyperbilirubinemia, 9.2% of children, and 4.5% of adults with hemolytic anemia. In both the newborn group and the group of children, G6PD deficiency was significantly more frequent in males. In the combined group of children (groups I and II), the proportion of males was 74% and 67% in all groups (P = .0008). In conclusion, in northern region of Turkey, G6PD deficiency is an important cause of neonatal hyperbilirubinemia and hemolytic crisis in children and adults. This study suggests that most pediatricians thought that G6PD deficiency is exclusively a male disease. For this reason, some female patients may have been undiagnosed.

  19. Review of key knowledge gaps in glucose-6-phosphate dehydrogenase deficiency detection with regard to the safe clinical deployment of 8-aminoquinoline treatment regimens: a workshop report

    PubMed Central

    2013-01-01

    The diagnosis and management of glucose-6-phosphate dehydrogenase (G6PD) deficiency is a crucial aspect in the current phases of malaria control and elimination, which will require the wider use of 8-aminoquinolines for both reducing Plasmodium falciparum transmission and achieving the radical cure of Plasmodium vivax. 8-aminoquinolines, such as primaquine, can induce severe haemolysis in G6PD-deficient individuals, potentially creating significant morbidity and undermining confidence in 8-aminoquinoline prescription. On the other hand, erring on the side of safety and excluding large numbers of people with unconfirmed G6PD deficiency from treatment with 8-aminoquinolines will diminish the impact of these drugs. Estimating the remaining G6PD enzyme activity is the most direct, accessible, and reliable assessment of the phenotype and remains the gold standard for the diagnosis of patients who could be harmed by the administration of primaquine. Genotyping seems an unambiguous technique, but its use is limited by cost and the large range of recognized G6PD genotypes. A number of enzyme activity assays diagnose G6PD deficiency, but they require a cold chain, specialized equipment, and laboratory skills. These assays are impractical for care delivery where most patients with malaria live. Improvements to the diagnosis of G6PD deficiency are required for the broader and safer use of 8-aminoquinolines to kill hypnozoites, while lower doses of primaquine may be safely used to kill gametocytes without testing. The discussions and conclusions of a workshop conducted in Incheon, Korea in May 2012 to review key knowledge gaps in G6PD deficiency are reported here. PMID:23537118

  20. Transition of metabolisms in living popular bark from growing to wintering stages and vice versa: changes in glucose 6-phosphate and 6-phosphogluconate dehydrogenase activities and in the levels of sugar phosphates.

    PubMed

    Sagisaka, S

    1974-10-01

    Activities of glucose 6-phosphate, 6-phosphogluconate, and isocitrate dehydrogenases, together with intermediate levels of the glycolytic pathway and the pentose phosphate cycle, were measured throughout a year in the living bark of poplar (Populus gelrica). Shoots, immediately after budding (early May), contained very high levels of the three enzyme activities, which fell gradually by early or mid-July to a level, roughly equivalent to budding (May) or growing (July) 2-year-old twigs. In September, the former two dehydrogenase activities of the new shoots and 2-year-old twigs began to rise, while the latter activity started to decrease. The rise of the two dehydrogenase activities continued until late November (or early December). The high level of the two dehydrogenase activities lasted until early in April of the following year and then the decrease in the activities began prior to the onset of budding, reaching a low, basal level in early May. The profile of changes in the two dehydrogenase activities appeared to coincide with the increase and decrease of soluble proteins.Normal concentrations of total hexose phosphates in the glycolytic pathway plus 6-phosphogluconate were found to be 288 to 895 mumoles/kilogram dry weight. During the metabolism transition (September and April), a transient and striking increase of 6-phosphogluconate was observed. In September, 6-phosphogluconate reached a level on the order of 10(-4)m and was 4 times that of fructose 6-phosphate. The increase in 6-phosphogluconate coincided with the increase in the glucose 6-phosphate dehydrogenase activity. Coincidentally, with the change of 6-phosphogluconate level, a large deviation of the in vivo ratio of fructose 6-phosphate to glucose 6-phosphate from the known equilibrium constant was observed, showing the relation of pentose phosphate cycle enzyme activity to the control of glycolysis. The ratio of glucose 6-phosphate to glucose 1-phosphate deviated from that predicted. These ratios

  1. Transition of metabolisms in living popular bark from growing to wintering stages and vice versa: changes in glucose 6-phosphate and 6-phosphogluconate dehydrogenase activities and in the levels of sugar phosphates.

    PubMed

    Sagisaka, S

    1974-10-01

    Activities of glucose 6-phosphate, 6-phosphogluconate, and isocitrate dehydrogenases, together with intermediate levels of the glycolytic pathway and the pentose phosphate cycle, were measured throughout a year in the living bark of poplar (Populus gelrica). Shoots, immediately after budding (early May), contained very high levels of the three enzyme activities, which fell gradually by early or mid-July to a level, roughly equivalent to budding (May) or growing (July) 2-year-old twigs. In September, the former two dehydrogenase activities of the new shoots and 2-year-old twigs began to rise, while the latter activity started to decrease. The rise of the two dehydrogenase activities continued until late November (or early December). The high level of the two dehydrogenase activities lasted until early in April of the following year and then the decrease in the activities began prior to the onset of budding, reaching a low, basal level in early May. The profile of changes in the two dehydrogenase activities appeared to coincide with the increase and decrease of soluble proteins.Normal concentrations of total hexose phosphates in the glycolytic pathway plus 6-phosphogluconate were found to be 288 to 895 mumoles/kilogram dry weight. During the metabolism transition (September and April), a transient and striking increase of 6-phosphogluconate was observed. In September, 6-phosphogluconate reached a level on the order of 10(-4)m and was 4 times that of fructose 6-phosphate. The increase in 6-phosphogluconate coincided with the increase in the glucose 6-phosphate dehydrogenase activity. Coincidentally, with the change of 6-phosphogluconate level, a large deviation of the in vivo ratio of fructose 6-phosphate to glucose 6-phosphate from the known equilibrium constant was observed, showing the relation of pentose phosphate cycle enzyme activity to the control of glycolysis. The ratio of glucose 6-phosphate to glucose 1-phosphate deviated from that predicted. These ratios

  2. Purification of glucose-6-phosphate dehydrogenase and glutathione reductase enzymes from the gill tissue of Lake Van fish and analyzing the effects of some chalcone derivatives on enzyme activities.

    PubMed

    Kuzu, Muslum; Aslan, Abdulselam; Ahmed, Ishtiaq; Comakli, Veysel; Demirdag, Ramazan; Uzun, Naim

    2016-04-01

    Glucose-6-phosphate dehydrogenase (G6PD) and glutathione reductase (GR) are metabolically quite important enzymes. Within this study, these two enzymes were purified for the first time from the gills of Lake Van fish. In the purifying process, ammonium sulfate precipitation and 2',5'-ADP Sepharose 4B affinity column chromatography techniques for glucose-6-phosphate dehydrogenase, temperature degradation and 2',5'-ADP Sepharose 4B affinity column chromatography for glutathione reductase enzyme were used. The control of the enzyme purity and determination of molecular weight were done with sodium dodecyl sulfate polyacrylamide gel electrophoresis. K(M) and V(max) values were determined with Lineweaver-Burk plot. Besides, the effects of some chalcone derivatives on the purified enzymes were analyzed. For the ones showing inhibition effect, % activity-[I] figures were drawn and IC50 values were determined. K(i) value was calculated by using Cheng-Prusoff equation.

  3. Purification of glucose-6-phosphate dehydrogenase and glutathione reductase enzymes from the gill tissue of Lake Van fish and analyzing the effects of some chalcone derivatives on enzyme activities.

    PubMed

    Kuzu, Muslum; Aslan, Abdulselam; Ahmed, Ishtiaq; Comakli, Veysel; Demirdag, Ramazan; Uzun, Naim

    2016-04-01

    Glucose-6-phosphate dehydrogenase (G6PD) and glutathione reductase (GR) are metabolically quite important enzymes. Within this study, these two enzymes were purified for the first time from the gills of Lake Van fish. In the purifying process, ammonium sulfate precipitation and 2',5'-ADP Sepharose 4B affinity column chromatography techniques for glucose-6-phosphate dehydrogenase, temperature degradation and 2',5'-ADP Sepharose 4B affinity column chromatography for glutathione reductase enzyme were used. The control of the enzyme purity and determination of molecular weight were done with sodium dodecyl sulfate polyacrylamide gel electrophoresis. K(M) and V(max) values were determined with Lineweaver-Burk plot. Besides, the effects of some chalcone derivatives on the purified enzymes were analyzed. For the ones showing inhibition effect, % activity-[I] figures were drawn and IC50 values were determined. K(i) value was calculated by using Cheng-Prusoff equation. PMID:26676512

  4. Single-Dose Primaquine in a Preclinical Model of Glucose-6-Phosphate Dehydrogenase Deficiency: Implications for Use in Malaria Transmission-Blocking Programs.

    PubMed

    Wickham, Kristina S; Baresel, Paul C; Marcsisin, Sean R; Sousa, Jason; Vuong, Chau T; Reichard, Gregory A; Campo, Brice; Tekwani, Babu L; Walker, Larry A; Rochford, Rosemary

    2016-10-01

    Individuals with glucose-6-phosphate dehydrogenase (G6PD) deficiency (G6PDd) are at risk for developing hemolytic anemia when given the antimalarial drug primaquine (PQ). The WHO Evidence Review Group released a report suggesting that mass administration of a single dose of PQ at 0.25 mg of base/kg of body weight (mpk) (mouse equivalent of 3.125 mpk) could potentially reduce malaria transmission based on its gametocytocidal activity and could be safely administered to G6PD-deficient individuals, but there are limited safety data available confirming the optimum single dose of PQ. A single-dose administration of PQ was therefore assessed in our huRBC-SCID mouse model used to predict hemolytic toxicity with respect to G6PD deficiency. In this model, nonobese diabetic (NOD)/SCID mice are engrafted with human red blood cells (huRBC) from donors with the African or Mediterranean variant of G6PDd (A-G6PDd or Med-G6PDd, respectively) and demonstrate dose-dependent sensitivity to PQ. In mice engrafted with A-G6PD-deficient huRBC, single-dose PQ at 3.125, 6.25, or 12.5 mpk had no significant loss of huRBC compared to the vehicle control group. In contrast, in mice engrafted with Med-G6PDd huRBC, a single dose of PQ at 3.125, 6.25, or 12.5 mpk resulted in a significant, dose-dependent loss of huRBC compared to the value for the vehicle control group. Our data suggest that administration of a single low dose of 0.25 mpk of PQ could induce hemolytic anemia in Med-G6PDd individuals but that use of single-dose PQ at 0.25 mpk as a gametocytocidal drug to block transmission would be safe in areas where A-G6PDd predominates. PMID:27458212

  5. Comparative analysis of glucose-6-phosphate dehydrogenase levels in pre-term and term babies delivered at University of Ilorin Teaching Hospital

    PubMed Central

    Obasa, Temitope Olorunsola; Adesiyun, Omotayo Olukemi; Mokuolu, Olugbenga Ayodeji; Ojuawo, Ayodele Isaac

    2012-01-01

    Glucose-6-phosphate (G6P) is an enzyme in the hexose monophosphate shunt required for the production of reducing equivalents needed to mop up free radicals. thereby keeping hemoglobin in its free state. Deficiency of the enzyme can cause severe neonatal jaundice. The aim of this study was to compare G6PD levels in pre-term and term babies, and evaluate the extent to which G6PD deficiency determines the severity of jaundice in various gestational age groups. Samples of cord blood collected from consecutively delivered babies in the University of Ilorin Teaching Hospital, Nigeria, were assayed for G6PD levels, and the babies were observed for jaundice during the first week of life. Those who developed jaundice had serial serum bilirubin measured. Nine hundred and thirty-three babies had G6PD assayed, with 348 being G6PD deficient, giving a hospital based prevalence of 37.3%. Of the 644 who were followed up, 143 (22.2%) were pre-term and 501(77.8%) were term babies. Babies with gestational age (GA) 27–29 weeks had the highest G6PD levels. However, there was no significant variation among the different gestational age groups (F=0.64, P=0.64). Jaundice occurred more in pre-term compared to term babies with a relative risk of 2.41 (χ2=60.95, P=0.00001). Occurrence of jaundice in pre-term babies was irrespective of G6PD status (χ2=0.2, P=0.66, RR=1.09, CI=0.83

  6. Frequency of glucose-6-phosphate dehydrogenase deficiency in malaria patients from six African countries enrolled in two randomized anti-malarial clinical trials

    PubMed Central

    2011-01-01

    Background Glucose-6-phosphate dehydrogenase (G6PD) deficiency is common in populations living in malaria endemic areas. G6PD genotype and phenotype were determined for malaria patients enrolled in the chlorproguanil-dapsone-artesunate (CDA) phase III clinical trial programme. Methods Study participants, aged > 1 year, with microscopically confirmed uncomplicated Plasmodium falciparum malaria, and haemoglobin ≥ 70 g/L or haematocrit ≥ 25%, were recruited into two clinical trials conducted in six African countries (Burkina Faso, Ghana, Kenya, Nigeria, Tanzania, Mali). G6PD genotype of the three most common African forms, G6PD*B, G6PD*A (A376G), and G6PD*A- (G202A, A542T, G680T and T968C), were determined and used for frequency estimation. G6PD phenotype was assessed qualitatively using the NADPH fluorescence test. Exploratory analyses investigated the effect of G6PD status on baseline haemoglobin concentration, temperature, asexual parasitaemia and anti-malarial efficacy after treatment with CDA 2/2.5/4 mg/kg or chlorproguanil-dapsone 2/2.5 mg/kg (both given once daily for three days) or six-dose artemether-lumefantrine. Results Of 2264 malaria patients enrolled, 2045 had G6PD genotype available and comprised the primary analysis population (1018 males, 1027 females). G6PD deficiency prevalence was 9.0% (184/2045; 7.2% [N = 147] male hemizygous plus 1.8% [N = 37] female homozygous), 13.3% (273/2045) of patients were heterozygous females, 77.7% (1588/2045) were G6PD normal. All deficient G6PD*A- genotypes were A376G/G202A. G6PD phenotype was available for 64.5% (1319/2045) of patients: 10.2% (134/1319) were G6PD deficient, 9.6% (127/1319) intermediate, and 80.2% (1058/1319) normal. Phenotype test specificity in detecting hemizygous males was 70.7% (70/99) and 48.0% (12/25) for homozygous females. Logistic regression found no significant effect of G6PD genotype on adjusted mean baseline haemoglobin (p = 0.154), adjusted mean baseline temperature (p = 0.9617), or

  7. Glucose-6-phosphate dehydrogenase and leptin are related to marbling differences among Limousin and Angus or Japanese Black x Angus steers.

    PubMed

    Bonnet, M; Faulconnier, Y; Leroux, C; Jurie, C; Cassar-Malek, I; Bauchart, D; Boulesteix, P; Pethick, D; Hocquette, J F; Chilliard, Y

    2007-11-01

    This work investigated the metabolic basis for the variability of carcass and i.m. adiposity in cattle. Our hypothesis was that the comparison of extreme breeds for adiposity might allow for the identification of some metabolic pathways determinant for carcass and i.m. adiposity. Thus, 23- to 28-mo-old steers of 3 breeds, 2 with high [Angus or Japanese Black x Angus (J. Black cross)] and 1 with low (Limousin) i.m. and carcass adiposity, were used to measure activities or mRNA levels, or both, of enzymes involved in de novo lipogenesis [acetyl-coA carboxylase, fatty acid synthase (FAS), glucose-6-phosphate dehydrogenase (G6PDH), malic enzyme], circulating triacylglycerol (TAG) uptake (lipoprotein lipase), and fatty acid esterification (glycerol-3-phosphate dehydrogenase), as well as the mRNA level of leptin, an adiposity-related factor. In a first study, enzyme activities were assayed in the s.c. adipose tissue (AT), the oxidative rectus abdominis, and the glycolytic semitendinosus muscles from steers finished for 6 mo. Compared with Angus or J. Black cross, Limousin steers had a 27% less (P = 0.003) rib fat thickness, and 23 and 29% less (P < or = 0.02) FAS and G6PDH activities in s.c. AT. In rectus abdominis and semitendinosus, the 75% less (P < 0.001) TAG content was concomitant with 50% less (P < 0.001) G6PDH activity. In a second study, enzyme activities plus mRNA levels were assayed in an oxido-glycolytic muscle, the longissimus thoracis (LT), in the i.m. AT dissected from LT, and in s.c. AT from the same Limousin steers and from Angus steers finished for 10 mo. Compared with Angus, the 50% less (P < 0.001) rib fat thickness in Limousin contrasted with the 1.1- to 5.8-fold greater (P < or = 0.02) mRNA levels or activities, or both, of acetyl-coA carboxylase, G6PDH, lipoprotein lipase, and glycerol-3-phosphate dehydrogenase in s.c. AT. Conversely, the 90% less (P < 0.001) TAG content in Limousin LT was concomitant to the 79 and 83% less (P < or = 0.002) G6PDH

  8. Glucose-6-phosphate dehydrogenase deficiency and the risk of malaria and other diseases in children in Kenya: a case-control and a cohort study

    PubMed Central

    Uyoga, Sophie; Ndila, Carolyne M; Macharia, Alex W; Nyutu, Gideon; Shah, Shivang; Peshu, Norbert; Clarke, Geraldine M; Kwiatkowski, Dominic P; Rockett, Kirk A; Williams, Thomas N

    2015-01-01

    Summary Background The global prevalence of X-linked glucose-6-phosphate dehydrogenase (G6PD) deficiency is thought to be a result of selection by malaria, but epidemiological studies have yielded confusing results. We investigated the relationships between G6PD deficiency and both malaria and non-malarial illnesses among children in Kenya. Methods We did this study in Kilifi County, Kenya, where the G6PD c.202T allele is the only significant cause of G6PD deficiency. We tested the associations between G6PD deficiency and severe and complicated Plasmodium falciparum malaria through a case-control study of 2220 case and 3940 control children. Cases were children aged younger than 14 years, who visited the high dependency ward of Kilifi County Hospital with severe malaria between March 1, 1998, and Feb 28, 2010. Controls were children aged between 3–12 months who were born within the same study area between August 2006, and September 2010. We assessed the association between G6PD deficiency and both uncomplicated malaria and other common diseases of childhood in a cohort study of 752 children aged younger than 10 years. Participants of this study were recruited from a representative sample of households within the Ngerenya and Chonyi areas of Kilifi County between Aug 1, 1998, and July 31, 2001. The primary outcome measure for the case-control study was the odds ratio for hospital admission with severe malaria (computed by logistic regression) while for the cohort study it was the incidence rate ratio for uncomplicated malaria and non-malaria illnesses (computed by Poisson regression), by G6PD deficiency category. Findings 2863 (73%) children in the control group versus 1643 (74%) in the case group had the G6PD normal genotype, 639 (16%) versus 306 (14%) were girls heterozygous for G6PD c.202T, and 438 (11%) versus 271 (12%) children were either homozygous girls or hemizygous boys. Compared with boys and girls without G6PD deficiency, we found significant

  9. Alterations in energy/redox metabolism induced by mitochondrial and environmental toxins: a specific role for glucose-6-phosphate-dehydrogenase and the pentose phosphate pathway in paraquat toxicity.

    PubMed

    Lei, Shulei; Zavala-Flores, Laura; Garcia-Garcia, Aracely; Nandakumar, Renu; Huang, Yuting; Madayiputhiya, Nandakumar; Stanton, Robert C; Dodds, Eric D; Powers, Robert; Franco, Rodrigo

    2014-09-19

    Parkinson's disease (PD) is a multifactorial disorder with a complex etiology including genetic risk factors, environmental exposures, and aging. While energy failure and oxidative stress have largely been associated with the loss of dopaminergic cells in PD and the toxicity induced by mitochondrial/environmental toxins, very little is known regarding the alterations in energy metabolism associated with mitochondrial dysfunction and their causative role in cell death progression. In this study, we investigated the alterations in the energy/redox-metabolome in dopaminergic cells exposed to environmental/mitochondrial toxins (paraquat, rotenone, 1-methyl-4-phenylpyridinium [MPP+], and 6-hydroxydopamine [6-OHDA]) in order to identify common and/or different mechanisms of toxicity. A combined metabolomics approach using nuclear magnetic resonance (NMR) and direct-infusion electrospray ionization mass spectrometry (DI-ESI-MS) was used to identify unique metabolic profile changes in response to these neurotoxins. Paraquat exposure induced the most profound alterations in the pentose phosphate pathway (PPP) metabolome. 13C-glucose flux analysis corroborated that PPP metabolites such as glucose-6-phosphate, fructose-6-phosphate, glucono-1,5-lactone, and erythrose-4-phosphate were increased by paraquat treatment, which was paralleled by inhibition of glycolysis and the TCA cycle. Proteomic analysis also found an increase in the expression of glucose-6-phosphate dehydrogenase (G6PD), which supplies reducing equivalents by regenerating nicotinamide adenine dinucleotide phosphate (NADPH) levels. Overexpression of G6PD selectively increased paraquat toxicity, while its inhibition with 6-aminonicotinamide inhibited paraquat-induced oxidative stress and cell death. These results suggest that paraquat "hijacks" the PPP to increase NADPH reducing equivalents and stimulate paraquat redox cycling, oxidative stress, and cell death. Our study clearly demonstrates that alterations in

  10. An examination of the role of asp-177 in the His-Asp catalytic dyad of Leuconostoc mesenteroides glucose 6-phosphate dehydrogenase: X-ray structure and pH dependence of kinetic parameters of the D177N mutant enzyme.

    PubMed

    Cosgrove, M S; Gover, S; Naylor, C E; Vandeputte-Rutten, L; Adams, M J; Levy, H R

    2000-12-12

    The role of Asp-177 in the His-Asp catalytic dyad of glucose 6-phosphate dehydrogenase from Leuconostoc mesenteroides has been investigated by a structural and functional characterization of the D177N mutant enzyme. Its three-dimensional structure has been determined by X-ray cryocrystallography in the presence of NAD(+) and in the presence of glucose 6-phosphate plus NADPH. The structure of a glucose 6-phosphate complex of a mutant (Q365C) with normal enzyme activity has also been determined and substrate binding compared. To understand the effect of Asp-177 on the ionization properties of the catalytic base His-240, the pH dependence of kinetic parameters has been determined for the D177N mutant and compared to that of the wild-type enzyme. The structures give details of glucose 6-phosphate binding and show that replacement of the Asp-177 of the catalytic dyad with asparagine does not affect the overall structure of glucose 6-phosphate dehydrogenase. Additionally, the evidence suggests that the productive tautomer of His-240 in the D177N mutant enzyme is stabilized by a hydrogen bond with Asn-177; hence, the mutation does not affect tautomer stabilization. We conclude, therefore, that the absence of a negatively charged aspartate at 177 accounts for the decrease in catalytic activity at pH 7.8. Structural analysis suggests that the pH dependence of the kinetic parameters of D177N glucose 6-phosphate dehydrogenase results from an ionized water molecule replacing the missing negative charge of the mutated Asp-177 at high pH. Glucose 6-phosphate binding orders and orients His-178 in the D177N-glucose 6-phosphate-NADPH ternary complex and appears to be necessary to form this water-binding site.

  11. A dynamic and stationary rheological study of erythrocytes incubated in a glucose medium.

    PubMed

    Riquelme, Bibiana; Foresto, Patricia; D'Arrigo, Mabel; Valverde, Juana; Rasia, Rodolfo

    2005-02-28

    A higher than normal glucose concentration in a suspending medium may produce non-enzymatic glycosylation of erythrocyte proteins. This process can modify the viscoelastic properties of erythrocytes. In this paper, we studied the possible relationship between glucose concentration in a suspending medium and erythrocyte rheological parameters. Human venous blood was obtained from the antecubital veins of 10 healthy volunteers. Blood samples were anticoagulated with EDTA and centrifuged. Red blood cells (RBCs) were washed and subsequently divided in aliquots, which were incubated in vitro with glucose solutions of different concentrations. Dynamic and stationary viscoelastic parameters of RBCs were determined by laser diffractometry in an Erythrodeformeter. Aggregate shape parameter (ASP) of the RBCs was determined by digital image processing. Significant changes were observed both in ASP and in rheological parameters when the glucose concentration in the medium was increased, demonstrating that a glucose concentration as low as 1% induces alterations in the mechanical properties of RBCs. PMID:15680283

  12. Silencing trehalose-6-phosphate synthase incapacitates adult mosquitoes by interfering with the biosynthetic pathway for flight fuel

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Trehalose is a disaccharide comprised of two glucose molecules. It is the main blood sugar of insects and is essential for flight. Trehalose is synthesized by two enzymes: trehalose-6-phosphate synthase (T6PS) converts glucose-6-phosphate to trehalose-6-phosphate, and trehalose-6-phosphate phosphata...

  13. Crystal Structures of An F420-Dependent Glucose-6-Phosphate Dehydrogenase Fgd1 Involved in the Activation of the Anti-Tb Drug Candidate Pa-824 Reveal the Basis of Coenzyme And Substrate Binding

    SciTech Connect

    Bashiri, G.; Squire, C.J.; Moreland, N.J.; Baker, E.N.

    2009-05-11

    The modified flavin coenzyme F{sub 420} is found in a restricted number of microorganisms. It is widely distributed in mycobacteria, however, where it is important in energy metabolism, and in Mycobacterium tuberculosis (Mtb) is implicated in redox processes related to non-replicating persistence. In Mtb, the F{sub 420}-dependent glucose-6-phosphate dehydrogenase FGD1 provides reduced F{sub 420} for the in vivo activation of the nitroimidazopyran prodrug PA-824, currently being developed for anti-tuberculosis therapy against both replicating and persistent bacteria. The structure of M. tuberculosis FGD1 has been determined by x-ray crystallography both in its apo state and in complex with F{sub 420} and citrate at resolutions of 1.90 and 1.95{angstrom}, respectively. The structure reveals a highly specific F{sub 420} binding mode, which is shared with several other F{sub 420}-dependent enzymes. Citrate occupies the substrate binding pocket adjacent to F{sub 420} and is shown to be a competitive inhibitor (IC{sub 50} 43 {micro}m). Modeling of the binding of the glucose 6-phosphate (G6P) substrate identifies a positively charged phosphate binding pocket and shows that G6P, like citrate, packs against the isoalloxazine moiety of F{sub 420} and helps promote a butterfly bend conformation that facilitates F{sub 420} reduction and catalysis.

  14. Triggering of suicidal erythrocyte death by penta-O-galloyl-β-D-glucose.

    PubMed

    Alzoubi, Kousi; Honisch, Sabina; Abed, Majed; Lang, Florian

    2013-12-24

    The polyphenolic 1,2,3,4,6-penta-O-galloyl-beta-D-glucose from several medicinal herbs triggers apoptosis and has, thus, been proposed for treatment of malignancy. The substance is at least partially effective through caspase activation. In analogy to apoptosis of nucleated cells, erythrocytes may enter suicidal death or eryptosis, which is characterized by cell shrinkage and by phosphatidylserine translocation to the erythrocyte surface. Eryptosis is triggered by increase of cytosolic Ca2+-activity ([Ca2+]i). The sensitivity to [Ca2+]i is enhanced by ceramide. The present study explored whether penta-O-galloyl-β-D-glucose stimulates eryptosis. Cell volume was estimated from forward scatter, phosphatidylserine exposure from annexin V binding, hemolysis from hemoglobin-release, [Ca2+]i from Fluo3-fluorescence and ceramide abundance from fluorescent antibodies. A 48-h exposure of human erythrocytes to penta-O-galloyl-β-D-glucose significantly decreased forward scatter (50 µM) and significantly increased annexin V binding (10 µM). Up to 50 µM penta-O-galloyl-β-D-glucose did not significantly modify [Ca2+]i. However, the effect of penta-O-galloyl-β-D-glucose (25 µM) induced annexin V binding was slightly, but significantly, blunted by removal of extracellular Ca2+, pointing to sensitization of erythrocytes to the scrambling effect of Ca2+. Penta-O-galloyl-β-D-glucose (25 µM) further increased ceramide formation. In conclusion, penta-O-galloyl-β-D-glucose stimulates suicidal erythrocyte death or eryptosis, an effect partially due to stimulation of ceramide formation with subsequent sensitization of erythrocytes to Ca2+.

  15. Stress-Induced GSK3 Regulates the Redox Stress Response by Phosphorylating Glucose-6-Phosphate Dehydrogenase in Arabidopsis[C][W][OA

    PubMed Central

    Dal Santo, Silvia; Stampfl, Hansjörg; Krasensky, Julia; Kempa, Stefan; Gibon, Yves; Petutschnig, Elena; Rozhon, Wilfried; Heuck, Alexander; Clausen, Tim; Jonak, Claudia

    2012-01-01

    Diverse stresses such as high salt conditions cause an increase in reactive oxygen species (ROS), necessitating a redox stress response. However, little is known about the signaling pathways that regulate the antioxidant system to counteract oxidative stress. Here, we show that a Glycogen Synthase Kinase3 from Arabidopsis thaliana (ASKα) regulates stress tolerance by activating Glc-6-phosphate dehydrogenase (G6PD), which is essential for maintaining the cellular redox balance. Loss of stress-activated ASKα leads to reduced G6PD activity, elevated levels of ROS, and enhanced sensitivity to salt stress. Conversely, plants overexpressing ASKα have increased G6PD activity and low levels of ROS in response to stress and are more tolerant to salt stress. ASKα stimulates the activity of a specific cytosolic G6PD isoform by phosphorylating the evolutionarily conserved Thr-467, which is implicated in cosubstrate binding. Our results reveal a novel mechanism of G6PD adaptive regulation that is critical for the cellular stress response. PMID:22885737

  16. Dynamics of morphofunctional erythrocyte properties during intravenous glucose injection in patients with coronary heart disease

    NASA Astrophysics Data System (ADS)

    Malinova, Lidia I.; Simonenko, Georgy V.; Denisova, Tatyana P.; Tuchin, Valery V.

    2007-02-01

    Dynamics of glucose concentration in human organism is an important diagnostic characteristic for it's parameters correlate significantly with the severity of metabolic, vessel and perfusion disorders. 36 patients with stable angina pectoris of II and III functional classes were involved in this study. All of them were men in age range of 45-59 years old. 7 patients hospitalized with acute myocardial infarction (aged from 49 to 59 years old) form the group of compare. Control group (n = 5) was of practically healthy men in comparable age. To all patients intravenous glucose solution (40%) in standard loading dose was injected. Capillary and vein blood samples were withdrawn before, and 5, 60, 120, 180 and 240 minutes after glucose load. At these time points blood pressure and glucose concentration were measured. In prepared blood smears shape, deformability and sizes of erythrocytes, quantity and degree of shear stress resistant erythrocyte aggregates were studied. Received data were approximated by polynomial of high degree to receive concentration function of studied parameters, which first derivative elucidate velocity characteristics of morphofunctional erythrocyte properties during intravenous glucose injection in patients with coronary heart disease and practically healthy persons. Received data show principle differences in dynamics of morphofunctional erythrocyte properties during intravenous glucose injection in patients with coronary heart disease as a possible mechanism of coronary blood flow destabilization.

  17. Derivativation of the human erythrocyte glucose transporter using a novel forskolin photoaffinity label

    SciTech Connect

    Wadzinski, B.; Shanahan, M.; Ruoho, A.

    1987-05-01

    An iodinated photoaffinity label for the glucose transporter, 3-iodo-4-azidophenethylamido-7-0-succinyldeacetyl-forskolin (IAPS-Fsk), has been synthesized, purified, and characterized. The K/sub i/ for inhibition of 3-0-methylglucose transport by TAPS-Fsk in human erythrocytes was found to be 0.1 uM. The carrier-free radioiodinated label has been shown to be a highly specific photoaffinity label for the human erythrocyte glucose transporter. Photolysis of erythrocyte membranes with 1-10 nM (I-125)IAPS-Fsk and analysis by SDS-PAGE showed specific derivatization of a broad band with an apparent molecular weight of 40-70 kDa. Photoincorporation using 2 nM (I-125)IAPS-Fsk was protected with D-glucose, cytochalasin B, and forskolin. No protection was observed with L-glucose. Endo-B-galactosidase digestion and trypsinization of (I-125)IAPS-Fsk labelled erythrocytes reduced the specifically radiolabelled transporter to 40 kDa and 18 kDa respectively. (I-125)-IAPS-Fsk will be used to study the structural aspects of the glucose transporter.

  18. Purification and Characterization of Glucose 6-Phosphate Dehydrogenase, 6-Phosphogluconate Dehydrogenase, and Glutathione Reductase from Rat Heart and Inhibition Effects of Furosemide, Digoxin, and Dopamine on the Enzymes Activities.

    PubMed

    Adem, Sevki; Ciftci, Mehmet

    2016-06-01

    The present study was aimed to investigate characterization and purification of glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, and glutathione reductase from rat heart and the inhibitory effect of three drugs. The purification of the enzymes was performed using 2',5'-ADP sepharose 4B affinity material. The subunit and the natural molecular weights were analyzed by SDS-PAGE and gel filtration. Biochemical characteristics such as the optimum temperature, pH, stable pH, and salt concentration were examined for each enzyme. Types of product inhibition and Ki values with Km and Vmax values of the substrates and coenzymes were determined. According to the obtained Ki and IC50 values, furosemide, digoxin, and dopamine showed inhibitory effect on the enzyme activities at low millimolar concentrations in vitro conditions. Dopamine inhibited the activity of these enzymes as competitive, whereas furosemide and digoxin inhibited the activity of the enzyme as noncompetitive.

  19. Effect of glucose concentration on formation of AGEs in erythrocytes in vitro.

    PubMed

    Nagai, Ryoji; Deemer, Elizabeth K; Brock, Jonathan W; Thorpe, Suzanne R; Baynes, John W

    2005-06-01

    Posttranslational modifications, such as advanced glycoxidation and lipoxidation end products (AGE/ALEs), are implicated in the pathogenesis of diabetic complications and atherosclerosis. Recent studies have demonstrated that AGE/ALEs are generated not only in extracellular matrix proteins, but also in intracellular proteins from metabolic intermediates. In this study we investigate the effect of glucose concentration on the formation of the AGE/ALEs, Nepsilon-(carboxymethyl)lysine (CML), Nepsilon-(carboxyethyl)lysine (CEL), S-(carboxymethyl)cysteine (CMC), and S-(2-succinyl)cysteine (2SC) in erythrocytes as a function of glucose concentration. Human erythrocytes (10% hematocrit) were incubated in Dulbecco's modified Eagle's medium (DMEM) containing 5 mM or 30 mM glucose for 5 days at 37 degrees C. Globin was recovered by precipitation with 0.25 M HCl in acetone. Following acid hydrolysis, amino acids were converted to their trifluoroacetyl methyl ester derivatives and analyzed by GC/MS/MS. The CML and CEL content of globin increased in a time- and glucose-dependent manner and also increased 1.3- and 1.8-fold, respectively, in incubations containing 30 mM glucose; whereas CMC and 2SC content did not change during the five-day incubations. Furthermore, CEL content of globin in erythrocytes incubated with 30 mM was the highest in the other AGEs, indicating that methylglyoxal may play a major role in AGE formation in erythrocytes. The erythrocyte system should be useful for cellular screening of the efficacy of inhibitors of AGE/ALE formation.

  20. Influence of glucose concentration on the membrane stability of human erythrocytes.

    PubMed

    Lemos, Guilherme Santos Duarte; Márquez-Bernardes, Liandra Freitas; Arvelos, Letícia Ramos; Paraíso, Lara Ferreira; Penha-Silva, Nilson

    2011-12-01

    The action of glucose as an osmolyte in relation to blood cells is not well-characterized in the literature. This study aimed to study the influence of glucose concentration on the stability of red blood cells. The stability of erythrocytes was evaluated by the half-transition point obtained from the curves of lysis induced by glucose in the absence of salt or by increase in medium hypotonicity in the absence and the presence of different concentrations of glucose. In the presence of 0.9 g/dl NaCl, there was no hemolysis with increasing concentration of glucose from 0 to 10 g/dl. In the absence of NaCl, the dependence of hemolysis with the 0-10 g/dl glucose was described by a decreasing sigmoid, with fully lysed and fully protected cells being encountered in the presence of 0-2 and 4-10 g/dl glucose, respectively. The possible origin of such stabilization effect is discussed with base of what is known about osmostabilization of biological complexes and about the influence of glucose on the rheological properties of erythrocytes. PMID:21735128

  1. Impairment of erythrocytes incubated in glucose medium: a wavelet-information theory analysis.

    PubMed

    Korol, A M; Rosso, O A; Martín, M T; D'Arrigo, M; Riquelme, B D

    2011-07-01

    This study investigates the effects produced by an increased concentration of glucose in a suspending medium on the erythrocytes Information Theory quantifiers. Erythrocytes, which were obtained from eight healthy volunteers, were washed and incubated in vitro with glucose solutions at different concentrations. The measured Wavelet-based Information Theory quantifiers include the Relative Wavelet Energy (RWE), the Normalized Total Wavelet Shannon Entropy (NTWS), MPR-Statistical Complexity Measure (SCM) and entropy-complexity plane. The results show that the increase in glucose concentration does not produce significant changes on the RWE, while significant ones on the NTSE, which combined with SCM values allow to identify different behaviour for all the different populations in the entropy-complexity plane. Modification in the hemorheological properties of cells could be clearly detected with these Wavelet-based Information Theory quantifiers. PMID:21301991

  2. Effects of test spills of chemically dispersed and nondispersed oil on the activity of aspartate aminotransferase and glucose-6-phosphate dehydrogenase in two intertidal bivalves, Mya arenaria and Mytilus edulis

    SciTech Connect

    Gilfillan, E.S.; Foster, J.; Gerber, R.; Hanson, S.A.; Page, D.S.; Vallas, D.

    1982-10-01

    In 1981, two test oil spills were made in Maine. One spill was 975 L (250 gal) of Murban crude oil; the other was 975 L of Murban crude oil premixed with 97 L (25 gal) of Corexit 9527. The uptake of the oil and its effects on enzymatic activity in two species of common intertidal bivalve mollusks, Mya arenaria and Mytilus edulis, were studied. Data were obtained on uptake and depuration of the oil for each species; data were also obtained on the activity of glucose-6-phosphate dehydrogenase and aspartate aminotransferase for each species. Data were collected both before and after each of the spills. Much less oil was taken up by the populations of animals exposed to chemically dispersed oil than by those exposed to nondispersed oil. Rates of depuration were the same for each species; they were also the same regardless of oil exposure. Significant long-term effects on enzyme activity were detected only in those animals exposed to nondispersed oil.

  3. Glucose-6-Phosphate Dehydrogenase Deficiency Overview

    MedlinePlus

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  4. Effects of Red Wine Tannat on Oxidative Stress Induced by Glucose and Fructose in Erythrocytes in Vitro

    PubMed Central

    Pazzini, Camila Eliza Fernandes; Colpo, Ana Ceolin; Poetini, Márcia Rósula; Pires, Cauê Ferreira; de Camargo, Vanessa Brum; Mendez, Andreas Sebastian Loureiro; Azevedo, Miriane Lucas; Soares, Júlio César Mendes; Folmer, Vanderlei

    2015-01-01

    The literature indicates that red wine presents in its composition several substances that are beneficial to health. This study has investigated the antioxidant effects of Tannat red wine on oxidative stress induced by glucose and fructose in erythrocytes in vitro, with the purpose to determine some of its majoritarian phenolic compounds and its antioxidant capacity. Erythrocytes were incubated using different concentrations of glucose and fructose in the presence or absence of wine. From these erythrocytes were determined the production of thiobarbituric acid reactive species (TBARS), glucose consumption, and osmotic fragility. Moreover, quantification of total phenolic, gallic acid, caffeic acid, epicatechin, resveratrol, and DPPH scavenging activity in wine were also assessed. Red wine showed high levels of polyphenols analyzed, as well as high antioxidant potential. Erythrocytes incubated with glucose and fructose had an increase in lipid peroxidation and this was prevented by the addition of wine. The wine increased glucose uptake into erythrocytes and was able to decrease the osmotic fragility of erythrocytes incubated with fructose. Altogether, these results suggest that wine leads to a reduction of the oxidative stress induced by high concentrations of glucose and fructose. PMID:26078708

  5. Effects of Red Wine Tannat on Oxidative Stress Induced by Glucose and Fructose in Erythrocytes in Vitro.

    PubMed

    Pazzini, Camila Eliza Fernandes; Colpo, Ana Ceolin; Poetini, Márcia Rósula; Pires, Cauê Ferreira; de Camargo, Vanessa Brum; Mendez, Andreas Sebastian Loureiro; Azevedo, Miriane Lucas; Soares, Júlio César Mendes; Folmer, Vanderlei

    2015-01-01

    The literature indicates that red wine presents in its composition several substances that are beneficial to health. This study has investigated the antioxidant effects of Tannat red wine on oxidative stress induced by glucose and fructose in erythrocytes in vitro, with the purpose to determine some of its majoritarian phenolic compounds and its antioxidant capacity. Erythrocytes were incubated using different concentrations of glucose and fructose in the presence or absence of wine. From these erythrocytes were determined the production of thiobarbituric acid reactive species (TBARS), glucose consumption, and osmotic fragility. Moreover, quantification of total phenolic, gallic acid, caffeic acid, epicatechin, resveratrol, and DPPH scavenging activity in wine were also assessed. Red wine showed high levels of polyphenols analyzed, as well as high antioxidant potential. Erythrocytes incubated with glucose and fructose had an increase in lipid peroxidation and this was prevented by the addition of wine. The wine increased glucose uptake into erythrocytes and was able to decrease the osmotic fragility of erythrocytes incubated with fructose. Altogether, these results suggest that wine leads to a reduction of the oxidative stress induced by high concentrations of glucose and fructose.

  6. Rapid GLUT-1 mediated glucose transport in erythrocytes from the grey-headed fruit bat (Pteropus poliocephalus).

    PubMed

    Craik, J D; Markovich, D

    2000-05-01

    D-Glucose entry into erythrocytes from adult grey-headed flying fox fruit bats (Pteropus poliocephalus) was rapid and showed saturation at high substrate concentrations. Kinetic parameters were estimated from the concentration dependence of initial rates of zero-trans D-glucose entry at 5.5 degrees C as Michaelis constant (K(m)) 1. 64+/-0.56 mM, and maximal velocity (V(max)) 1162+/-152 micromol.l. cell water(-1).min(-1). D-Glucose entry was inhibited by cytochalasin B; mass law analysis of D-glucose-displaceable cytochalasin B binding gave values of K(d) 37.1+/-5.0 nM and B(max) 361.2+/-9.1 pmol/mg membrane protein. Entry of 2-deoxy-D-glucose, and 3-O-methyl-D-glucose, into P. poliocephalus red cells was rapid, entry of D-fructose was very slow. Glucose transporter polypeptides were identified on immunoblots as a band M(r) 47000-54000 and their identity confirmed by D-glucose-sensitive photolabeling of membranes with [3H]-cytochalasin B. Peptide-N-glycanase F digestion of both human and bat erythrocyte membranes generated GLUT-1-derived bands M(r) 37000. Trypsin digestion of human and fruit bat erythrocyte membranes generated fragmentation patterns consistent with similar GLUT-1 polypeptide structures in both species. Erythrocytes from adult Australian ghost bats (Macroderma gigas), a carnivorous microchiropteran bat, also expressed high levels of GLUT-1.

  7. Glucose-6-phosphate dehydrogenase from brewers' yeast. The effects of pH and temperature on the steady-state kinetic parameters of the two-chain protein species.

    PubMed

    Kuby, S A; Roy, R N

    1976-05-01

    A systematic study has been made of the pH- and temperature-dependency of the steady-state kinetic parameters of the stabilized two-subunit enzyme species of glucose-6-phosphate dehydrogenase, in the absence of superimposed association-dissociation reactions. The Vmax(app) data obtained in several buffers between pH 5 and 10 and at 18-32 degrees C lead to the postulate that at least two sets of protonic equilibria may govern the catalysis (one near pH 5.7 AT 25 DEGREES C and another near pH 9.2); furthermore, two pathways for product formation (i.e., two Vmax's) appear to be required to explain the biphasic nature of the log Vmax(app) vs. pH curves, with Vmax(basic) greater than Vmax(acidic + neutral). Of the several buffers explored, either a uniform degree of interaction or a minimal degree of buffer species interaction could be assessed from the enthalpy changes associated with the derived values for ionization constants attributed to the protonic equilibria in the enzyme-substrates ternary complexes for the case of Tris-acetate-EDTA buffers, at constant ionic strength. With the selection of this buffer at 0.1 (T/2) and at 25 and 32 degrees C, a self-consistent kinetic mechanism has emerged which allows for the random binding of the two fully ionized substrates to the enzyme via two major pathways, and product formation by both E-A--B- and HE-A--B-. As before (Kuby et al. Arch. Biochem, Biophys. 165, 153-178, 1974), a quasi-equilibrium is presumed, with rate-limiting steps (k + 5 and k + 5') at the interconversion of the ternary complexes. Values for the two sets of protonic equilibria defined by this mechanism (viz., pKk, pKH2 for the first ionizations, and pKk', pKH' for the second) could then be estimated. From their numerical values (e.g., at 25 degrees C: pKK = 5.7 PKH2 = 5.2; and pKK' = 9.1, PKH' = 8.2) and from the values for delta H degrees ioniz (e.g., delta H degrees pKK APPROXIMATELY 5.1 KCAL/MOL; DELTA H degrees pKK' APPROXIMATELY 11 KCAL/MOL), A

  8. Lower reference limits of quantitative cord glucose-6-phosphate dehydrogenase estimated from healthy term neonates according to the clinical and laboratory standards institute guidelines: a cross sectional retrospective study

    PubMed Central

    2013-01-01

    Background Previous studies have reported the lower reference limit (LRL) of quantitative cord glucose-6-phosphate dehydrogenase (G6PD), but they have not used approved international statistical methodology. Using common standards is expecting to yield more true findings. Therefore, we aimed to estimate LRL of quantitative G6PD detection in healthy term neonates by using statistical analyses endorsed by the International Federation of Clinical Chemistry (IFCC) and the Clinical and Laboratory Standards Institute (CLSI) for reference interval estimation. Methods This cross sectional retrospective study was performed at King Abdulaziz Hospital, Saudi Arabia, between March 2010 and June 2012. The study monitored consecutive neonates born to mothers from one Arab Muslim tribe that was assumed to have a low prevalence of G6PD-deficiency. Neonates that satisfied the following criteria were included: full-term birth (37 weeks); no admission to the special care nursery; no phototherapy treatment; negative direct antiglobulin test; and fathers of female neonates were from the same mothers’ tribe. The G6PD activity (Units/gram Hemoglobin) was measured spectrophotometrically by an automated kit. This study used statistical analyses endorsed by IFCC and CLSI for reference interval estimation. The 2.5th percentiles and the corresponding 95% confidence intervals (CI) were estimated as LRLs, both in presence and absence of outliers. Results 207 males and 188 females term neonates who had cord blood quantitative G6PD testing met the inclusion criteria. Method of Horn detected 20 G6PD values as outliers (8 males and 12 females). Distributions of quantitative cord G6PD values exhibited a normal distribution in absence of the outliers only. The Harris-Boyd method and proportion criteria revealed that combined gender LRLs were reliable. The combined bootstrap LRL in presence of the outliers was 10.0 (95% CI: 7.5-10.7) and the combined parametric LRL in absence of the outliers was 11

  9. [The regulation of glucose-6-phosphate dehydrogenase and glycogen synthase activities by insulin superfamily peptides in myometrium of pregnant women and its impairments under different types of diabetes mellitus].

    PubMed

    Kuznetsova, L A; Chistiakova, O V

    2009-01-01

    The regulatory effects of insulin, insulin-like growth factor 1 (IGF-1), and relaxin on glucose-6-phosphate dehydrogenase (G6PDH) and glycogen synthase (GS) activities have been studied in myometrium of pregnant women of control group and with diabetes mellitus of different etiology. In patients with type 1 diabetes G6PDH activity did not differ from the control group, but the enzyme activity was sharply decreased in pregnant women with type 2 diabetes and gestational diabetes. In the control group maximal stimulation of G6PDH activity was observed at 10(-9) M of peptides and their stimulating effect decreased in the following order: insulin > relaxin > IGF-1. In pregnant women with types 1 diabetes insulin effect on the enzyme activity was lower than in the control, and the effects of IGF-1 and relaxin were absent. In the group of pregnant women with type 2 diabetes and gestational diabetes the effects of insulin and IGF-1 were decreased, but the effect of relaxin was somewhat higher thus giving the following order in their efficiency relaxin > IGF-1 = insulin. At 10(-9) M peptides exhibited similar stimulating effects on the active form of GS-I, but had no influence on the total enzyme activity in the control group of pregnant women. In patients with type 1 diabetes GS activity remained unchanged (versus control), and peptides did not stimulate the enzyme activity. In patients with type 2 diabetes a significant decrease in GS activity was accompanied by the decrease in the effect of peptides, giving the following order of their efficiency: insulin = IGF-1 > relaxin. In myometrium of pregnant women with gestational (treated and untreated) diabetes GS activity decreased, the effect of insulin was weaker, whereas the effects of relaxin and IGF-1 increased thus giving the following order of their efficiency: relaxin > IGF-1 > insulin. Insulin therapy of type 1 diabetes incompletely restored sensitivity of the enzymes to the peptide actions. At the same time, in women

  10. Glucose-6-phosphate dehydrogenase-dependent hydrogen peroxide production is involved in the regulation of plasma membrane H+-ATPase and Na+/H+ antiporter protein in salt-stressed callus from Carex moorcroftii.

    PubMed

    Li, Jisheng; Chen, Guichen; Wang, Xiaomin; Zhang, Yanli; Jia, Honglei; Bi, Yurong

    2011-03-01

    Glucose-6-phosphate dehydrogenase (G6PDH) is important for the activation of plant resistance to environmental stresses, and ion homeostasis is the physiological foundation for living cells. In this study, we investigated G6PDH roles in modulating ion homeostasis under salt stress in Carex moorcroftii callus. G6PDH activity increased to its maximum in 100 mM NaCl treatment and decreased with further increased NaCl concentrations. K+/Na+ ratio in 100 mM NaCl treatment did not exhibit significant difference compared with the control; however, in 300 mM NaCl treatment, it decreased. Low-concentration NaCl (100 mM) stimulated plasma membrane (PM) H+-ATPase and NADPH oxidase activities as well as Na+/H+ antiporter protein expression, whereas high-concentration NaCl (300 mM) decreased their activity and expression. When G6PDH activity and expression were reduced by glycerol treatments, PM H+-ATPase and NADPH oxidase activities, Na+/H+ antiporter protein level and K+/Na+ ratio dramatically decreased. Simultaneously, NaCl-induced hydrogen peroxide (H₂O₂) accumulation was abolished. Exogenous application of H₂O₂ increased G6PDH, PM H+-ATPase and NADPH oxidase activities, Na+/H+ antiporter protein expression and K+/Na+ ratio in the control and glycerol treatments. Diphenylene iodonium (DPI), the NADPH oxidase inhibitor, which counteracted NaCl-induced H₂O₂ accumulation, decreased G6PDH, PM H+-ATPase and NADPH oxidase activities, Na+/H+ antiporter protein level and K+/Na+ ratio. Western blot result showed that G6PDH expression was stimulated by NaCl and H₂O₂, and blocked by DPI. Taken together, G6PDH is involved in H₂O₂ accumulation under salt stress. H₂O₂, as a signal, upregulated PM H+-ATPase activity and Na+/H+ antiporter protein level, which subsequently resulted in the enhanced K+/Na+ ratio. G6PDH played a central role in the process.

  11. Purification and characterization of human erythrocyte glucose transporter in decylmaltoside detergent solution.

    PubMed

    Boulter, J M; Wang, D N

    2001-07-01

    The facilitative glucose transporter from human erythrocyte membrane, Glut1, was purified by a novel method. The nonionic detergent decylmaltoside was selected for solubilization on the basis of its efficiency to extract Glut1 from the erythrocyte membrane and its ability to maintain the protein in a monodisperse state. A positive, anion-exchange chromatography protocol produced a Glut1 preparation of 95% purity with little copurified lipid. This protein preparation exhibited cytochalasin B binding in detergent solution, as measured by tryptophan fluorescence quenching. The transporter existed as a monomer in decylmaltoside, with a Stokes radius of 50 A and a molecular mass of 147 kDa for the protein-detergent complex. We screened detergent, pH, additive, and lipid and have found conditions to maintain Glut1 monodispersity for 8 days at 25 degrees C or over 5 weeks at 4 degrees C. This Glut1 preparation represents the best available material for two- and three-dimensional crystallization trials of the human glucose transporter protein.

  12. [3H]forskolin. Direct photoaffinity labeling of the erythrocyte D-glucose transporter.

    PubMed

    Shanahan, M F; Morris, D P; Edwards, B M

    1987-05-01

    Irradiation of erythrocyte ghosts in the presence of [3H]forskolin resulted in a concentration-dependent, covalent incorporation of radiolabel into several of the major membrane protein bands. Most of the incorporation occurred in four regions of the gel. Peak 1 (216 kDa) was a sharp peak near the top of the gel in the region corresponding to spectrin. Peak 2 appeared to be associated with band 3 (89 kDa), while a third peak occurred around the position of band 4.2 (76 kDa). The fourth region of labeling was a broad area between 43-75 kDa which corresponds to the region of the glucose transporter. Forskolin labeling of this region was inhibited by cytochalasin B and D-glucose, but not L-glucose. Extraction of extrinsic membrane proteins resulted in a loss of radiolabeled protein from the 216- and 76-kDa regions. Treatment of membranes labeled with either cytochalasin B or forskolin with endo-beta-galactosidase resulted in identical shifts of the 43 to 75-kDa peaks to 42 kDa. Similarly, trypsinization of membranes photolabeled with either cytochalasin B or forskolin resulted in the generation of a 17-kDa radiolabeled fragment in both cases. Photoincorporation of [3H]cytochalasin B into the glucose transporter was blocked in a concentration-dependent manner by unlabeled forskolin. PMID:3106349

  13. /sup 3/H)forskolin. Direct photoaffinity labeling of the erythrocyte D-glucose transporter

    SciTech Connect

    Shanahan, M.F.; Morris, D.P.; Edwards, B.M.

    1987-05-05

    Irradiation of erythrocyte ghosts in the presence of (/sup 3/H)forskolin resulted in a concentration-dependent, covalent incorporation of radiolabel into several of the major membrane protein bands. Most of the incorporation occurred in four regions of the gel. Peak 1 (216 kDa) was a sharp peak near the top of the gel in the region corresponding to spectrin. Peak 2 appeared to be associated with band 3 (89 kDa), while a third peak occurred around the position of band 4.2 (76 kDa). The fourth region of labeling was a broad area between 43-75 kDa which corresponds to the region of the glucose transporter. Forskolin labeling of this region was inhibited by cytochalasin B and D-glucose, but not L-glucose. Extraction of extrinsic membrane proteins resulted in a loss of radiolabeled protein from the 216- and 76-kDa regions. Treatment of membranes labeled with either cytochalasin B or forskolin with endo-beta-galactosidase resulted in identical shifts of the 43 to 75-kDa peaks to 42 kDa. Similarly, trypsinization of membranes photolabeled with either cytochalasin B or forskolin resulted in the generation of a 17-kDa radiolabeled fragment in both cases. Photoincorporation of (/sup 3/H)cytochalasin B into the glucose transporter was blocked in a concentration-dependent manner by unlabeled forskolin.

  14. Discovery of a Plasmodium falciparum glucose-6-phosphate dehydrogenase 6- phosphogluconolactonase inhibitor (R,Z)-N-((1-ethylpyrrolidin-2-yl)methyl)-2-(2-fluorobenzylidene)-3-oxo-3,4-dihydro-2H-benzo[b][1,4]thiazine-6-carboxamide (ML276) that reduces parasite growth in vitro

    PubMed Central

    Preuss, Janina; Maloney, Patrick; Peddibhotla, Satyamaheshwar; Hedrick, Michael P.; Hershberger, Paul; Gosalia, Palak; Milewski, Monika; Li, Yujie Linda; Sugarman, Eliot; Hood, Becky; Suyama, Eigo; Nguyen, Kevin; Vasile, Stefan; Sergienko, Eduard; Mangravita-Novo, Arianna; Vicchiarelli, Michael; McAnally, Danielle; Smith, Layton H.; Roth, Gregory P.; Diwan, Jena; Chung, Thomas D.Y.; Jortzik, Esther; Rahlfs, Stefan; Becker, Katja; Pinkerton, Anthony B.; Bode, Lars

    2012-01-01

    A high throughput screen of the NIH’s MLSMR collection of ~340,000 compounds was undertaken to identify compounds that inhibit Plasmodium falciparum glucose-6-phosphate dehydrogenase (PfG6PD). PfG6PD is essential for proliferating and propagating P. falciparum and differs structurally and mechanistically from the human ortholog. The reaction catalyzed by glucose-6-phosphate dehydrogenase (G6PD) is the first, rate-limiting step in the pentose phosphate pathway (PPP), a key metabolic pathway sustaining anabolic needs in reductive equivalents and synthetic materials in fastgrowing cells. In P. falciparum the bifunctional enzyme glucose-6-phosphate dehydrogenase-6- phosphogluconolactonase (PfGluPho) catalyzes the first two steps of the PPP. Because P. falciparum and infected host red blood cells rely on accelerated glucose flux, they depend on the G6PD activity of PfGluPho. The lead compound identified from this effort, (R,Z)-N-((1-ethylpyrrolidin-2-yl)methyl)-2- (2-fluorobenzylidene)-3-oxo-3,4-dihydro-2H-benzo[b][1,4]thiazine-6-carboxamide, 11, (ML276), is a submicromolar inhibitor of PfG6PD (IC50 = 889 nM). It is completely selective for the enzyme’s human isoform, displays micromolar potency (IC50 = 2.6 μM) against P. falciparum in culture, and has good drug-like properties, including high solubility and moderate microsomal stability. Studies testing the potential advantage of inhibiting PfG6PD in vivo are in progress. PMID:22813531

  15. Regulation of glucosamine-6-phosphate deaminase synthesis in yeast.

    PubMed

    Singh, B; Datta, A

    1979-02-19

    A basal level of glucosamine-6-phosphate deaminase is detected in yeast cells grown on glucose. However, a burst of enzyme production occurs in the presence of N-acetylglucosamine in pathogenic Candida albicans and non-pathogenic Saccharomyces cervisiae. The enzyme synthesis stops and its concentration in the cells declines rapidly as soon as N-acetylglucosamine is removed from the medium. Experiments with RNA- and protein-synthesis inhibitors indicate that the appearance of new enzyme activity is dependent on concomitant new protein synthesis and the inducer operates at a transcriptional level. However, inhibition of DNA synthesis either by hydroxyurea or by mitomycin-C does not impair the synthesis of glucosamine-6-phosphate deaminase. PMID:369615

  16. Interactions of androgens, green tea catechins and the antiandrogen flutamide with the external glucose-binding site of the human erythrocyte glucose transporter GLUT1.

    PubMed

    Naftalin, Richard J; Afzal, Iram; Cunningham, Philip; Halai, Mansur; Ross, Clare; Salleh, Naguib; Milligan, Stuart R

    2003-10-01

    This study investigates the effects of androgens, the antiandrogen flutamide and green tea catechins on glucose transport inhibition in human erythrocytes. These effects may relate to the antidiabetogenic effects of green tea. Testosterone, 4-androstene-3,17-dione, dehydroepiandrosterone (DHEA) and DHEA-3-acetate inhibit glucose exit from human erythrocytes with half-maximal inhibitions (Ki) of 39.2+/-8.9, 29.6+/-3.7, 48.1+/-10.2 and 4.8+/-0.98 microM, respectively. The antiandrogen flutamide competitively relieves these inhibitions and of phloretin. Dehydrotestosterone has no effect on glucose transport, indicating the differences between androgen interaction with GLUT1 and human androgen receptor (hAR). Green tea catechins also inhibit glucose exit from erythrocytes. Epicatechin 3-gallate (ECG) has a Ki ECG of 0.14+/-0.01 microM, and epigallocatechin 3-gallate (EGCG) has a Ki EGCG of 0.97+/-0.13 microM. Flutamide reverses these effects. Androgen-screening tests show that the green tea catechins do not act genomically. The high affinities of ECG and EGCG for GLUT1 indicate that this might be their physiological site of action. There are sequence homologies between GLUT1 and the ligand-binding domain (LBD) of hAR containing the amino-acid triads Arg 126, Thr 30 and Asn 288, and Arg 126, Thr 30 and Asn 29, with similar 3D topology to the polar groups binding 3-keto and 17-beta OH steroid groups in hAR LBD. These triads are appropriately sited for competitive inhibition of glucose import at the external opening of the hydrophilic pore traversing GLUT1.

  17. Enzymatic and regulatory properties of the trehalose-6-phosphate synthase from the thermoacidophilic archaeon Thermoplasma acidophilum.

    PubMed

    Gao, Yanyan; Jiang, Ying; Liu, Qiulei; Wang, Ruiming; Liu, Xinli; Liu, Bo

    2014-06-01

    Trehalose-6-phosphate synthase plays an important role in trehalose metabolism. It catalyzes the transfer of glucose from UDP-glucose (UDPG) to glucose 6-phosphate to produce trehalose-6-phosphate. Herein we describe the characterization of a trehalose-6-phosphate synthase from the thermoacidophilic archaeon Thermoplasma acidophilum. The dimeric enzyme could utilize UDPG, ADP-Glucose (ADPG) and GDP-Glucose (GDPG) as glycosyl donors and various phosphorylated monosaccharides as glycosyl acceptors. The optimal temperature and pH were found to be 60 °C and pH 6, and the enzyme exhibited notable pH and thermal stability. The enzymatic activity could be stimulated by divalent metal ions and polyanions heparin and chondroitin sulfate. Moreover, the protein was considerably resistant to additives ethanol, EDTA, urea, DTT, SDS, β-mercaptoethanol, methanol, isopropanol and n-butanol. Molecular modeling and mutagenesis analysis revealed that the N-loop region was important for the catalytic efficiency of the enzyme, indicating different roles of N-loop sequences in different trehalose-6-phosphate synthases. PMID:24508535

  18. Stimulation of glucose uptake by insulin-like growth factor II in human muscle is not mediated by the insulin-like growth factor II/mannose 6-phosphate receptor.

    PubMed Central

    Burguera, B; Elton, C W; Caro, J F; Tapscott, E B; Pories, W J; Dimarchi, R; Sakano, K; Dohm, G L

    1994-01-01

    Although the growth-promoting effects of insulin-like growth factor II (IGF-II) have been intensively studied, the acute actions of this hormone on glucose metabolism have been less well evaluated, especially in skeletal muscle of humans. We and other groups have shown that IGFs reduce glycaemic levels in humans and stimulate glucose uptake in rat muscle. The purpose of the present study was to evaluate the effect of IGF-II on glucose transport in muscle of normal and obese patients with and without non-insulin-dependent diabetes mellitus (NIDDM), as well as to identify the receptor responsible for this action. 2-Deoxyglucose transport was determined in vitro using a muscle-fibre strip preparation. IGF-II were investigated in biopsy material of rectus abdominus muscle taken from lean and obese patients and obese patients with NIDDM at the time of surgery. In the lean group, IGF-II (100 nM) stimulated glucose transport 2.1-fold, which was slightly less than stimulation by insulin (2.8-fold) at the same concentration. Binding of IGF-II was approx. 25% of that of insulin at 1 nM concentrations of both hormones. Obesity with or without NIDDM significantly reduced IGF-II-stimulated glucose uptake compared with the lean group. In order to explore which receptor mediated the IGF-II effect, we compared glucose uptake induced by IGF-II and two IGF-II analogues: [Leu27]IGF-II, with high affinity for the IGF-II/Man 6-P receptor but markedly reduced affinity for the IGF-I and insulin receptors, and [Arg54,Arg55]IGF-II was similar to that of IGF-II, whereas [Leu27]IGF-II had a very diminished effect. Results show that IGF-II is capable of stimulating muscle glucose uptake in lean but not in obese subjects and this effect seems not to be mediated via an IGF-II/Man 6-P receptor. Images Figure 2 PMID:8010960

  19. Suicide for survival--death of infected erythrocytes as a host mechanism to survive malaria.

    PubMed

    Föller, Michael; Bobbala, Diwakar; Koka, Saisudha; Huber, Stephan M; Gulbins, Erich; Lang, Florian

    2009-01-01

    The pathogen of malaria, Plasmodium, enters erythrocytes and thus escapes recognition by the immune system. The pathogen induces oxidative stress to the host erythrocyte, which triggers eryptosis, the suicidal death of erythrocytes. Eryptosis is characterized by cell shrinkage, membrane blebbing and cell membrane phospholipid scrambling with phosphatidylserine exposure at the cell surface. Phosphatidylserine-exposing erythrocytes are identified by macrophages which engulf and degrade the eryptotic cells. To the extent that infected erythrocytes undergo eryptosis prior to exit of Plasmodiaand subsequent infection of other erythrocytes, the premature eryptosis may protect against malaria. Accordingly, any therapeutical intervention accelerating suicidal death of infected erythrocytes has the potential to foster elimination of infected erythrocytes, delay the development of parasitemia and favorably influence the course of malaria. Eryptosis is stimulated by a wide variety of triggers including osmotic shock, oxidative stress, energy depletion and a wide variety of xenobiotics. Diseases associated with accelerated eryptosis include sepsis, haemolytic uremic syndrome, malaria, sickle-cell anemia, beta-thalassemia, glucose-6-phosphate dehydrogenase (G6PD)-deficiency, phosphate depletion, iron deficiency and Wilson's disease. Among the known stimulators of eryptosis, paclitaxel, chlorpromazine, cyclosporine, curcumin, PGE2 and lead have indeed been shown to favourably influence the course of malaria. Moreover, sickle-cell trait, beta-thalassemia trait, glucose-6-phosphate dehydrogenase (G6PD)-deficiency and iron deficiency confer some protection against a severe course of malaria. Importantly, counteracting Plasmodia by inducing eryptosis is not expected to generate resistance of the pathogen, as the proteins involved in suicidal death of the host cell are not encoded by the pathogen and thus cannot be modified by mutations of its genes.

  20. Erythrocyte survival in sheep exposed to ozone

    SciTech Connect

    Moore, G.S.; Calabrese, E.J.; Labato, F.J.

    1981-07-01

    Erythrocyte survival studies in the Dorset ewe using chromium 51 were performed. The purpose of the study was to determine if ozone exposure produces decreased cell survival which may be the result of premature erythrocyte aging. This strain of sheep has an erythrocyte glucose-6-phosphate dehydrogenase (G6PD) activity that is very low, being comparable to human A-variants with G6PD deficiency. Ozone exposure may produce hemolytic effects in G6PD deficients more readily than in erythrocytes with normal activity. A decrease in hematocrit was observed in the ozone exposed groups. With respect to red cell destruction, ozone does not appear to act immediately, but rather there appears to be a delayed effect. At 0.25 ppM ozone, the group reached the 50% remaining level an average of 1 day sooner than the control group. There was no significant difference between control and exposed groups at the 0.50 ppM and 0.70 ppM levels. Also, the results demonstrate a net decrease in hematocrit which is greater for 0.25 ppM ozone than any other exposure level. (RJC)

  1. Triggers, Inhibitors, Mechanisms, and Significance of Eryptosis: The Suicidal Erythrocyte Death

    PubMed Central

    Lang, Elisabeth

    2015-01-01

    Suicidal erythrocyte death or eryptosis is characterized by erythrocyte shrinkage, cell membrane blebbing, and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Triggers of eryptosis include Ca2+ entry, ceramide formation, stimulation of caspases, calpain activation, energy depletion, oxidative stress, and dysregulation of several kinases. Eryptosis is triggered by a wide variety of xenobiotics. It is inhibited by several xenobiotics and endogenous molecules including NO and erythropoietin. The susceptibility of erythrocytes to eryptosis increases with erythrocyte age. Phosphatidylserine exposing erythrocytes adhere to the vascular wall by binding to endothelial CXC-Motiv-Chemokin-16/Scavenger-receptor for phosphatidylserine and oxidized low density lipoprotein (CXCL16). Phosphatidylserine exposing erythrocytes are further engulfed by phagocytosing cells and are thus rapidly cleared from circulating blood. Eryptosis eliminates infected or defective erythrocytes thus counteracting parasitemia in malaria and preventing detrimental hemolysis of defective cells. Excessive eryptosis, however, may lead to anemia and may interfere with microcirculation. Enhanced eryptosis contributes to the pathophysiology of several clinical disorders including metabolic syndrome and diabetes, malignancy, cardiac and renal insufficiency, hemolytic uremic syndrome, sepsis, mycoplasma infection, malaria, iron deficiency, sickle cell anemia, thalassemia, glucose 6-phosphate dehydrogenase deficiency, and Wilson's disease. Facilitating or inhibiting eryptosis may be a therapeutic option in those disorders. PMID:25821808

  2. Crystal Structure Analysis of Human Glutamine : Fructose 6-Phosphate Amidotransferase, a Key Regulator in Type 2 Diabetes

    NASA Astrophysics Data System (ADS)

    Nakaishi, Yuichiro; Bando, Masahiko

    Glutamine : fructose 6-phosphate amidotransferase (GFAT) is a rate-limiting enzyme in the hexoamine biosythetic pathway and plays an important role in type 2 diabetes. We now report the first structures of the isomerase domain of the human GFAT in the presence of cyclic glucose 6-phosphate and linear glucosamine 6-phosphate. The C-terminal tail including the active site displays a rigid conformation, similar to the corresponding Escherichia coli enzyme. The diversity of the CF helix near the active site suggests the helix is a major target for drug design. Our study provides insights into the development of therapeutic drugs for type 2 diabetes.

  3. Novel steroid inhibitors of glucose 6-phosphate dehydrogenase.

    PubMed

    Hamilton, Niall M; Dawson, Martin; Fairweather, Emma E; Hamilton, Nicola S; Hitchin, James R; James, Dominic I; Jones, Stuart D; Jordan, Allan M; Lyons, Amanda J; Small, Helen F; Thomson, Graeme J; Waddell, Ian D; Ogilvie, Donald J

    2012-05-10

    Novel derivatives of the steroid DHEA 1, a known uncompetitive inhibitor of G6PD, were designed, synthesized, and tested for their ability to inhibit this dehydrogenase enzyme. Several compounds with approximately 10-fold improved potency in an enzyme assay were identified, and this improved activity translated to efficacy in a cellular assay. The SAR for steroid inhibition of G6PD has been substantially developed; the 3β-alcohol can be replaced with 3β-H-bond donors such as sulfamide, sulfonamide, urea, and carbamate. Improved potency was achieved by replacing the androstane nucleus with a pregnane nucleus, provided a ketone at C-20 is present. For pregnan-20-ones incorporation of a 21-hydroxyl group is often beneficial. The novel compounds generally have good physicochemical properties and satisfactory in vitro DMPK parameters. These derivatives may be useful for examining the role of G6PD inhibition in cells and will assist the future design of more potent steroid inhibitors with potential therapeutic utility. PMID:22506561

  4. Genetics Home Reference: glucose-6-phosphate dehydrogenase deficiency

    MedlinePlus

    ... as some antibiotics and medications used to treat malaria). Hemolytic anemia can also occur after eating fava ... a G6PD mutation may be partially protected against malaria, an infectious disease carried by a certain type ...

  5. G6PD Deficiency (Glucose-6-Phosphate Dehydrogenase) (For Parents)

    MedlinePlus

    ... are high-risk areas for the infectious disease malaria . Researchers have found evidence that the parasite that ... deficiency may have developed as a protection against malaria. continue G6PD Deficiency Symptom Triggers Kids with G6PD ...

  6. Comparative erythrocyte metabolism in marsupials and monotremes.

    PubMed

    Parkinson, A L; Whittington, A T; Spencer, P B; Grigg, G; Hinds, L; Gallagher, C; Kuchel, P; Agar, N S

    1995-03-01

    Concentrations of ATP and DPG, activities of 10 enzymes and the glycolytic rates were measured in the erythrocytes of 11 species of marsupials and two species of monotremes. Mean DPG concentrations were greater in the erythrocytes of marsupials than those of eutherian mammals. The opposite is true of ATP. Significant findings from the results of enzyme activities were: high activity of hexokinase (7.39 +/- 0.82 EU/g Hb) in the short-beaked echidna, pyruvate kinase (37.49 +/- 1.0 EU/g) Hb in bridled nailtail wallaby and glucose-6-phosphate dehydrogenase (G6PD; 41.66 +/- 1.24 EU/g Hb) in black-striped wallaby. About 6- to 7-fold difference in the activity of G6PD levels between the two species of wombats was confirmed. Glucose phosphate isomerase activity was also shown to be twice as high in the red cells of the common wombat compared with those of the southern hairy nosed wombat. There were wide variations in the glycolytic rate among the species examined.

  7. Comparative erythrocyte metabolism in marsupials and monotremes.

    PubMed

    Parkinson, A L; Whittington, A T; Spencer, P B; Grigg, G; Hinds, L; Gallagher, C; Kuchel, P; Agar, N S

    1995-03-01

    Concentrations of ATP and DPG, activities of 10 enzymes and the glycolytic rates were measured in the erythrocytes of 11 species of marsupials and two species of monotremes. Mean DPG concentrations were greater in the erythrocytes of marsupials than those of eutherian mammals. The opposite is true of ATP. Significant findings from the results of enzyme activities were: high activity of hexokinase (7.39 +/- 0.82 EU/g Hb) in the short-beaked echidna, pyruvate kinase (37.49 +/- 1.0 EU/g) Hb in bridled nailtail wallaby and glucose-6-phosphate dehydrogenase (G6PD; 41.66 +/- 1.24 EU/g Hb) in black-striped wallaby. About 6- to 7-fold difference in the activity of G6PD levels between the two species of wombats was confirmed. Glucose phosphate isomerase activity was also shown to be twice as high in the red cells of the common wombat compared with those of the southern hairy nosed wombat. There were wide variations in the glycolytic rate among the species examined. PMID:7599974

  8. Mannose-6-phosphate regulates destruction of lipid-linked oligosaccharides

    PubMed Central

    Gao, Ningguo; Shang, Jie; Huynh, Dang; Manthati, Vijaya L.; Arias, Carolina; Harding, Heather P.; Kaufman, Randal J.; Mohr, Ian; Ron, David; Falck, John R.; Lehrman, Mark A.

    2011-01-01

    Mannose-6-phosphate (M6P) is an essential precursor for mannosyl glycoconjugates, including lipid-linked oligosaccharides (LLO; glucose3mannose9GlcNAc2-P-P-dolichol) used for protein N-glycosylation. In permeabilized mammalian cells, M6P also causes specific LLO cleavage. However, the context and purpose of this paradoxical reaction are unknown. In this study, we used intact mouse embryonic fibroblasts to show that endoplasmic reticulum (ER) stress elevates M6P concentrations, leading to cleavage of the LLO pyrophosphate linkage with recovery of its lipid and lumenal glycan components. We demonstrate that this M6P originates from glycogen, with glycogenolysis activated by the kinase domain of the stress sensor IRE1-α. The apparent futility of M6P causing destruction of its LLO product was resolved by experiments with another stress sensor, PKR-like ER kinase (PERK), which attenuates translation. PERK's reduction of N-glycoprotein synthesis (which consumes LLOs) stabilized steady-state LLO levels despite continuous LLO destruction. However, infection with herpes simplex virus 1, an N-glycoprotein-bearing pathogen that impairs PERK signaling, not only caused LLO destruction but depleted LLO levels as well. In conclusion, the common metabolite M6P is also part of a novel mammalian stress-signaling pathway, responding to viral stress by depleting host LLOs required for N-glycosylation of virus-associated polypeptides. Apparently conserved throughout evolution, LLO destruction may be a response to a variety of environmental stresses. PMID:21737679

  9. Phosphorylation of the human erythrocyte glucose transporter by protein kinase C: localization of the site of in vivo and in vitro phosphorylation.

    PubMed

    Deziel, M R; Lippes, H A; Rampal, A L; Jung, C Y

    1989-01-01

    1. The human erythrocyte glucose transporter was phosphorylated in vitro by protein kinase C. 2. Tryptic cleavage of phosphorylated native transporter produced two major unphosphorylated membrane-embedded fragments weighing 23 and 19 kDa and released numerous water-soluble peptides. 3. Ion-exchange FPLC of the soluble tryptic peptides resolved the mixture into two phosphopeptide peaks. 4. Tryptic digestion of glucose transporter that was phosphorylated in vivo in response to phorbol esters produced soluble phosphopeptides that eluted at identical salt concentrations. 5. Proteolytic digestion and peptide mapping of the transporter revealed that the site(s) of phosphorylation lie within the large cytoplasmic domain that bisects the molecule.

  10. Structural Basis for Morpheein-type Allosteric Regulation of Escherichia coli Glucosamine-6-phosphate Synthase

    PubMed Central

    Mouilleron, Stéphane; Badet-Denisot, Marie-Ange; Pecqueur, Ludovic; Madiona, Karine; Assrir, Nadine; Badet, Bernard; Golinelli-Pimpaneau, Béatrice

    2012-01-01

    The amino-terminal cysteine of glucosamine-6-phosphate synthase (GlmS) acts as a nucleophile to release and transfer ammonia from glutamine to fructose 6-phosphate through a channel. The crystal structure of the C1A mutant of Escherichia coli GlmS, solved at 2.5 Å resolution, is organized as a hexamer, where the glutaminase domains adopt an inactive conformation. Although the wild-type enzyme is active as a dimer, size exclusion chromatography, dynamic and quasi-elastic light scattering, native polyacrylamide gel electrophoresis, and ultracentrifugation data show that the dimer is in equilibrium with a hexameric state, in vitro and in cellulo. The previously determined structures of the wild-type enzyme, alone or in complex with glucosamine 6-phosphate, are also consistent with a hexameric assembly that is catalytically inactive because the ammonia channel is not formed. The shift of the equilibrium toward the hexameric form in the presence of cyclic glucosamine 6-phosphate, together with the decrease of the specific activity with increasing enzyme concentration, strongly supports product inhibition through hexamer stabilization. Altogether, our data allow us to propose a morpheein model, in which the active dimer can rearrange into a transiently stable form, which has the propensity to form an inactive hexamer. This would account for a physiologically relevant allosteric regulation of E. coli GlmS. Finally, in addition to cyclic glucose 6-phosphate bound at the active site, the hexameric organization of E. coli GlmS enables the binding of another linear sugar molecule. Targeting this sugar-binding site to stabilize the inactive hexameric state is therefore suggested for the development of specific antibacterial inhibitors. PMID:22851174

  11. Stiffness of normal and pathological erythrocytes studied by means of atomic force microscopy.

    PubMed

    Dulińska, Ida; Targosz, Marta; Strojny, Wojciech; Lekka, Małgorzata; Czuba, Paweł; Balwierz, Walentyna; Szymoński, Marek

    2006-03-31

    During recent years, atomic force microscopy has become a powerful technique for studying the mechanical properties (such as stiffness, viscoelasticity, hardness and adhesion) of various biological materials. The unique combination of high-resolution imaging and operation in physiological environment made it useful in investigations of cell properties. In this work, the microscope was applied to measure the stiffness of human red blood cells (erythrocytes). Erythrocytes were attached to the poly-L-lysine-coated glass surface by fixation using 0.5% glutaraldehyde for 1 min. Different erythrocyte samples were studied: erythrocytes from patients with hemolytic anemias such as hereditary spherocytosis and glucose-6-phosphate-dehydrogenase deficiency patients with thalassemia, and patients with anisocytosis of various causes. The determined Young's modulus was compared with that obtained from measurements of erythrocytes from healthy subjects. The results showed that the Young's modulus of pathological erythrocytes was higher than in normal cells. Observed differences indicate possible changes in the organization of cell cytoskeleton associated with various diseases.

  12. Catalase and glutathione peroxidase are equally active in detoxification of hydrogen peroxide in human erythrocytes

    SciTech Connect

    Gaetani, G.F.; Galiano, S.; Canepa, L.; Ferraris, A.M.; Kirkman, H.N.

    1989-01-01

    Genetic deficiencies of glucose-6-phosphate dehydrogenase (G6PD) and NADPH predispose affected erythrocytes to destruction from peroxides. Conversely, genetic deficiencies of catalase do not predispose affected erythrocytes to peroxide-induced destruction. These observations have served to strengthen the assumption that the NADPH/glutathione/glutathione peroxidase pathway is the principal means for disposal of H/sub 2/O/sub 2/ in human erythrocytes. Recently, however, mammalian catalase was found to have tightly bound NADPH and to require NADPH for the prevention and reversal of inactivation by its toxic substrate (H/sub 2/O/sub 2/). Since both catalase and the glutathione pathway are dependent on NADPH for function, this finding raises the possibility that both mechanisms destroy H/sub 2/O/sub 2/ in human erythrocytes. A comparison of normal and acatalasemic erythrocytes in the present study indicated that catalase accounts for more than half of the destruction of H/sub 2/O/sub 2/ when H/sub 2/O/sub 2/ is generated at a rate comparable to that which leads to hemolysis in G6PD- deficient erythrocytes.

  13. Antioxidant status of erythrocytes and their response to oxidative challenge in humans with argemone oil poisoning

    SciTech Connect

    Babu, Challagundla K.; Khanna, Subhash K.; Das, Mukul

    2008-08-01

    Oxidative damage of biomolecules and antioxidant status in erythrocytes of humans from an outbreak of argemone oil (AO) poisoning in Kannauj (India) and AO intoxicated experimental animals was investigated. Erythrocytes of the dropsy patients and AO treated rats were found to be more susceptible to 2,2'-azobis (2-amidinopropane) dihydrochloride (AAPH) induced peroxidative stress. Significant decrease in RBC glutathione (GSH) levels (46, 63%) with concomitant enhancement in oxidized glutathione (172, 154%) levels was noticed in patients and AO intoxicated animals. Further, depletion of glutathione reductase (GR), glucose-6-phosphate dehydrogenase (G-6-PDH) and glutathione-S-transferase (GST) (42-52%) was observed in dropsy patients. Oxidation of erythrocyte membrane lipids and proteins was increased (120-144%) in patients and AO treated animals (112-137%) along with 8-OHdG levels in whole blood (180%) of dropsy patients. A significant reduction in {alpha}-tocopherol content (68%) was noticed in erythrocytes of dropsy patients and hepatic, plasma and RBCs of AO treated rats (59-70%) thereby indicating the diminished antioxidant potential to scavenge free radicals or the limited transport of {alpha}-tocopherol from liver to RBCs leading to enhanced oxidation of lipids and proteins in erythrocytes. These studies implicate an important role of erythrocyte degradation in production of anemia and breathlessness in epidemic dropsy.

  14. Glucose and nucleoside transporters of human erythrocytes: effects of detergents on immunoadsorption of a membrane protein to its monoclonal antibody.

    PubMed

    Jhun, B H; Berenski, C J; Craik, J D; Paterson, A R; Cass, C E; Jung, C Y

    1991-01-30

    Immunoadsorption of membrane proteins solubilized in detergents has been used widely for identification, purification and quantitation of transporters and receptors. In an effort to separate the glucose and nucleoside nucleoside transporters of human erythrocytes (GT and NT, respectively) that copurify in a membrane protein fraction band 4.5, we examined in the present study the effects of seven different detergents on the immunoadsorption of GT to its monoclonal antibody, 65D4 (Craik, et al. (1988) Biochem. Cell Biol. 66, 839-852). The following results were obtained. (1) The maximum extent of the immunoadsorption of GT by 65D4 varied between 52 to 98% in different detergents. For non-ionic detergents, there was an apparent inverse correlation between the maximum immunoreactivity of GT and the aggregation number or micellar size of detergents. (2) The immunoprecipitate of GT by 65D4 was contaminated with nucleoside transporter to an extent that varied from 2 to 35 mol% in different detergents. There is an inverse correlation between the extent of the contamination and the detergent aggregation number. However, this contamination was quantitatively accounted for by a time-dependent, non-specific aggregation of NT with GT in detergents. (3) A high degree of purification of NT in band 4.5 by immunoadsorptive removal of GT with 65D4 was achieved in C12E8 as predicted by the observed low NT-GT aggregation and the relatively high epitope-accessibility of GT in this detergent. Based on these findings, we conclude that certain detergents can reduce the immunoreactivity of membrane proteins significantly by modulating epitope accessibility, and may also produce a false immuno-cross-reactivity by inducing nonspecific protein aggregation.

  15. Interactions of ATP, oestradiol, genistein and the anti-oestrogens, faslodex (ICI 182780) and tamoxifen, with the human erythrocyte glucose transporter, GLUT1.

    PubMed Central

    Afzal, Iram; Cunningham, Philip; Naftalin, Richard J

    2002-01-01

    17 beta-Oestradiol (ED when subscript to K) and the phytoestrogen isoflavone genistein (GEN) inhibit glucose transport in human erythrocytes and erythrocyte ghosts. The selective oestrogen receptor modulators or anti-oestrogens, faslodex (ICI 182780) (FAS) and tamoxifen (TAM), competitively antagonize oestradiol inhibition of glucose exit from erythrocytes (K(i(ED/FAS))=2.84+/-0.16 microM and K(i(ED/TAM))=100+/-2 nM). Faslodex has no significant inhibitory effect on glucose exit, but tamoxifen alone inhibits glucose exit (K(i(TAM))=300+/-100 nM). In ghosts, ATP (1-4 mM) competitively antagonizes oestradiol, genistein and cytochalasin B (CB)-dependent inhibitions of glucose exit, (K(i(ATP/ED))=2.5+/-0.23 mM, K(i(ATP/GEN))=0.99+/-0.17 mM and K(i(ATP/CB))=0.76+/-0.08 mM). Tamoxifen and faslodex reverse oestradiol-dependent inhibition of glucose exit with ATP>1 mM (K(i(ED/TAM))=130+/-5 nM and K(i(ED/FAS))=2.7+/-0.9 microM). The cytoplasmic surface of the glucose transporter (GLUT)1 contains four sequences with close homologies to sequences in the ligand-binding domain of human oestrogen receptor beta (hesr-2). One homology is adjacent to the Walker ATP-binding motif II (GLUT1, residues 225-229) in the large cytoplasmic segment linking transmembrane helices 6 and 7; another GLUT (residues 421-423) contains the Walker ATP-binding motif III. Mapping of these regions on to a three-dimensional template of GLUT indicates that a possible oestrogen-binding site lies between His(337), Arg(349) and Glu(249) at the cytoplasmic entrance to the hydrophilic pore spanning GLUT, which have a similar topology to His(475), Glu(305) and Arg(346) in hesr-2 that anchor the head and tail hydroxy groups of oestradiol and genistein, and thus are suitably placed to provide an ATP-sensitive oestrogen binding site that could modulate glucose export. PMID:12133004

  16. Sodium Nitrate Induces Reactive Oxygen Species That Lower the Antioxidant Power, Damage the Membrane, and Alter Pathways of Glucose Metabolism in Human Erythrocytes.

    PubMed

    Ansari, Fariheen Aisha; Mahmood, Riaz

    2015-12-01

    Nitrate salts are widely used as food additives and nitrogenous fertilizers and are present as contaminants in drinking water supplies. The effect of different concentrations (1-15 mM) of sodium nitrate (NaNO3) on human erythrocytes was studied under in vitro conditions. Treatment of erythrocytes with NaNO3 resulted in increases in methemoglobin levels, lipid peroxidation, and protein oxidation and a decrease in glutathione content. There were changes in the activities of all major antioxidant defense enzymes, and the pathways of glucose metabolism were also affected. Increased generation of reactive oxygen species (ROS) took place while the antioxidant power was impaired. The osmotic fragility of cells was increased, and membrane-bound enzymes were greatly inhibited. All changes were statistically significant at a probability level of P < 0.05 at all concentrations of NaNO3 except the lowest (1 mM). Thus, NaNO3 generates ROS that cause significant damage to human erythrocytes and interfere in normal cellular pathways. PMID:26586154

  17. Mechanisms and pathophysiological significance of eryptosis, the suicidal erythrocyte death.

    PubMed

    Lang, Elisabeth; Lang, Florian

    2015-03-01

    Eryptosis, the suicidal erythrocyte death characterized by cell shrinkage and cell membrane scrambling, is stimulated by Ca(2+) entry through Ca(2+)-permeable, PGE2-activated cation channels, by ceramide, caspases, calpain, complement, hyperosmotic shock, energy depletion, oxidative stress, and deranged activity of several kinases (e.g. AMPK, GK, PAK2, CK1α, JAK3, PKC, p38-MAPK). Eryptosis is triggered by intoxication, malignancy, hepatic failure, diabetes, chronic renal insufficiency, hemolytic uremic syndrome, dehydration, phosphate depletion, fever, sepsis, mycoplasma infection, malaria, iron deficiency, sickle cell anemia, thalassemia, glucose 6-phosphate dehydrogenase deficiency, and Wilson's disease. Eryptosis may precede and protect against hemolysis but by the same token result in anemia and deranged microcirculation. PMID:25636585

  18. Acute consumption of organic and conventional tropical grape juices (Vitis labrusca L.) increases antioxidants in plasma and erythrocytes, but not glucose and uric acid levels, in healthy individuals.

    PubMed

    Toaldo, Isabela Maia; Cruz, Fernanda Alves; da Silva, Edson Luiz; Bordignon-Luiz, Marilde T

    2016-08-01

    Bioactive polyphenols in grapes are influenced by grape variety and cultivation conditions. The Vitis labrusca L. varieties are cultivated in tropical regions and used for grape juice production. We hypothesized that polyphenols from tropical grape juices would beneficially affect redox homeostasis in humans. Therefore, the effects of acute consumption of organic and conventional grape juices from V labrusca L. on antioxidants biomarkers were investigated in healthy individuals. In a controlled, randomized, crossover, intervention trial, 24 individuals were assigned to drink 400 mL of conventional juice, organic juice, or water. Each intervention was followed by a 14-day washout period. Blood samples were obtained before and 1 hour after acute intake and analyzed for erythrocyte reduced glutathione, serum total antioxidant capacity, antioxidant enzymes in erythrocytes, and glucose and uric acid in serum. The ingestion of both grape juices resulted in elevated levels of reduced glutathione (P< .001) and serum total antioxidant capacity (P< .05) and increased activity of catalase (P< .001), superoxide dismutase (P< .001), and glutathione peroxidase (P< .05) compared with the control intervention, with no significant differences between grape juices (P< .05). The intake of juices did not affect significantly the concentrations of glucose or uric acid. Grape juice polyphenols were associated with increased antioxidants, and the chemical differences between organic and conventional juices were not predictive of the observed responses. The results suggest a bioactive potential of V labrusca L. juices to improve redox homeostasis, which is involved in defense against oxidative stress in humans. PMID:27440535

  19. Acute consumption of organic and conventional tropical grape juices (Vitis labrusca L.) increases antioxidants in plasma and erythrocytes, but not glucose and uric acid levels, in healthy individuals.

    PubMed

    Toaldo, Isabela Maia; Cruz, Fernanda Alves; da Silva, Edson Luiz; Bordignon-Luiz, Marilde T

    2016-08-01

    Bioactive polyphenols in grapes are influenced by grape variety and cultivation conditions. The Vitis labrusca L. varieties are cultivated in tropical regions and used for grape juice production. We hypothesized that polyphenols from tropical grape juices would beneficially affect redox homeostasis in humans. Therefore, the effects of acute consumption of organic and conventional grape juices from V labrusca L. on antioxidants biomarkers were investigated in healthy individuals. In a controlled, randomized, crossover, intervention trial, 24 individuals were assigned to drink 400 mL of conventional juice, organic juice, or water. Each intervention was followed by a 14-day washout period. Blood samples were obtained before and 1 hour after acute intake and analyzed for erythrocyte reduced glutathione, serum total antioxidant capacity, antioxidant enzymes in erythrocytes, and glucose and uric acid in serum. The ingestion of both grape juices resulted in elevated levels of reduced glutathione (P< .001) and serum total antioxidant capacity (P< .05) and increased activity of catalase (P< .001), superoxide dismutase (P< .001), and glutathione peroxidase (P< .05) compared with the control intervention, with no significant differences between grape juices (P< .05). The intake of juices did not affect significantly the concentrations of glucose or uric acid. Grape juice polyphenols were associated with increased antioxidants, and the chemical differences between organic and conventional juices were not predictive of the observed responses. The results suggest a bioactive potential of V labrusca L. juices to improve redox homeostasis, which is involved in defense against oxidative stress in humans.

  20. The erythrocyte calcium pump is inhibited by non-enzymic glycation: studies in situ and with the purified enzyme.

    PubMed Central

    González Flecha, F L; Castello, P R; Caride, A J; Gagliardino, J J; Rossi, J P

    1993-01-01

    In a previous paper we demonstrated that incubation of either intact erythrocytes or erythrocytes membranes with glucose decreases the activity of the membrane Ca(2+)-ATPase [González Flecha, Bermúdez, Cédola, Gagliardino and Rossi (1990) Diabetes 39, 707-711]. The aim of the present work was to obtain information about the mechanism of this inhibition. For this purpose, experiments were carried out with purified Ca(2+)-ATPase, inside-out vesicles and membranes from human erythrocytes. Incubation of the purified Ca(2+)-ATPase with glucose led to a decay in the enzyme activity of up to 50% of the control activity under the conditions used. The decrease in ATPase activity was concomitant with labelling by [6-3H]glucose of the purified Ca2+ pump; the kinetic properties of both processes were almost identical, suggesting that inhibition is a consequence of the incorporation of glucose into the Ca(2+)-ATPase molecule. In inside-out vesicles, glucose also promoted inhibition of Ca(2+)-ATPase activity as well as of active Ca2+ transport. Arabinose, xylose, mannose, ribose, fructose and glucose 6-phosphate (but not mannitol) were also able to inactive the ATPase. The activation energy for both the decrease in ATPase activity by glucose and the labelling of the pump with [6-3H]glucose was about 65 kJ/mol. Furthermore, inorganic phosphate enhanced the inactivation of the Ca(2+)-ATPase by glucose. This evidence strongly suggests that inhibition is a non-enzymically catalysed process. Inactivation of the Ca(2+)-ATPase by glucose was enhanced by reductive alkylation with sodium borohydride. Aminoguanidine, an inhibitor of the formation of the advanced end products of glycosylation, did not prevent the deleterious effect of glucose on the enzyme activity. Therefore it is concluded that inactivation of the Ca2+ pump is a consequence of the glycation of this protein. PMID:8393658

  1. The erythrocyte calcium pump is inhibited by non-enzymic glycation: studies in situ and with the purified enzyme.

    PubMed

    González Flecha, F L; Castello, P R; Caride, A J; Gagliardino, J J; Rossi, J P

    1993-07-15

    In a previous paper we demonstrated that incubation of either intact erythrocytes or erythrocytes membranes with glucose decreases the activity of the membrane Ca(2+)-ATPase [González Flecha, Bermúdez, Cédola, Gagliardino and Rossi (1990) Diabetes 39, 707-711]. The aim of the present work was to obtain information about the mechanism of this inhibition. For this purpose, experiments were carried out with purified Ca(2+)-ATPase, inside-out vesicles and membranes from human erythrocytes. Incubation of the purified Ca(2+)-ATPase with glucose led to a decay in the enzyme activity of up to 50% of the control activity under the conditions used. The decrease in ATPase activity was concomitant with labelling by [6-3H]glucose of the purified Ca2+ pump; the kinetic properties of both processes were almost identical, suggesting that inhibition is a consequence of the incorporation of glucose into the Ca(2+)-ATPase molecule. In inside-out vesicles, glucose also promoted inhibition of Ca(2+)-ATPase activity as well as of active Ca2+ transport. Arabinose, xylose, mannose, ribose, fructose and glucose 6-phosphate (but not mannitol) were also able to inactive the ATPase. The activation energy for both the decrease in ATPase activity by glucose and the labelling of the pump with [6-3H]glucose was about 65 kJ/mol. Furthermore, inorganic phosphate enhanced the inactivation of the Ca(2+)-ATPase by glucose. This evidence strongly suggests that inhibition is a non-enzymically catalysed process. Inactivation of the Ca(2+)-ATPase by glucose was enhanced by reductive alkylation with sodium borohydride. Aminoguanidine, an inhibitor of the formation of the advanced end products of glycosylation, did not prevent the deleterious effect of glucose on the enzyme activity. Therefore it is concluded that inactivation of the Ca2+ pump is a consequence of the glycation of this protein.

  2. Impact of umbelliferone on erythrocyte redox status in STZ-diabetic rats.

    PubMed Central

    Ramesh, B.; Pugalendi, K. V.

    2005-01-01

    Oxidative stress is currently hypothesized to be a mechanism underlying diabetes. The present study was designed to evaluate the effect of umbelliferone (UMB), a derivative of coumarin, on erythrocyte lipid peroxidation, antioxidants, and lipid profile in normal and streptozotocin (STZ) diabetic rats. Diabetes was induced in adult male albino rats of Wistar strain, weighing 180 to 200 g, by the administration of STZ (40 mg/kg/b-wt) intraperitonially. The normal and diabetic rats were treated with UMB in 10 percent dimethyl sulfoxide (DMSO) dissolved in water for 45 days. The diabetic rats had elevated levels of blood glucose and lipid peroxidation markers such as thiobarbituric acid reactive substances (TBARS), conjugated dienes (CD), and lipid hydroperoxide (HP) and decreased levels of nonenzymatic antioxidants (Vitamin C and reduced glutathione [GSH]), elevated levels of vitamin E, and elevated levels of enzymatic antioxidants (superoxide dismutase [SOD], catalase [CAT], glutathione peroxidase [GPx]), elevated glucose-6-phosphate dehydrogenase activity, and altered lipid profile (cholesterol and phospholipids) in erythrocytes. These changes were reversed by treatment with UMB. Thus, our results indicate that the administration of UMB shows promising potential for the restoration of normal blood glucose levels, erythrocyte lipid peroxidation, antioxidants, and lipid profile in STZ-diabetic. PMID:16464311

  3. Attenuation of erythrocyte membrane oxidative stress by Sesbania grandiflora in streptozotocin-induced diabetic rats.

    PubMed

    Sureka, Chandrabose; Ramesh, Thiyagarajan; Begum, Vavamohaideen Hazeena

    2015-08-01

    The aim of the present study was to investigate the protective effects of Sesbania grandiflora flower (SGF) extract on erythrocyte membrane in Streptozotocin (STZ)-induced diabetic rats. Adult male albino rats of Wistar strain, weighing 190-220 g, were made diabetic by an intraperitonial administration of STZ (45 mg/kg). Normal and diabetic rats were treated with SGF, and diabetic rats were also treated with glibenclamide as drug control, for 45 days. In this study plasma insulin and haemoglobin levels were decreased and blood glucose, glycosylated haemoglobin, protein oxidation, lipid peroxidation markers, and osmotic fragility levels were increased in diabetic rats. Moreover, erythrocytes antioxidant enzymes such as superoxide dismutase, catalase, glutathione peroxide, glutathione reductase, glutathione-S-transferase, and glucose-6-phosphate dehydrogenase activities and non-enzymatic antioxidants such as vitamin C, vitamin E, reduced glutathione (GSH), and oxidized glutathione (GSSG) levels were altered. Similarly, the activities of total ATPases, Na(+)/K(+)-ATPase, Ca(2+)-ATPase, and Mg(2+)-ATPase were also decreased in the erythrocytes of diabetic rats. Administration of SGF to STZ-induced diabetic rats reduced blood glucose and glycosylated haemoglobin levels with increased levels of insulin and haemoglobin. Moreover, SGF reversed the protein and lipid peroxidation markers, osmotic fragility, membrane-bound ATPases activities, and antioxidant status in STZ-induced diabetic rats. These results suggest that SGF could provide a protective effect on diabetes by decreasing oxidative stress-associated diabetic complications.

  4. Attenuation of erythrocyte membrane oxidative stress by Sesbania grandiflora in streptozotocin-induced diabetic rats.

    PubMed

    Sureka, Chandrabose; Ramesh, Thiyagarajan; Begum, Vavamohaideen Hazeena

    2015-08-01

    The aim of the present study was to investigate the protective effects of Sesbania grandiflora flower (SGF) extract on erythrocyte membrane in Streptozotocin (STZ)-induced diabetic rats. Adult male albino rats of Wistar strain, weighing 190-220 g, were made diabetic by an intraperitonial administration of STZ (45 mg/kg). Normal and diabetic rats were treated with SGF, and diabetic rats were also treated with glibenclamide as drug control, for 45 days. In this study plasma insulin and haemoglobin levels were decreased and blood glucose, glycosylated haemoglobin, protein oxidation, lipid peroxidation markers, and osmotic fragility levels were increased in diabetic rats. Moreover, erythrocytes antioxidant enzymes such as superoxide dismutase, catalase, glutathione peroxide, glutathione reductase, glutathione-S-transferase, and glucose-6-phosphate dehydrogenase activities and non-enzymatic antioxidants such as vitamin C, vitamin E, reduced glutathione (GSH), and oxidized glutathione (GSSG) levels were altered. Similarly, the activities of total ATPases, Na(+)/K(+)-ATPase, Ca(2+)-ATPase, and Mg(2+)-ATPase were also decreased in the erythrocytes of diabetic rats. Administration of SGF to STZ-induced diabetic rats reduced blood glucose and glycosylated haemoglobin levels with increased levels of insulin and haemoglobin. Moreover, SGF reversed the protein and lipid peroxidation markers, osmotic fragility, membrane-bound ATPases activities, and antioxidant status in STZ-induced diabetic rats. These results suggest that SGF could provide a protective effect on diabetes by decreasing oxidative stress-associated diabetic complications. PMID:26176361

  5. Characterization of a Mannose-6-Phosphate Isomerase from Bacillus amyloliquefaciens and Its Application in Fructose-6-Phosphate Production

    PubMed Central

    Sigdel, Sujan; Singh, Ranjitha; Kim, Tae-Su; Li, Jinglin; Kim, Sang-Yong; Kim, In-Won; Jung, Woo-Suk; Pan, Cheol-Ho; Kang, Yun Chan; Lee, Jung-Kul

    2015-01-01

    The BaM6PI gene encoding a mannose-6-phosphate isomerase (M6PI, EC 5.3.1.8) was cloned from Bacillus amyloliquefaciens DSM7 and overexpressed in Escherichia coli. The enzyme activity of BaM6PI was optimal at pH and temperature of 7.5 and 70°C, respectively, with a kcat/Km of 13,900 s-1 mM-1 for mannose-6-phosphate (M6P). The purified BaM6PI demonstrated the highest catalytic efficiency of all characterized M6PIs. Although M6PIs have been characterized from several other sources, BaM6PI is distinguished from other M6PIs by its wide pH range and high catalytic efficiency for M6P. The binding orientation of the substrate M6P in the active site of BaM6PI shed light on the molecular basis of its unusually high activity. BaM6PI showed 97% substrate conversion from M6P to fructose-6-phosphate demonstrating the potential for using BaM6PI in industrial applications. PMID:26171785

  6. Characterization of fructose 6 phosphate phosphoketolases purified from Bifidobacterium species.

    PubMed

    Grill, J P; Crociani, J; Ballongue, J

    1995-07-01

    Fructose 6 phosphate phosphoketolases (F6PPKs) were purified from Bifidobacterium longum BB536, B. dentium ATCC 27534, B. globosum ATCC 25864, and Bifidobacterium animalis ATCC 25527. Concerning ions (Cu++, Zn++, Ca++, Mg++, Fe++, Co++, Mn++) and common enzyme inhibitors (fructose, ammonium sulfate, iodoacetate, and parachloromercuribenzoic acid), no difference appeared between the enzymes. Cu++, parachloromercuribenzoic acid (pCMB), and mercuric acetate induced high enzymatic inhibition. The study of pCMB demonstrated a noncompetitive inhibition. Additional results showed that the sulfhydryl group was not involved in catalytic reaction. Photooxidation experiments and determination of ionizable group pKas (5.16-7.17) suggested the presence of one or more histidines necessary for the catalytic reaction and explained the inhibition observed with pCMB. In light of the noncompetitive inhibition, this group was not directly involved in substrate binding. Determination of Km demonstrated that the affinities for fructose 6 phosphate in the case of animal and human origin strains were close. In addition, the same enzymatic efficiency (Kcat/Km) was obtained for each strain. The F6PPK activity was regulated by sodium pyrophosphate, ATP, and especially by ADP.

  7. Physical mapping of the human glutamine:fructose-6-phosphate amidotransferase gene (GFPT) to chromosome 2p13

    SciTech Connect

    Whitmore, T.E.; Mudri, S.L.; McKnight, G.L.

    1995-03-20

    Diabetic hyperglycemia influences insulin resistance through a process termed glucose toxicity. Implicated as a source of the mediators of this toxicity is an increased intracellular glucose metabolism through the hexosamine pathway. The hexosamine pathway itself is controlled by the rate-limiting enzyme glutamine:fructose-6-phosphate amidotransferase (GFAT), which is the first enzyme of the pathway. It has been shown that there is a close correlation between the glucose-mediated reduction of GFAT activity and the onset of insulin desensitization of the glucose transport system, a condition associated with insulin-resistant states of non-insulin-dependent diabetes mellitus and obesity. To gain a better understanding of the molecular regulation of GFAT and its role in the induction of insulin resistance, we previously isolated and cloned the cDNA for the human form of this enzyme and expressed the functional protein in Escherichia coli. 9 refs., 1 fig.

  8. [Sorbitol-6-Phosphate Dehydrogenase Gene Polymorhism in Malus Mill. (Rosaceae)].

    PubMed

    Boris, K V; Kudryavtsev, A M; Kochieva, E Z

    2015-11-01

    The sorbitol-6-phosphate dehydrogenase gene (S6PDH) sequences of six representatives of the genus Malus, which belong to five different taxonomic sections, were examined for the first time. The exon-intron structure and polymorphism of the nucleotide and amino acid sequences of these genes was characterized. The intraspecific polymorphism of the S6PDH gene was assessed for the first time in 40 Russian and foreign apple (Malus domestica) cultivars. It was demonstrated that the interspecific polymorphism level of the S6PDH coding sequences in the studied. representatives of the genus Malus was 4%, and the intraspecific polymorphism level of M. domestica cultivars was very low, constituting 0.96%. PMID:26845854

  9. [Sorbitol-6-Phosphate Dehydrogenase Gene Polymorhism in Malus Mill. (Rosaceae)].

    PubMed

    Boris, K V; Kudryavtsev, A M; Kochieva, E Z

    2015-11-01

    The sorbitol-6-phosphate dehydrogenase gene (S6PDH) sequences of six representatives of the genus Malus, which belong to five different taxonomic sections, were examined for the first time. The exon-intron structure and polymorphism of the nucleotide and amino acid sequences of these genes was characterized. The intraspecific polymorphism of the S6PDH gene was assessed for the first time in 40 Russian and foreign apple (Malus domestica) cultivars. It was demonstrated that the interspecific polymorphism level of the S6PDH coding sequences in the studied. representatives of the genus Malus was 4%, and the intraspecific polymorphism level of M. domestica cultivars was very low, constituting 0.96%.

  10. Mannose 6-phosphate receptors in an ancient vertebrate, zebrafish.

    PubMed

    Nolan, Catherine M; McCarthy, Karena; Eivers, Edward; Jirtle, Randy L; Byrnes, Lucy

    2006-03-01

    The endosome/lysosome system plays key roles in embryonic development, but difficulties posed by inaccessible mammalian embryos have hampered detailed studies. The accessible, transparent embryos of Danio rerio, together with the genetic and experimental approaches possible with this organism, provide many advantages over rodents. In mammals, mannose 6-phosphate receptors (MPRs) target acid hydrolases to endosomes and lysosomes, but nothing is known of acid hydrolase targeting in zebrafish. Here, we describe the sequence of the zebrafish cation-dependent MPR (CD-MPR) and cation-independent MPR (CI-MPR), and compare them with their mammalian orthologs. We show that all residues critical for mannose 6-phosphate (M6P) recognition are present in the extracellular domains of the zebrafish receptors, and that trafficking signals in the cytoplasmic tails are also conserved. This suggests that the teleost receptors possess M6P binding sites with properties similar to those of mammalian MPRs, and that targeting of lysosomal enzymes by MPRs represents an ancient pathway in vertebrate cell biology. We also determined the expression patterns of the CD-MPR and CI-MPR during embryonic development in zebrafish. Both genes are expressed from the one-cell stage through to the hatching period. In early embryos, expression is ubiquitous, but in later stages, expression of both receptors is restricted to the anterior region of the embryo, covering the forebrain, midbrain and hindbrain. The expression patterns suggest time- and tissue-specific functions for the receptors, with particular evidence for roles in neural development. Our study establishes zebrafish as a novel, genetically tractable model for in vivo studies of MPR function and lysosome biogenesis. PMID:16411117

  11. Mannose 6-phosphate receptors in an ancient vertebrate, zebrafish.

    PubMed

    Nolan, Catherine M; McCarthy, Karena; Eivers, Edward; Jirtle, Randy L; Byrnes, Lucy

    2006-03-01

    The endosome/lysosome system plays key roles in embryonic development, but difficulties posed by inaccessible mammalian embryos have hampered detailed studies. The accessible, transparent embryos of Danio rerio, together with the genetic and experimental approaches possible with this organism, provide many advantages over rodents. In mammals, mannose 6-phosphate receptors (MPRs) target acid hydrolases to endosomes and lysosomes, but nothing is known of acid hydrolase targeting in zebrafish. Here, we describe the sequence of the zebrafish cation-dependent MPR (CD-MPR) and cation-independent MPR (CI-MPR), and compare them with their mammalian orthologs. We show that all residues critical for mannose 6-phosphate (M6P) recognition are present in the extracellular domains of the zebrafish receptors, and that trafficking signals in the cytoplasmic tails are also conserved. This suggests that the teleost receptors possess M6P binding sites with properties similar to those of mammalian MPRs, and that targeting of lysosomal enzymes by MPRs represents an ancient pathway in vertebrate cell biology. We also determined the expression patterns of the CD-MPR and CI-MPR during embryonic development in zebrafish. Both genes are expressed from the one-cell stage through to the hatching period. In early embryos, expression is ubiquitous, but in later stages, expression of both receptors is restricted to the anterior region of the embryo, covering the forebrain, midbrain and hindbrain. The expression patterns suggest time- and tissue-specific functions for the receptors, with particular evidence for roles in neural development. Our study establishes zebrafish as a novel, genetically tractable model for in vivo studies of MPR function and lysosome biogenesis.

  12. Fructose metabolism in the human erythrocyte. Phosphorylation to fructose 3-phosphate.

    PubMed Central

    Petersen, A; Kappler, F; Szwergold, B S; Brown, T R

    1992-01-01

    In human erythrocytes, the first step in the metabolism of fructose is generally thought to be phosphorylation to fructose 6-phosphate catalysed by hexokinase. In variance with this assumption, we show here that fructose in these cells is metabolized primarily to fructose 3-phosphate by a specific 3-phosphokinase. This process has an overall estimated Km of 30 mM with respect to extracellular fructose and an apparent Vmax. of 0.6 mumol/h per ml. At a fixed concentration of fructose in the medium, the accumulation of fructose 3-phosphate was linearly dependent on the duration of incubation up to 5 h and was not affected by glucose. Once accumulated, fructose 3-phosphate appears to be degraded and/or relatively slowly metabolized, decreasing by only approximately 30% after a 12 h incubation in a fructose-free medium. PMID:1599419

  13. Structural and Chemical Basis for Glucosamine 6-Phosphate Binding and Activation of the glmS Ribozyme

    SciTech Connect

    Cochrane, J.; Lipchock, S; Smith, K; Strobel, S

    2009-01-01

    The glmS ribozyme is the first naturally occurring catalytic RNA that relies on an exogenous, nonnucleotide cofactor for reactivity. From a biochemical perspective, the glmS ribozyme derived from Bacillus anthracis is the best characterized. However, much of the structural work to date has been done on a variant glmS ribozyme, derived from Thermoanaerobacter tengcongensis. Here we present structures of the B. anthracis glmS ribozyme in states before the activating sugar, glucosamine 6-phosphate (GlcN6P), has bound and after the reaction has occurred. These structures show an active site preorganized to bind GlcN6P that retains some affinity for the sugar even after cleavage of the RNA backbone. A structure of an inactive glmS ribozyme with a mutation distal from the ligand-binding pocket highlights a nucleotide critical to the reaction that does not affect GlcN6P binding. Structures of the glmS ribozyme bound to a naturally occurring inhibitor, glucose 6-phosphate (Glc6P), and a nonnatural activating sugar, mannosamine 6-phosphate (MaN6P), reveal a binding mode similar to that of GlcN6P. Kinetic analyses show a pH dependence of ligand binding that is consistent with titration of the cofactor's phosphate group and support a model in which the major determinant of activity is the sugar amine independent of its stereochemical presentation.

  14. {open_quotes}The effects of diabetes on the activity of the enzyme glutamine: fructose-6-phosphate amindotransferase{close_quotes}

    SciTech Connect

    Nelson, S.P.

    1994-12-31

    Hexsoamine synthetic pathway (HexNSP) controls the supply of essential substrates for glycoprotein synthesis. In vitro studies suggest that increased flux of glucose via the hexsoamine synthetic pathway may play a role in glucose induced insulin resistance of glucose transport. Glutamine: fructose-6-phosphate amindotransferase (GFAT) controls flux into the hexsoamine synthetic pathway; the major products are UDPN-acetylhexosamines (UDP.HexNac=UDP.GlcNAc= UDP.GalNac). I examined whether diabetes ({approximately} 7 days post intravenous streptozotocin, and genetically linked) affects the activity of glutamine: fructose-6-phosphate in rat and mouse skeletal muscle in vivo. Nucleotide linked HexNAc were analyzed by high pressure liquid chromatography(HPLC) in deproteinized hind limb muscle extracts.

  15. Glucosamine and Glucosamine-6-phosphate Derivatives: Catalytic Cofactor Analogs for the glmS Ribozyme

    PubMed Central

    Posakony, Jeffrey J.; Ferré-D'Amaré, Adrian R.

    2013-01-01

    Two analogues of glucosamine-6-phosphate (GlcN6P, 1) and five of glucosamine (GlcN, 2) were prepared for evaluation as catalytic cofactor of the glmS ribozyme, a bacterial gene-regulatory RNA that controls cell wall biosynthesis. Glucosamine and allosamine with 3-azido substitutions were prepared by SN2 reactions of the respective 1,2,4,6-protected sugars; final acidic hydrolysis afforded the fully deprotected compounds as their TFA salts. A 6-phospho-2-aminoglucolactam (31) was prepared from glucosamine in a 13-step synthesis, which included a late-stage POCl3-phosphorylation. A simple and widely applicable 2-step procedure with the triethylsilyl (TES) protecting group was developed to selectively expose the 6-OH group in N-protected glucosamine analogs, which provided another route to chemical phosphorylation. Mitsunobu chemistry afforded 6-cyano (35) and 6-azido (36) analogues of GlcN-(Cbz) and the selectivity for the 6-position was confirmed by NMR (COSY, HMBC, HMQC) experiments. Compound 36 was converted to the fully deprotected 6-azido-GlcN (37) and 2,6-diaminoglucose (38) analogs. A 2-hydroxylamino glucose (42) analogue was prepared via an oxaziridine (41). Enzymatic phosphorylation of 42 and chemical phosphorylation of its 6-OH precursor (43) were possible, but 42 and the 6-phospho product (44) were unstable under neutral or basic conditions. Chemical phosphorylation of the previously described 2-guanidinyl-glucose (46) afforded its 6-phospho analogue (49) after final deprotection. PMID:23578404

  16. Cytochrome P{sub 450}-dependent toxic effects of primaquine on human erythrocytes

    SciTech Connect

    Ganesan, Shobana; Tekwani, Babu L.; Sahu, Rajnish; Tripathi, Lalit M.; Walker, Larry A.

    2009-11-15

    Primaquine, an 8-aminoquinoline, is the drug of choice for radical cure of relapsing malaria. Use of primaquine is limited due to its hemotoxicity, particularly in populations with glucose-6-phosphate dehydrogenase deficiency [G6PD(-)]. Biotransformation appears to be central to the anti-infective and hematological toxicities of primaquine, but the mechanisms are still not well understood. Metabolic studies with primaquine have been hampered due to the reactive nature of potential hemotoxic metabolites. An in vitro metabolism-linked hemotoxicity assay has been developed. Co-incubation of the drug with normal or G6PD(-) erythrocytes, microsomes or recombinant cytochrome P{sub 450} (CYP) isoforms has allowed in situ generation of potential hemotoxic metabolite(s), which interact with the erythrocytes to generate hemotoxicity. Methemoglobin formation, real-time generation of reactive oxygen intermediates (ROIs) and depletion of reactive thiols were monitored as multiple biochemical end points for hemotoxicity. Primaquine alone did not produce any hemotoxicity, while a robust increase was observed in methemoglobin formation and generation of ROIs by primaquine in the presence of human or mouse liver microsomes. Multiple CYP isoforms (CYP2E1, CYP2B6, CYP1A2, CYP2D6 and CYP3A4) variably contributed to the hemotoxicity of primaquine. This was further confirmed by significant inhibition of primaquine hemotoxicity by the selective CYP inhibitors, namely thiotepa (CYP2B6), fluoxetine (CYP2D6) and troleandomycin (CYP3A4). Primaquine caused similar methemoglobin formation in G6PD(-) and normal human erythrocytes. However, G6PD(-) erythrocytes suffered higher oxidative stress and depletion of thiols than normal erythrocytes due to primaquine toxicity. The results provide significant insights regarding CYP isoforms contributing to hemotoxicity and may be useful in controlling toxicity of primaquine to increase its therapeutic utility.

  17. Cellular properties of human erythrocytes preserved in saline-adenine-glucose-mannitol in the presence of L-carnitine.

    PubMed

    Arduini, Arduino; Minetti, Giampaolo; Ciana, Annarita; Seppi, Claudio; Brovelli, Augusta; Profumo, Antonella; Vercellati, Cristina; Zappa, Manuela; Zanella, Alberto; Dottori, Secondo; Bonomini, Mario

    2007-01-01

    L-Carnitine (LC) in the preservation medium during storage of red blood cells (RBC) can improve the mean 24-hr percent recovery in vivo and increase RBC life-span after reinfusion. The purpose of the study was to investigate the differences in the biochemical properties of RBCs stored in the presence or absence of LC, and the cell-age related responses to storage conditions and to LC. RBC concentrates in saline-adenine-glucose-mannitol (SAG-M) were stored in the presence or absence of 5 mM LC at 4 degrees C for up to 8 weeks. RBC subpopulations of different densities were prepared by centrifugation on Stractan density gradient. Cells were sampled at 0, 3, 6, and 8 weeks, and hematological and cellular properties analyzed (MCV, MCHC, 4.1a/4.1b ratio as a cell age parameter, intracellular Na(+) and K(+)). After 6 weeks, MCV of RBC stored in the presence of LC was lower than that of controls (6 weeks MCV: controls 95.4 +/- 1.8 fl; LC 91.5 +/- 2.0 fl; n = 6; P < 0.005). This was due to swelling of control cells, and affected mainly older RBCs. LC appeared to reduce or retard cell swelling. Among the osmotically active substances whose changes during storage could contribute to cell swelling, only intracellular Na(+) and K(+) differed between stored control RBCs and LC-treated cells. LC reduces the swelling of older cells during storage at 4 degrees C in SAG-M, possibly by acting on the permeability of cell membrane to monovalent cations. PMID:16947328

  18. A Tale of Two Sugars: Trehalose 6-Phosphate and Sucrose.

    PubMed

    Figueroa, Carlos M; Lunn, John E

    2016-09-01

    Trehalose 6-phosphate (Tre6P), the intermediate of trehalose biosynthesis, is an essential signal metabolite in plants, linking growth and development to carbon status. The Suc-Tre6P nexus model postulates that Tre6P is both a signal and negative feedback regulator of Suc levels, forming part of a mechanism to maintain Suc levels within an optimal range and functionally comparable to the insulin-glucagon system for regulating blood Glc levels in animals. The target range and sensitivity of the Tre6P-Suc feedback control circuit can be adjusted according to the cell type, developmental stage, and environmental conditions. In source leaves, Tre6P modulates Suc levels by affecting Suc synthesis, whereas in sink organs it regulates Suc consumption. In illuminated leaves, Tre6P influences the partitioning of photoassimilates between Suc, organic acids, and amino acids via posttranslational regulation of phosphoenolpyruvate carboxylase and nitrate reductase. At night, Tre6P regulates the remobilization of leaf starch reserves to Suc, potentially linking starch turnover in source leaves to carbon demand from developing sink organs. Use of Suc for growth in developing tissues is strongly influenced by the antagonistic activities of two protein kinases: SUC-NON-FERMENTING-1-RELATED KINASE1 (SnRK1) and TARGET OF RAPAMYCIN (TOR). The relationship between Tre6P and SnRK1 in developing tissues is complex and not yet fully resolved, involving both direct and indirect mechanisms, and positive and negative effects. No direct connection between Tre6P and TOR has yet been described. The roles of Tre6P in abiotic stress tolerance and stomatal regulation are also discussed. PMID:27482078

  19. Sucrose induces expression of the sorbitol-6-phosphate dehydrogenase gene in source leaves of loquat.

    PubMed

    Suzuki, Yasuo; Dandekar, Abhaya M

    2014-03-01

    Rosaceae fruit trees use sorbitol and sucrose as translocating sugars and the sorbitol-to-sucrose ratio in source leaves determines apple fruit quality. Here, we investigate the effects of sugars on the expression of genes encoding key photosynthetic enzymes, including sorbitol-6-phosphate dehydrogenase (S6PDH, EC 1.1.1.200), sucrose phosphate synthase (SPS, EC 2.4.1.14), and ADP-glucose pyrophosphorylase (ADPGPPase, EC 2.7.7.27) to understand the sugar-signaling mechanism in Rosaceae fruit trees. Mature leaf-petiole cuttings of loquat (Eriobotrya japonica Lindl. cv. Mogi) were supplied with a water, sorbitol or sucrose solution for 2 days at 20°C. The relative levels of the transcripts were analyzed by real-time polymerase chain reaction (PCR). S6PDH transcription was decreased by sorbitol but drastically increased by sucrose. SPS and ADPGPPase large subunit transcription were decreased by sucrose and sorbitol. The simultaneous application of sorbitol and sucrose revealed that S6PDH transcription increased in a dose-dependent manner with sucrose. These results show that both sorbitol and sucrose work as signaling molecules in source organs of Rosaceae fruit trees. These trees have mechanisms to positively keep sorbitol as the dominant translocating sugar, suggesting that sorbitol plays an important role in their survival strategy. Effects of various sugars on S6PDH expression were investigated. Palatinose, a sucrose analog, increased S6PDH transcription much more drastically than sucrose. Mannose and 3-O-methylglucose, glucose analogs, also increased S6PDH transcription; however, glucose did not. Models of sugar signaling in source organs of Rosaceae fruit trees are discussed.

  20. Non-oxidative synthesis of pentose 5-phosphate from hexose 6-phosphate and triose phosphate by the L-type pentose pathway.

    PubMed

    Williams, J F; Blackmore, P F

    1983-01-01

    1. Ribose 5-phosphate was non-oxidatively synthesized from glucose 6-phosphate and triose phosphate by an enzyme extract prepared from rat liver (RLEP). Analysis of the intermediates by GLC, ion-exchange chromatography and specific enzymatic analysis, revealed the presence of the following intermediates of the L-type pentose pathway: altro-heptulose 1,7-bisphosphate, arabinose 5-phosphate and D-glycero D-ido octulose 8-phosphate. 2. With either [1-14C] or [2-14C]glucose 6-phosphate as diagnostic substrates, the distribution of 14C in ribose 5-phosphate was determined. At early time intervals (0.5-8 hr), [1-14C]glucose 6-phosphate introduced 14C into C-1, C-3 and C-5 of ribose 5-phosphate, at 17 hr 14C was confined to C-1. With [2-14C]glucose 6-phosphate as substrate, 14C was confined to C-2, C-3 and C-5 of ribose 5-phosphate during early times (0.5-8 hr), while at 17 hr 14C was located in C-2. 3. The transketolase exchange reaction, [14C]ribose 5-phosphate + altro-heptulose 7-phosphate in equilibrium ribose 5-phosphate + [14C]altro-heptulose 7-phosphate, was demonstrated for the first time using purified transketolase, its activity was measured and it is proposed to play a major role in the relocation of 14C into C-3 and C-5 or ribose 5-phosphate during the prediction labelling experiments. 4. The coupled transketolase-transaldolase reactions, 2 fructose 6-phosphate in equilibrium altro-heptulose 7-phosphate + xylulose 5-phosphate and 2 altro-heptulose 7-phosphate in equilibrium fructose 6-phosphate + D-glycero D-altro octulose 8-phosphate were demonstrated with purified enzymes, but are concluded to play a minor role in the non-oxidative synthesis of pentose 5-phosphate and octulose phosphate by (RLEP). 5. The formation of gem diol and dimers of erythrose 4-phosphate is proposed to account in part for the failure to detect monomeric erythrose 4-phosphate in the carbon balance studies. 6. The equilibrium value for the pentose pathway acting by the reverse mode in

  1. Contribution of fructose-6-phosphate to glucocorticoid activation in the endoplasmic reticulum: possible implication in the metabolic syndrome.

    PubMed

    Senesi, Silvia; Legeza, Balázs; Balázs, Zoltán; Csala, Miklós; Marcolongo, Paola; Kereszturi, Eva; Szelényi, Péter; Egger, Christine; Fulceri, Rosella; Mandl, József; Giunti, Roberta; Odermatt, Alex; Bánhegyi, Gábor; Benedetti, Angelo

    2010-10-01

    Both fructose consumption and increased intracellular glucocorticoid activation have been implicated in the pathogenesis of the metabolic syndrome. Glucocorticoid activation by 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) depends on hexose-6-phosphate dehydrogenase (H6PD), which physically interacts with 11β-HSD1 at the luminal surface of the endoplasmic reticulum (ER) membrane and generates reduced nicotinamide adenine dinucleotide phosphate for the reduction of glucocorticoids. The reducing equivalents for the reaction are provided by glucose-6-phosphate (G6P) that is transported by G6P translocase into the ER. Here, we show that fructose-6-phosphate (F6P) can substitute for G6P and is sufficient to maintain reductase activity of 11β-HSD1 in isolated microsomes. Our findings indicate that the mechanisms of F6P and G6P transport across the ER membrane are distinct and provide evidence that F6P is converted to G6P in the ER lumen, thus yielding substrate for H6PD-dependent reduced nicotinamide adenine dinucleotide phosphate generation. Using the purified enzyme, we show that F6P cannot be directly dehydrogenated by H6PD, and we also excluded H6PD as a phosphohexose isomerase. Therefore, we postulate the existence of an ER luminal hexose-phosphate isomerase different from the cytosolic enzyme. The results suggest that cytosolic F6P promotes prereceptor glucocorticoid activation in white adipose tissue, which might have a role in the pathophysiology of the metabolic syndrome.

  2. [Studies of the blood antioxidant system and oxygen-transporting properties of human erythrocytes during 105-day isolation].

    PubMed

    Brazhe, N A; Baĭzhumanov, A A; Parshina, E Iu; Iusipovich, A I; Akhalaia, M Ia; Iarlykova, Iu V; Labetskaia, O I; Ivanova, S M; Morukov, B V; Maksimov, G V

    2011-01-01

    Effects of strict 105-d isolation on blood antioxidant status, erythrocyte membrane processes and oxygen-binding properties of hemoglobin were studied in 6 male volunteers (25 to 40 y.o.) in ground-based simulation of a mission to Mars (experiment Mars-105). The parameters were measured using venous blood samples collected during BDC, on days 35, 70 and 105 of the experiment and on days 7 and 14-15 after its completion. Methods of biochemistry (determination of enzyme activity and thin-layer chromatography) and biophysical (laser interference microscopy, Raman spectroscopy) showed changes in relative content of lipid and phospholipid fractions suggesting growth of membrane microviscosity and increase in TBA-AP (active products of lipids peroxidation interacting with thiobarbituric acid). A significant increase in glucose-6-phosphate dehydrogenase and superoxide dismutase activities against reduction of catalase activity points to both reparative processes in erythrocytes and disbalance between the number of evolving active forms of oxygen and antioxidant protection mechanisms in cells. Hemoglobin sensitivity of oxygen and blood level of oxyhemoglobin were found to increase, too. It is presumed that adaptation of organism to stresses experienced during and after the experiment may destroy balance of the antioxidant protection systems which is conducive to oxidation of membrane phospholipids, alteration of their content, increase of membrane microviscosity and eventual failure of the gas-exchange function of erythrocytes. PMID:21675192

  3. Structural basis for morpheein-type allosteric regulation of Escherichia coli glucosamine-6-phosphate synthase: equilibrium between inactive hexamer and active dimer.

    PubMed

    Mouilleron, Stéphane; Badet-Denisot, Marie-Ange; Pecqueur, Ludovic; Madiona, Karine; Assrir, Nadine; Badet, Bernard; Golinelli-Pimpaneau, Béatrice

    2012-10-01

    The amino-terminal cysteine of glucosamine-6-phosphate synthase (GlmS) acts as a nucleophile to release and transfer ammonia from glutamine to fructose 6-phosphate through a channel. The crystal structure of the C1A mutant of Escherichia coli GlmS, solved at 2.5 Å resolution, is organized as a hexamer, where the glutaminase domains adopt an inactive conformation. Although the wild-type enzyme is active as a dimer, size exclusion chromatography, dynamic and quasi-elastic light scattering, native polyacrylamide gel electrophoresis, and ultracentrifugation data show that the dimer is in equilibrium with a hexameric state, in vitro and in cellulo. The previously determined structures of the wild-type enzyme, alone or in complex with glucosamine 6-phosphate, are also consistent with a hexameric assembly that is catalytically inactive because the ammonia channel is not formed. The shift of the equilibrium toward the hexameric form in the presence of cyclic glucosamine 6-phosphate, together with the decrease of the specific activity with increasing enzyme concentration, strongly supports product inhibition through hexamer stabilization. Altogether, our data allow us to propose a morpheein model, in which the active dimer can rearrange into a transiently stable form, which has the propensity to form an inactive hexamer. This would account for a physiologically relevant allosteric regulation of E. coli GlmS. Finally, in addition to cyclic glucose 6-phosphate bound at the active site, the hexameric organization of E. coli GlmS enables the binding of another linear sugar molecule. Targeting this sugar-binding site to stabilize the inactive hexameric state is therefore suggested for the development of specific antibacterial inhibitors.

  4. A role for AMPK in the inhibition of glucose-6-phosphate dehydrogenase by polyunsaturated fatty acids

    SciTech Connect

    Kohan, Alison B.; Talukdar, Indrani; Walsh, Callee M.; Salati, Lisa M.

    2009-10-09

    Both polyunsaturated fatty acids and AMPK promote energy partitioning away from energy consuming processes, such as fatty acid synthesis, towards energy generating processes, such as {beta}-oxidation. In this report, we demonstrate that arachidonic acid activates AMPK in primary rat hepatocytes, and that this effect is p38 MAPK-dependent. Activation of AMPK mimics the inhibition by arachidonic acid of the insulin-mediated induction of G6PD. Similar to intracellular signaling by arachidonic acid, AMPK decreases insulin signal transduction, increasing Ser{sup 307} phosphorylation of IRS-1 and a subsequent decrease in AKT phosphorylation. Overexpression of dominant-negative AMPK abolishes the effect of arachidonic acid on G6PD expression. These data suggest a role for AMPK in the inhibition of G6PD by polyunsaturated fatty acids.

  5. The glucose 6-phosphate shunt around the Calvin-Benson cycle.

    PubMed

    Sharkey, Thomas D; Weise, Sean E

    2016-07-01

    It is just over 60 years since a cycle for the regeneration of the CO2-acceptor used in photosynthesis was proposed. In this opinion paper, we revisit the origins of the Calvin-Benson cycle that occurred at the time that the hexose monophosphate shunt, now called the pentose phosphate pathway, was being worked out. Eventually the pentose phosphate pathway was separated into two branches, an oxidative branch and a non-oxidative branch. It is generally thought that the Calvin-Benson cycle is the reverse of the non-oxidative branch of the pentose phosphate pathway but we describe crucial differences and also propose that some carbon routinely passes through the oxidative branch of the pentose phosphate pathway. This creates a futile cycle but may help to stabilize photosynthesis. If it occurs it could explain a number of enigmas including the lack of complete labelling of the Calvin-Benson cycle intermediates when carbon isotopes are fed to photosynthesizing leaves.

  6. Glycosidases Interact Selectively With Mannose-6-Phosphate Receptors of Bull Spermatozoa.

    PubMed

    Aguilera, Andrea C; Boschin, Verónica; Carvelli, Lorena; Cavicchia, Juan C; Sosa, Miguel A

    2016-11-01

    Glycosidases may play a role in sperm maturation during epididymal transit. In this work, we describe the interaction of these enzymes with bull spermatozoa. We found that β-galactosidase associated to spermatozoa can be released under low ionic strength conditions, whereas the interaction of N-acetyl-β-D-glucosaminidase and β-glucuronidase with spermatozoa appeared to be stronger. On the other hand, α-mannosidase and α-fucosidase cannot be removed from the gametes. In addition, part of N-acetyl-β-D-glucosaminidase, β-galactosidase, and β-glucuronidase can also be released by mannose-6-phosphate. Taking into account these data, we explored the presence of cation-independent- and cation-dependent-mannose-6-phosphate receptors in the spermatozoa and found that cation-independent mannose-6-phosphate receptor is highly expressed in bull spermatozoa and cation-dependent-mannose-6-phosphate receptor is expressed at a lesser extent. In addition, by immunofluorescence, we observed that cation-independent-mannose-6-phosphate receptor is mostly located at the acrosomal zone, whereas cation-dependent-mannose-6-phosphate receptor presents a different distribution pattern on spermatozoa during the epididymal transit. N-acetyl-β-D-glucosaminidase and β-glucuronidase isolated from epididymal fluid interacted mostly with cation-independent-mannose-6-phosphate receptor, while β-galactosidase was recognized by both receptors. We concluded that glycosidases might play different roles in bull spermatozoa and that mannos-6-phosphate receptors may act as recruiters of some enzymes. J. Cell. Biochem. 117: 2464-2472, 2016. © 2016 Wiley Periodicals, Inc.

  7. Feedback inhibition of starch degradation in Arabidopsis leaves mediated by trehalose 6-phosphate.

    PubMed

    Martins, Marina Camara Mattos; Hejazi, Mahdi; Fettke, Joerg; Steup, Martin; Feil, Regina; Krause, Ursula; Arrivault, Stéphanie; Vosloh, Daniel; Figueroa, Carlos María; Ivakov, Alexander; Yadav, Umesh Prasad; Piques, Maria; Metzner, Daniela; Stitt, Mark; Lunn, John Edward

    2013-11-01

    Many plants accumulate substantial starch reserves in their leaves during the day and remobilize them at night to provide carbon and energy for maintenance and growth. In this paper, we explore the role of a sugar-signaling metabolite, trehalose-6-phosphate (Tre6P), in regulating the accumulation and turnover of transitory starch in Arabidopsis (Arabidopsis thaliana) leaves. Ethanol-induced overexpression of trehalose-phosphate synthase during the day increased Tre6P levels up to 11-fold. There was a transient increase in the rate of starch accumulation in the middle of the day, but this was not linked to reductive activation of ADP-glucose pyrophosphorylase. A 2- to 3-fold increase in Tre6P during the night led to significant inhibition of starch degradation. Maltose and maltotriose did not accumulate, suggesting that Tre6P affects an early step in the pathway of starch degradation in the chloroplasts. Starch granules isolated from induced plants had a higher orthophosphate content than granules from noninduced control plants, consistent either with disruption of the phosphorylation-dephosphorylation cycle that is essential for efficient starch breakdown or with inhibition of starch hydrolysis by β-amylase. Nonaqueous fractionation of leaves showed that Tre6P is predominantly located in the cytosol, with estimated in vivo Tre6P concentrations of 4 to 7 µm in the cytosol, 0.2 to 0.5 µm in the chloroplasts, and 0.05 µm in the vacuole. It is proposed that Tre6P is a component in a signaling pathway that mediates the feedback regulation of starch breakdown by sucrose, potentially linking starch turnover to demand for sucrose by growing sink organs at night.

  8. N-acetylglucosamine 6-Phosphate Deacetylase (nagA) Is Required for N-acetyl Glucosamine Assimilation in Gluconacetobacter xylinus

    PubMed Central

    Yadav, Vikas; Panilaitis, Bruce; Shi, Hai; Numuta, Keiji; Lee, Kyongbum; Kaplan, David L.

    2011-01-01

    Metabolic pathways for amino sugars (N-acetylglucosamine; GlcNAc and glucosamine; Gln) are essential and remain largely conserved in all three kingdoms of life, i.e., microbes, plants and animals. Upon uptake, in the cytoplasm these amino sugars undergo phosphorylation by phosphokinases and subsequently deacetylation by the enzyme N-acetylglucosamine 6-phosphate deacetylase (nagA) to yield glucosamine-6-phosphate and acetate, the first committed step for both GlcNAc assimilation and amino-sugar-nucleotides biosynthesis. Here we report the cloning of a DNA fragment encoding a partial nagA gene and its implications with regard to amino sugar metabolism in the cellulose producing bacterium Glucoacetobacter xylinus (formally known as Acetobacter xylinum). For this purpose, nagA was disrupted by inserting tetracycline resistant gene (nagA::tetr; named as ΔnagA) via homologous recombination. When compared to glucose fed conditions, the UDP-GlcNAc synthesis and bacterial growth (due to lack of GlcNAc utilization) was completely inhibited in nagA mutants. Interestingly, that inhibition occured without compromising cellulose production efficiency and its molecular composition under GlcNAc fed conditions. We conclude that nagA plays an essential role for GlcNAc assimilation by G. xylinus thus is required for the growth and survival for the bacterium in presence of GlcNAc as carbon source. Additionally, G. xylinus appears to possess the same molecular machinery for UDP-GlcNAc biosynthesis from GlcNAc precursors as other related bacterial species. PMID:21655093

  9. Sorbitol synthesis by an engineered Lactobacillus casei strain expressing a sorbitol-6-phosphate dehydrogenase gene within the lactose operon.

    PubMed

    Nissen, Lorenzo; Pérez-Martínez, Gaspar; Yebra, María J

    2005-08-01

    Sorbitol is claimed to have important health-promoting effects and Lactobacillus casei is a lactic acid bacterium relevant as probiotic and used as a cheese starter culture. A sorbitol-producing L. casei strain might therefore be of considerable interest in the food industry. A recombinant strain of L. casei was constructed by the integration of a d-sorbitol-6-phosphate dehydrogenase-encoding gene (gutF) in the chromosomal lactose operon (strain BL232). gutF expression in this strain followed the same regulation as that of the lac genes, that is, it was repressed by glucose and induced by lactose. (13)C-nuclear magnetic resonance analysis of supernatants of BL232 resting cells demonstrated that, when pre-grown on lactose, cells were able to synthesize sorbitol from glucose. Inactivation of the l-lactate dehydrogenase gene in BL232 led to an increase in sorbitol production, suggesting that the engineered route provides an alternative pathway for NAD(+) regeneration. PMID:16002237

  10. Disruption of the Candida albicans TPS1 gene encoding trehalose-6-phosphate synthase impairs formation of hyphae and decreases infectivity.

    PubMed

    Zaragoza, O; Blazquez, M A; Gancedo, C

    1998-08-01

    The TPS1 gene from Candida albicans, which encodes trehalose-6-phosphate synthase, has been cloned by functional complementation of a tps1 mutant from Saccharomyces cerevisiae. In contrast with the wild-type strain, the double tps1/tps1 disruptant did not accumulate trehalose at stationary phase or after heat shock. Growth of the tps1/tps1 disruptant at 30 degreesC was indistinguishable from that of the wild type. However, at 42 degreesC it did not grow on glucose or fructose but grew normally on galactose or glycerol. At 37 degreesC, the yeast-hypha transition in the mutant in glucose-calf serum medium did not occur. During growth at 42 degreesC, the mutant did not form hyphae in galactose or in glycerol. Some of the growth defects observed may be traced to an unbalanced sugar metabolism that reduces the cellular content of ATP. Mice inoculated with 10(6) CFU of the tps1/tps1 mutant did not show visible symptoms of infection 16 days after inoculation, while those similarly inoculated with wild-type cells were dead 12 days after inoculation.

  11. Disruption of the Candida albicans TPS1 Gene Encoding Trehalose-6-Phosphate Synthase Impairs Formation of Hyphae and Decreases Infectivity†

    PubMed Central

    Zaragoza, Oscar; Blazquez, Miguel A.; Gancedo, Carlos

    1998-01-01

    The TPS1 gene from Candida albicans, which encodes trehalose-6-phosphate synthase, has been cloned by functional complementation of a tps1 mutant from Saccharomyces cerevisiae. In contrast with the wild-type strain, the double tps1/tps1 disruptant did not accumulate trehalose at stationary phase or after heat shock. Growth of the tps1/tps1 disruptant at 30°C was indistinguishable from that of the wild type. However, at 42°C it did not grow on glucose or fructose but grew normally on galactose or glycerol. At 37°C, the yeast-hypha transition in the mutant in glucose-calf serum medium did not occur. During growth at 42°C, the mutant did not form hyphae in galactose or in glycerol. Some of the growth defects observed may be traced to an unbalanced sugar metabolism that reduces the cellular content of ATP. Mice inoculated with 106 CFU of the tps1/tps1 mutant did not show visible symptoms of infection 16 days after inoculation, while those similarly inoculated with wild-type cells were dead 12 days after inoculation. PMID:9683476

  12. Physiological roles of trehalose in Leptinotarsa larvae revealed by RNA interference of trehalose-6-phosphate synthase and trehalase genes.

    PubMed

    Shi, Ji-Feng; Xu, Qing-Yu; Sun, Qiang-Kun; Meng, Qing-Wei; Mu, Li-Li; Guo, Wen-Chao; Li, Guo-Qing

    2016-10-01

    Trehalose is proposed to serve multiple physiological roles in insects. However, its importance remains largely unconfirmed. In the present paper, we knocked down either a trehalose biosynthesis gene (trehalose-6-phosphate synthase, LdTPS) or each of three degradation genes (soluble trehalases LdTRE1a, LdTRE1b or membrane-bound LdTRE2) in Leptinotarsa decemlineata by RNA interference (RNAi). Knockdown of LdTPS decreased trehalose content and caused larval and pupal lethality. The LdTPS RNAi survivors consumed a greater amount of foliage, obtained a heavier body mass, accumulated more glycogen, lipid and proline, and had a smaller amount of chitin compared with the controls. Ingestion of trehalose but not glucose rescued the food consumption increase and larval mass rise, increased survivorship, and recovered glycogen, lipid and chitin to the normal levels. In contrast, silencing of LdTRE1a increased trehalose content and resulted in larval and pupal lethality. The surviving LdTRE1a RNAi hypomorphs fed a smaller quantity of food, had a lighter body weight, depleted lipid and several glucogenic amino acids, and contained a smaller amount of chitin. Neither trehalose nor glucose ingestion rescued these LdTRE1a RNAi defects. Silencing of LdTRE1b caused little effects. Knockdown of LdTRE2 caused larval death, increased trehalose contents in several tissues and diminished glycogen in the brain-corpora cardiaca-corpora allata complex (BCC). Feeding glucose but not trehalose partially rescued the high mortality rate and recovered glycogen content in the BCC. It seems that trehalose is involved in feeding regulation, sugar absorption, brain energy supply and chitin biosynthesis in L. decemlineata larvae. PMID:27524277

  13. Crystal Structure and Substrate Specificity of D-Galactose-6-Phosphate Isomerase Complexed with Substrates

    PubMed Central

    Lee, Jung-Kul; Pan, Cheol-Ho

    2013-01-01

    D-Galactose-6-phosphate isomerase from Lactobacillus rhamnosus (LacAB; EC 5.3.1.26), which is encoded by the tagatose-6-phosphate pathway gene cluster (lacABCD), catalyzes the isomerization of D-galactose-6-phosphate to D-tagatose-6-phosphate during lactose catabolism and is used to produce rare sugars as low-calorie natural sweeteners. The crystal structures of LacAB and its complex with D-tagatose-6-phosphate revealed that LacAB is a homotetramer of LacA and LacB subunits, with a structure similar to that of ribose-5-phosphate isomerase (Rpi). Structurally, LacAB belongs to the RpiB/LacAB superfamily, having a Rossmann-like αβα sandwich fold as has been identified in pentose phosphate isomerase and hexose phosphate isomerase. In contrast to other family members, the LacB subunit also has a unique α7 helix in its C-terminus. One active site is distinctly located at the interface between LacA and LacB, whereas two active sites are present in RpiB. In the structure of the product complex, the phosphate group of D-tagatose-6-phosphate is bound to three arginine residues, including Arg-39, producing a different substrate orientation than that in RpiB, where the substrate binds at Asp-43. Due to the proximity of the Arg-134 residue and backbone Cα of the α6 helix in LacA to the last Asp-172 residue of LacB with a hydrogen bond, a six-carbon sugar-phosphate can bind in the larger pocket of LacAB, compared with RpiB. His-96 in the active site is important for ring opening and substrate orientation, and Cys-65 is essential for the isomerization activity of the enzyme. Two rare sugar substrates, D-psicose and D-ribulose, show optimal binding in the LacAB-substrate complex. These findings were supported by the results of LacA activity assays. PMID:24015281

  14. Crystal structure and substrate specificity of D-galactose-6-phosphate isomerase complexed with substrates.

    PubMed

    Jung, Woo-Suk; Singh, Raushan Kumar; Lee, Jung-Kul; Pan, Cheol-Ho

    2013-01-01

    D-Galactose-6-phosphate isomerase from Lactobacillus rhamnosus (LacAB; EC 5.3.1.26), which is encoded by the tagatose-6-phosphate pathway gene cluster (lacABCD), catalyzes the isomerization of D-galactose-6-phosphate to D-tagatose-6-phosphate during lactose catabolism and is used to produce rare sugars as low-calorie natural sweeteners. The crystal structures of LacAB and its complex with D-tagatose-6-phosphate revealed that LacAB is a homotetramer of LacA and LacB subunits, with a structure similar to that of ribose-5-phosphate isomerase (Rpi). Structurally, LacAB belongs to the RpiB/LacAB superfamily, having a Rossmann-like αβα sandwich fold as has been identified in pentose phosphate isomerase and hexose phosphate isomerase. In contrast to other family members, the LacB subunit also has a unique α7 helix in its C-terminus. One active site is distinctly located at the interface between LacA and LacB, whereas two active sites are present in RpiB. In the structure of the product complex, the phosphate group of D-tagatose-6-phosphate is bound to three arginine residues, including Arg-39, producing a different substrate orientation than that in RpiB, where the substrate binds at Asp-43. Due to the proximity of the Arg-134 residue and backbone Cα of the α6 helix in LacA to the last Asp-172 residue of LacB with a hydrogen bond, a six-carbon sugar-phosphate can bind in the larger pocket of LacAB, compared with RpiB. His-96 in the active site is important for ring opening and substrate orientation, and Cys-65 is essential for the isomerization activity of the enzyme. Two rare sugar substrates, D-psicose and D-ribulose, show optimal binding in the LacAB-substrate complex. These findings were supported by the results of LacA activity assays.

  15. Identification of a low affinity mannose 6-phosphate-binding site in domain 5 of the cation-independent mannose 6-phosphate receptor.

    PubMed

    Reddy, Sreelatha T; Chai, Wengang; Childs, Robert A; Page, Jimmy D; Feizi, Ten; Dahms, Nancy M

    2004-09-10

    The 300-kDa cation-independent mannose 6-phosphate receptor (CI-MPR) and the 46-kDa cation-dependent MPR (CD-MPR) are type I integral membrane glycoproteins that play a critical role in the intracellular delivery of newly synthesized mannose 6-phosphate (Man-6-P)-containing acid hydrolases to the lysosome. The extracytoplasmic region of the CI-MPR contains 15 contiguous domains, and the two high affinity ( approximately 1 nm) Man-6-P-binding sites have been mapped to domains 1-3 and 9, with essential residues localized to domains 3 and 9. Domain 5 of the CI-MPR exhibits significant sequence homology to domains 3 and 9 as well as to the CD-MPR. A structure-based sequence alignment was performed that predicts that domain 5 contains the four conserved key residues (Gln, Arg, Glu, and Tyr) identified as essential for carbohydrate recognition by the CD-MPR and domains 3 and 9 of the CI-MPR, but lacks two cysteine residues predicted to form a disulfide bond within the binding pocket. To determine whether domain 5 harbors a carbohydrate-binding site, a construct that encodes domain 5 alone (Dom5His) was expressed in Pichia pastoris. Microarray analysis using 30 different oligosaccharides demonstrated that Dom5His bound specifically to a Man-6-P-containing oligosaccharide (pentamannosyl 6-phosphate). Frontal affinity chromatography showed that the affinity of Dom5His for Man-6-P was approximately 300-fold lower (K(i) = 5.3 mm) than that observed for domains 1-3 and 9. The interaction affinity for the lysosomal enzyme beta-glucuronidase was also much lower (K(d) = 54 microm) as determined by surface plasmon resonance analysis. Taken together, these results demonstrate that the CI-MPR contains a third Man-6-P recognition site that is located in domain 5 and that exhibits lower affinity than the carbohydrate-binding sites present in domains 1-3 and 9. PMID:15252023

  16. Purification from Fusobacterium mortiferum ATCC 25557 of a 6-phosphoryl-O-alpha-D-glucopyranosyl:6-phosphoglucohydrolase that hydrolyzes maltose 6-phosphate and related phospho-alpha-D-glucosides.

    PubMed Central

    Thompson, J; Gentry-Weeks, C R; Nguyen, N Y; Folk, J E; Robrish, S A

    1995-01-01

    6-Phosphoryl-O-alpha-D-glucopyranosyl:6-phosphoglucohydrolase (6-phospho-alpha-glucosidase) has been purified from Fusobacterium mortiferum ATCC 25557. p-Nitrophenyl-alpha-D-glucopyranoside 6-phosphate (pNP alpha Glc6P) served as the chromogenic substrate for detection and assay of enzyme activity. The O2-sensitive, metal-dependent phospho-alpha-glucosidase was stabilized during purification by inclusion of dithiothreitol and Mn2+ ion in chromatography buffers. Various 6-phosphoryl-O-alpha-linked glucosides, including maltose 6-phosphate, pNP alpha Glc6P, trehalose 6-phosphate, and sucrose 6-phosphate, were hydrolyzed by the enzyme to yield D-glucose 6-phosphate and aglycone moieties in a 1:1 molar ratio. 6-Phospho-alpha-glucosidase (M(r) of approximately 49,000; pI of approximately 4.9) is activated by Fe2+, Mn2+, Co2+, and Ni2+, and the maximum rate of pNP alpha Glc6P hydrolysis occurs at 40 degrees C within the pH range 7.0 to 7.5. The sequence of the first 32 amino acids of 6-phospho-alpha-glucosidase exhibits 67% identity (90% similarity) to that deduced for the N terminus of a putative phospho-beta-glucosidase (designated ORF f212) encoded by glvG in Escherichia coli. Western blots involving highly specific polyclonal antibody against 6-phospho-alpha-glucosidase and spectrophotometric analyses with pNP alpha Glc6P revealed only low levels of the enzyme in glucose-, mannose-, or fructose-grown cells of F. mortiferum. Synthesis of 6-phospho-alpha-glucosidase increased dramatically during growth of the organism on alpha-glucosides, such as maltose, alpha-methylglucoside, trehalose, turanose, and palatinose. PMID:7730284

  17. Metabolic properties of low ATP erythrocytes of the monotremes.

    PubMed

    Kim, H D; Zeidler, R B; Sallis, J; Nicol, S; Isaacks, R E

    1984-02-13

    The erythrocytes of the monotremes, having a trace amount of ATP, can metabolize glucose to lactate at a rate comparable to human and other mammalian erythrocytes. The echidna energy metabolism is unique in that adenosine can stimulate glycolytic carbon flow, resulting in a nearly 20-fold net synthesis of ATP.

  18. Characterizing the Hexose-6-Phosphate Transport System of Vibrio cholerae, a Utilization System for Carbon and Phosphate Sources

    PubMed Central

    Moisi, Manuel; Lichtenegger, Sabine; Tutz, Sarah; Seper, Andrea

    2013-01-01

    The facultative human pathogen Vibrio cholerae transits between the gastrointestinal tract of its host and aquatic reservoirs. V. cholerae adapts to different situations by the timely coordinated expression of genes during its life cycle. We recently identified a subclass of genes that are induced at late stages of infection. Initial characterization demonstrated that some of these genes facilitate the transition of V. cholerae from host to environmental conditions. Among these genes are uptake systems lacking detailed characterization or correct annotation. In this study, we comprehensively investigated the function of the VCA0682-to-VCA0687 gene cluster, which was previously identified as in vivo induced. The results presented here demonstrate that the operon encompassing open reading frames VCA0685 to VCA0687 encodes an ABC transport system for hexose-6-phosphates with Km values ranging from 0.275 to 1.273 μM for glucose-6P and fructose-6P, respectively. Expression of the operon is induced by the presence of hexose-6P controlled by the transcriptional activator VCA0682, representing a UhpA homolog. Finally, we provide evidence that the operon is essential for the utilization of hexose-6P as a C and P source. Thereby, a physiological role can be assigned to hexose-6P uptake, which correlates with increased fitness of V. cholerae after a transition from the host into phosphate-limiting environments. PMID:23417487

  19. Caffeine inhibits erythrocyte membrane derangement by antioxidant activity and by blocking caspase 3 activation.

    PubMed

    Tellone, Ester; Ficarra, Silvana; Russo, Annamaria; Bellocco, Ersilia; Barreca, Davide; Laganà, Giuseppina; Leuzzi, Ugo; Pirolli, Davide; De Rosa, Maria Cristina; Giardina, Bruno; Galtieri, Antonio

    2012-02-01

    The aim of this research was to investigate the effect of caffeine on band 3 (the anion exchanger protein), haemoglobin function, caspase 3 activation and glucose-6-phosphate metabolism during the oxygenation-deoxygenation cycle in human red blood cells. A particular attention has been given to the antioxidant activity by using in vitro antioxidant models. Caffeine crosses the erythrocyte membrane and interacts with the two extreme conformational states of haemoglobin (the T and the R-state within the framework of the simple two states allosteric model) with different binding affinities. By promoting the high affinity state (R-state), the caffeine-haemoglobin interaction does enhance the pentose phosphate pathway. This is of benefit for red blood cells since it leads to an increase of NADPH availability. Moreover, caffeine effect on band 3, mediated by haemoglobin, results in an extreme increase of the anion exchange, particularly in oxygenated erythrocytes. This enhances the transport of the endogenously produced CO(2) thereby avoiding the production of dangerous secondary radicals (carbonate and nitrogen dioxide) which are harmful to the cellular membrane. Furthermore caffeine destabilizes the haeme-protein interactions within the haemoglobin molecule and triggers the production of superoxide and met-haemoglobin. However this damaging effect is almost balanced by the surprising scavenger action of the alkaloid with respect to the hydroxyl radical. These experimental findings are supported by in silico docking and molecular dynamics studies and by what we may call the "caspase silence"; in fact, there is no evidence of any caspase 3 activity enhancement; this is likely due to the promotion of positive metabolic conditions which result in an increase of the cellular reducing power.

  20. Sporothrix schenckii: purification and partial biochemical characterization of glucosamine-6-phosphate synthase, a potential antifungal target.

    PubMed

    González-Ibarra, Joaquín; Milewski, Sławomir; Villagómez-Castro, Julio C; Cano-Canchola, Carmen; López-Romero, Everardo

    2010-02-01

    The first committed step of the biosynthetic pathway leading to uridine-5'-diphospho-N-acetyl-D-glucosamine (UDP-GlcNAc) is catalyzed by glucosamine-6-phosphate synthase (GlcN-6-P synthase), an enzyme proposed as a potential antifungal chemotherapy target. Here, we describe the purification and biochemical characterization of the native enzyme from the dimorphic pathogenic fungus Sporothrix schenckii. The availability of the pure protein facilitated its biochemical characterization. The enzyme exhibited subunit and native molecular masses of 79 and 350+/-5 kDa, respectively, suggesting a homotetrameric structure. Isoelectric point was 6.26 and K(m) values for fructose-6-phosphate and L-glutamine were 1.12+/-0.3 and 2.2+/-0.7 mM, respectively. Inhibition of activity by UDP-GlcNAc was enhanced by Glc-6-P and phosphorylation stimulated GlcN-6-P synthase activity without affecting the enzyme sensitivity to the aminosugar. A glutamine analogue, FMDP [N(3)-(4-methoxyfumaroyl)-L-2,3-diaminopropanoic acid] was a more potent inhibitor of activity than ADMP (2-Amino-2-deoxy-D-mannitol-6-phosphate) but the latter was a stronger inhibitor of growth in two culture media. To our knowledge, this is the first report on the purification and biochemical characterization of a non-recombinant GlcN-6-P synthase from a true dimorphic fungus. Inhibition of enzyme activity and fungal growth by specific inhibitors of GlcN-6-P synthase strongly reinforces the role of this enzyme as a potential target for antifungal chemotherapy. PMID:19353425

  1. Molecular cloning and characterization of a trehalose-6-phosphate synthase/phosphatase from Dunaliella viridis.

    PubMed

    Zhang, Nan; Wang, Fei; Meng, Xiangzong; Luo, Saifan; Li, Qiyun; Dong, Hongyun; Xu, Zhengkai; Song, Rentao

    2011-04-01

    Dunaliella is a group of green algae with exceptional stress tolerance capability, and is considered as an important model organism for stress tolerance study. Here we cloned a TPS (trehalose-6-phosphate synthase) gene from Dunaliella viridis and designated it as DvTPS (D. viridis trehalose-6-phosphate synthase/phosphatase).The DvTPS cDNA contained an ORF of 2793 bp encoding 930 aa. DvTPS had both TPS and TPP domain and belonged to the Group II TPS/TPP fusion gene family. Southern blots showed it has a single copy in the genome. Genome sequence analysis revealed that it has 18 exons and 17 introns. DvTPS had a constitutive high expression level under various NaCl culture conditions, however, could be induced by salt shock. Promoter analysis indicated there were ten STREs (stress response element) in its promoter region, giving a possible explanation of its inducible expression pattern upon salt shock. Yeast functional complementation analysis showed that DvTPS had neither TPS nor TPP activity. However, DvTPS could improve the salt tolerance of yeast salt sensitive mutant G19. Our results indicated that despite DvTPS showed significant similarity with TPS/TPP, its real biological function is still remained to be revealed. PMID:20878239

  2. Trehalose-6-phosphate and SnRK1 kinases in plant development and signaling: the emerging picture

    PubMed Central

    Tsai, Allen Y.-L.; Gazzarrini, Sonia

    2014-01-01

    Carbohydrates, or sugars, regulate various aspects of plant growth through modulation of cell division and expansion. Besides playing essential roles as sources of energy for growth and as structural components of cells, carbohydrates also regulate the timing of expression of developmental programs. The disaccharide trehalose is used as an energy source, as a storage and transport molecule for glucose, and as a stress-responsive compound important for cellular protection during stress in all kingdoms. Trehalose, however, is found in very low amounts in most plants, pointing to a signaling over metabolic role for this non-reducing disaccharide. In the last decade, trehalose-6-phosphate (T6P), an intermediate in trehalose metabolism, has been shown to regulate embryonic and vegetative development, flowering time, meristem determinacy, and cell fate specification in plants. T6P acts as a global regulator of metabolism and transcription promoting plant growth and triggering developmental phase transitions in response to sugar availability. Among the T6P targets are members of the Sucrose-non-fermenting1-related kinase1 (SnRK1) family, which are sensors of energy availability and inhibit plant growth and development during metabolic stress to maintain energy homeostasis. In this review, we will discuss the opposite roles of the sugar metabolite T6P and the SnRK1 kinases in the regulation of developmental phase transitions in response to carbohydrate levels. We will focus on how these two global regulators of metabolic processes integrate environmental cues and interact with hormonal signaling pathways to modulate plant development. PMID:24744765

  3. Deletion of Hexose-6-phosphate Dehydrogenase Activates the Unfolded Protein Response Pathway and Induces Skeletal Myopathy*S⃞

    PubMed Central

    Lavery, Gareth G.; Walker, Elizabeth A.; Turan, Nil; Rogoff, Daniela; Ryder, Jeffery W.; Shelton, John M.; Richardson, James A.; Falciani, Francesco; White, Perrin C.; Stewart, Paul M.; Parker, Keith L.; McMillan, Daniel R.

    2008-01-01

    Hexose-6-phosphate dehydrogenase (H6PD) is the initial component of a pentose phosphate pathway inside the endoplasmic reticulum (ER) that generates NADPH for ER enzymes. In liver H6PD is required for the 11-oxoreductase activity of 11β-hydroxysteroid dehydrogenase type 1, which converts inactive 11-oxo-glucocorticoids to their active 11-hydroxyl counterparts; consequently, H6PD null mice are relatively insensitive to glucocorticoids, exhibiting fasting hypoglycemia, increased insulin sensitivity despite elevated circulating levels of corticosterone, and increased basal and insulin-stimulated glucose uptake in muscles normally enriched in type II (fast) fibers, which have increased glycogen content. Here, we show that H6PD null mice develop a severe skeletal myopathy characterized by switching of type II to type I (slow) fibers. Running wheel activity and electrically stimulated force generation in isolated skeletal muscle are both markedly reduced. Affected muscles have normal sarcomeric structure at the electron microscopy level but contain large intrafibrillar membranous vacuoles and abnormal triads indicative of defects in structure and function of the sarcoplasmic reticulum (SR). SR proteins involved in calcium metabolism, including the sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA), calreticulin, and calsequestrin, show dysregulated expression. Microarray analysis and real-time PCR demonstrate overexpression of genes encoding proteins in the unfolded protein response pathway. We propose that the absence of H6PD induces a progressive myopathy by altering the SR redox state, thereby impairing protein folding and activating the unfolded protein response pathway. These studies thus define a novel metabolic pathway that links ER stress to skeletal muscle integrity and function. PMID:18222920

  4. Feedback Inhibition of Starch Degradation in Arabidopsis Leaves Mediated by Trehalose 6-Phosphate1[W][OPEN

    PubMed Central

    Martins, Marina Camara Mattos; Hejazi, Mahdi; Fettke, Joerg; Steup, Martin; Feil, Regina; Krause, Ursula; Arrivault, Stéphanie; Vosloh, Daniel; Figueroa, Carlos María; Ivakov, Alexander; Yadav, Umesh Prasad; Piques, Maria; Metzner, Daniela; Stitt, Mark; Lunn, John Edward

    2013-01-01

    Many plants accumulate substantial starch reserves in their leaves during the day and remobilize them at night to provide carbon and energy for maintenance and growth. In this paper, we explore the role of a sugar-signaling metabolite, trehalose-6-phosphate (Tre6P), in regulating the accumulation and turnover of transitory starch in Arabidopsis (Arabidopsis thaliana) leaves. Ethanol-induced overexpression of trehalose-phosphate synthase during the day increased Tre6P levels up to 11-fold. There was a transient increase in the rate of starch accumulation in the middle of the day, but this was not linked to reductive activation of ADP-glucose pyrophosphorylase. A 2- to 3-fold increase in Tre6P during the night led to significant inhibition of starch degradation. Maltose and maltotriose did not accumulate, suggesting that Tre6P affects an early step in the pathway of starch degradation in the chloroplasts. Starch granules isolated from induced plants had a higher orthophosphate content than granules from noninduced control plants, consistent either with disruption of the phosphorylation-dephosphorylation cycle that is essential for efficient starch breakdown or with inhibition of starch hydrolysis by β-amylase. Nonaqueous fractionation of leaves showed that Tre6P is predominantly located in the cytosol, with estimated in vivo Tre6P concentrations of 4 to 7 µm in the cytosol, 0.2 to 0.5 µm in the chloroplasts, and 0.05 µm in the vacuole. It is proposed that Tre6P is a component in a signaling pathway that mediates the feedback regulation of starch breakdown by sucrose, potentially linking starch turnover to demand for sucrose by growing sink organs at night. PMID:24043444

  5. Altering trehalose-6-phosphate content in transgenic potato tubers affects tuber growth and alters responsiveness to hormones during sprouting.

    PubMed

    Debast, Stefan; Nunes-Nesi, Adriano; Hajirezaei, Mohammad R; Hofmann, Jörg; Sonnewald, Uwe; Fernie, Alisdair R; Börnke, Frederik

    2011-08-01

    Trehalose-6-phosphate (T6P) is a signaling metabolite that regulates carbon metabolism, developmental processes, and growth in plants. In Arabidopsis (Arabidopsis thaliana), T6P signaling is, at least in part, mediated through inhibition of the SNF1-related protein kinase SnRK1. To investigate the role of T6P signaling in a heterotrophic, starch-accumulating storage organ, transgenic potato (Solanum tuberosum) plants with altered T6P levels specifically in their tubers were generated. Transgenic lines with elevated T6P levels (B33-TPS, expressing Escherichia coli osmoregulatory trehalose synthesis A [OtsA], which encodes a T6P synthase) displayed reduced starch content, decreased ATP contents, and increased respiration rate diagnostic for high metabolic activity. On the other hand, lines with significantly reduced T6P (B33-TPP, expressing E. coli OtsB, which encodes a T6P phosphatase) showed accumulation of soluble carbohydrates, hexose phosphates, and ATP, no change in starch when calculated on a fresh weight basis, and a strongly reduced tuber yield. [¹⁴C]glucose feeding to transgenic tubers indicated that carbon partitioning between starch and soluble carbohydrates was not altered. Transcriptional profiling of B33-TPP tubers revealed that target genes of SnRK1 were strongly up-regulated and that T6P inhibited potato tuber SnRK1 activity in vitro. Among the SnRK1 target genes in B33-TPP tubers, those involved in the promotion of cell proliferation and growth were down-regulated, while an inhibitor of cell cycle progression was up-regulated. T6P-accumulating tubers were strongly delayed in sprouting, while those with reduced T6P sprouted earlier than the wild type. Early sprouting of B33-TPP tubers correlated with a reduced abscisic acid content. Collectively, our data indicate that T6P plays an important role for potato tuber growth.

  6. Source/sink interactions underpin crop yield: the case for trehalose 6-phosphate/SnRK1 in improvement of wheat.

    PubMed

    Lawlor, David W; Paul, Matthew J

    2014-01-01

    Considerable interest has been evoked by the analysis of the regulatory pathway in carbohydrate metabolism and cell growth involving the non-reducing disaccharide trehalose (TRE). TRE is at small concentrations in mesophytes such as Arabidopsis thaliana and Triticum aestivum, excluding a role in osmoregulation once suggested for it. Studies of TRE metabolism, and genetic modification of it, have shown a very wide and more important role of the pathway in regulation of many processes in development, growth, and photosynthesis. It has now been established that rather than TRE, it is trehalose 6-phosphate (T6P) which has such profound effects. T6P is the intermediary in TRE synthesis formed from glucose-6-phosphate and UDP-glucose, derived from sucrose, by the action of trehalose phosphate synthase. The concentration of T6P is determined both by the rate of synthesis, which depends on the sucrose concentration, and also by the rate of breakdown by trehalose-6-phosphate phosphatase which produces TRE. Changing T6P concentrations by genetically modifying the enzymes of synthesis and breakdown has altered photosynthesis, sugar metabolism, growth, and development which affect responses to, and recovery from, environmental factors. Many of the effects of T6P on metabolism and growth occur via the interaction of T6P with the SnRK1 protein kinase system. T6P inhibits the activity of SnRK1, which de-represses genes encoding proteins involved in anabolism. Consequently, a large concentration of sucrose increases T6P and thereby inhibits SnRK1, so stimulating growth of cells and their metabolic activity. The T6P/SnRK1 mechanism offers an important new view of how the distribution of assimilates to organs, such as developing grains in cereal plants, is achieved. This review briefly summarizes the factors determining, and limiting, yield of wheat (particularly mass/grain which is highly conserved) and considers how T6P/SnRK1 might function to determine grain yield and might be

  7. Myb-binding site regulates the expression of glucosamine-6-phosphate isomerase in Dictyostelium discoideum.

    PubMed

    Tabata, K; Matsuda, Y; Viller, E; Masamune, Y; Katayama, T; Yasukawa, H

    2001-10-01

    A homolog of the glucosamine-6-phosphate isomerase in the cellular slime mold Dictyostelium discoideum has been analyzed. The gene disruption mutant was arrested at the mound stage, demonstrating that the gene is important for development. The gene was expressed in vegetatively growing cells, silenced on starvation and expressed again in prestalk cells during the multicellular stages. The upstream region of the gene (1376 bp relative to ATG) was cloned and sequenced to study the transcription control mechanisms. Analysis of deletion mutants and a site-directed mutant indicated that the Myb-binding sequence (5'-AACTG-3') localized in the upstream region is important for gene expression. The results of gel-shift assays showed the presence of an Myb-related protein binding to the sequence at the growing phase and another protein binding to the sequence at developmental stages. PMID:11576175

  8. Structural analysis of N-acetylglucosamine-6-phosphate deacetylase apoenzyme from Escherichia coli.

    PubMed

    Ferreira, Frederico M; Mendoza-Hernandez, Guillermo; Castañeda-Bueno, Maria; Aparicio, Ricardo; Fischer, Hannes; Calcagno, Mario L; Oliva, Glaucius

    2006-06-01

    We report the crystal structure of the apoenzyme of N-acetylglucosamine-6-phosphate (GlcNAc6P) deacetylase from Escherichia coli (EcNAGPase) and the spectrometric evidence of the presence of Zn2+ in the native protein. The GlcNAc6P deacetylase is an enzyme of the amino sugar catabolic pathway that catalyzes the conversion of the GlcNAc6P into glucosamine 6-phosphate (GlcN6P). The crystal structure was phased by the single isomorphous replacement with anomalous scattering (SIRAS) method using low-resolution (2.9 A) iodine anomalous scattering and it was refined against a native dataset up to 2.0 A resolution. The structure is similar to two other NAGPases whose structures are known from Thermotoga maritima (TmNAGPase) and Bacillus subtilis (BsNAGPase); however, it shows a phosphate ion bound at the metal-binding site. Compared to these previous structures, the apoenzyme shows extensive conformational changes in two loops adjacent to the active site. The E. coli enzyme is a tetramer and its dimer-dimer interface was analyzed. The tetrameric structure was confirmed in solution by small-angle X-ray scattering data. Although no metal ions were detected in the present structure, experiments of photon-induced X-ray emission (PIXE) spectra and of inductively coupled plasma emission spectroscopy (ICP-AES) with enzyme that was neither exposed to chelating agents nor metal ions during purification, revealed the presence of 1.4 atoms of Zn per polypeptide chain. Enzyme inactivation by metal-sequestering agents and subsequent reactivation by the addition of several divalent cations, demonstrate the role of metal ions in EcNAGPase structure and catalysis.

  9. Phosphatase-Inert Glucosamine 6-Phosphate Mimics Serve as Actuators of the glmS Riboswitch

    PubMed Central

    2015-01-01

    The glmS riboswitch is unique among gene-regulating riboswitches and catalytic RNAs. This is because its own metabolite, glucosamine-6-phosphate (GlcN6P), binds to the riboswitch and catalytically participates in the RNA self-cleavage reaction, thereby providing a novel negative feedback mechanism. Given that a number of pathogens harbor the glmS riboswitch, artificial actuators of this potential RNA target are of great interest. Structural/kinetic studies point to the 2-amino and 6-phosphate ester functionalities in GlcN6P as being crucial for this actuation. As a first step toward developing artificial actuators, we have synthesized a series of nine GlcN6P analogs bearing phosphatase-inert surrogates in place of the natural phosphate ester. Self-cleavage assays with the Bacillus cereusglmS riboswitch give a broad SAR. Two analogs display significant activity, namely, the 6-deoxy-6-phosphonomethyl analog (5) and the 6-O-malonyl ether (13). Kinetic profiles show a 22-fold and a 27-fold higher catalytic efficiency, respectively, for these analogs vs glucosamine (GlcN). Given their nonhydrolyzable phosphate surrogate functionalities, these analogs are arguably the most robust artificial glmS riboswitch actuators yet reported. Interestingly, the malonyl ether (13, extra O atom) is much more effective than the simple malonate (17), and the “sterically true” phosphonate (5) is far superior to the chain-truncated (7) or chain-extended (11) analogs, suggesting that positioning via Mg coordination is important for activity. Docking results are consistent with this view. Indeed, the viability of the phosphonate and 6-O-malonyl ether mimics of GlcN6P points to a potential new strategy for artificial actuation of the glmS riboswitch in a biological setting, wherein phosphatase-resistance is paramount. PMID:25254431

  10. Allosteric kinetics of the isoform 1 of human glucosamine-6-phosphate deaminase.

    PubMed

    Alvarez-Añorve, Laura I; Alonzo, Diego A; Mora-Lugo, Rodrigo; Lara-González, Samuel; Bustos-Jaimes, Ismael; Plumbridge, Jacqueline; Calcagno, Mario L

    2011-12-01

    The human genome contains two genes encoding for two isoforms of the enzyme glucosamine-6-phosphate deaminase (GNPDA, EC 3.5.99.6). Isoform 1 has been purified from several animal sources and the crystallographic structure of the human recombinant enzyme was solved at 1.75Å resolution (PDB ID: 1NE7). In spite of their great structural similarity, human and Escherichia coli GNPDAs show marked differences in their allosteric kinetics. The allosteric site ligand, N-acetylglucosamine 6-phosphate (GlcNAc6P), which is an activator of the K-type of E. coli GNPDA has an unusual mixed allosteric effect on hGNPDA1, behaving as a V activator and a K inhibitor (antiergistic or crossed mixed K(-)V(+) effect). In the absence of GlcNAc6P, the apparent k(cat) of the enzyme is so low, that GlcNAc6P behaves as an essential activator. Additionally, substrate inhibition, dependent on GlcNAc6P concentration, is observed. All these kinetic properties can be well described within the framework of the Monod allosteric model with some additional postulates. These unusual kinetic properties suggest that hGNPDA1 could be important for the maintenance of an adequate level of the pool of the UDP-GlcNAc6P, the N-acetylglucosylaminyl donor for many reactions in the cell. In this research we have also explored the possible functional significance of the C-terminal extension of hGNPDA1 enzyme, which is not present in isoform 2, by constructing and studying two mutants truncated at positions 268 and 275.

  11. Improvement of drought tolerance and grain yield in common bean by overexpressing trehalose-6-phosphate synthase in rhizobia.

    PubMed

    Suárez, Ramón; Wong, Arnoldo; Ramírez, Mario; Barraza, Aarón; Orozco, María Del Carmen; Cevallos, Miguel A; Lara, Miguel; Hernández, Georgina; Iturriaga, Gabriel

    2008-07-01

    Improving stress tolerance and yield in crops are major goals for agriculture. Here, we show a new strategy to increase drought tolerance and yield in legumes by overexpressing trehalose-6-phosphate synthase in the symbiotic bacterium Rhizobium etli. Phaseolus vulgaris (common beans) plants inoculated with R. etli overexpressing trehalose-6-phosphate synthase gene had more nodules with increased nitrogenase activity and higher biomass compared with plants inoculated with wild-type R. etli. In contrast, plants inoculated with an R. etli mutant in trehalose-6-phosphate synthase gene had fewer nodules and less nitrogenase activity and biomass. Three-week-old plants subjected to drought stress fully recovered whereas plants inoculated with a wild-type or mutant strain wilted and died. The yield of bean plants inoculated with R. etli overexpressing trehalose-6-phosphate synthase gene and grown with constant irrigation increased more than 50%. Macroarray analysis of 7,200 expressed sequence tags from nodules of plants inoculated with the strain overexpressing trehalose-6-phosphate synthase gene revealed upregulation of genes involved in stress tolerance and carbon and nitrogen metabolism, suggesting a signaling mechanism for trehalose. Thus, trehalose metabolism in rhizobia is key for signaling plant growth, yield, and adaptation to abiotic stress, and its manipulation has a major agronomical impact on leguminous plants.

  12. Glucose-6-phosphate dehydrogenase (G6PD) mutations database: review of the "old" and update of the new mutations.

    PubMed

    Minucci, Angelo; Moradkhani, Kamran; Hwang, Ming Jing; Zuppi, Cecilia; Giardina, Bruno; Capoluongo, Ettore

    2012-03-15

    In the present paper we have updated the G6PD mutations database, including all the last discovered G6PD genetic variants. We underline that the last database has been published by Vulliamy et al. [1] who analytically reported 140 G6PD mutations: along with Vulliamy's database, there are two main sites, such as http://202.120.189.88/mutdb/ and www.LOVD.nl/MR, where almost all G6PD mutations can be found. Compared to the previous mutation reports, in our paper we have included for each mutation some additional information, such as: the secondary structure and the enzyme 3D position involving by mutation, the creation or abolition of a restriction site (with the enzyme involved) and the conservation score associated with each amino acid position. The mutations reported in the present tab have been divided according to the gene's region involved (coding and non-coding) and mutations affecting the coding region in: single, multiple (at least with two bases involved) and deletion. We underline that for the listed mutations, reported in italic, literature doesn't provide all the biochemical or bio-molecular information or the research data. Finally, for the "old" mutations, we tried to verify features previously reported and, when subsequently modified, we updated the specific information using the latest literature data. PMID:22293322

  13. Characterization of three putative xylulose 5-phosphate/fructose 6-phosphate phosphoketolases in the cyanobacterium Anabaena sp. PCC 7120.

    PubMed

    Moriyama, Takashi; Tajima, Naoyuki; Sekine, Kohsuke; Sato, Naoki

    2015-01-01

    Xylulose 5-phosphate/fructose 6-phosphate phosphoketolase (Xfp) is a key enzyme in the central carbohydrate metabolism in heterofermentative bacteria, in which enzymatic property of Xfps is well characterized. This is not the case in other microbes. The cyanobacterium Anabaena sp. PCC 7120 possesses three putative genes encoding Xfp, all1483, all2567, and alr1850. We purified three putative Xfps as recombinant proteins. The results of gel filtration indicated that these proteins form homomultimer complex. All1483 and All2567 showed phosphoketolase activity, whereas Alr1850 did not show the activity. Kinetic analyses demonstrated that substrates, fructose 6-phosphate and inorganic phosphate, are cooperatively bound to enzymes positively and negatively, respectively.

  14. Mutations in the genes encoding 11beta-hydroxysteroid dehydrogenase type 1 and hexose-6-phosphate dehydrogenase interact to cause cortisone reductase deficiency.

    PubMed

    Draper, Nicole; Walker, Elizabeth A; Bujalska, Iwona J; Tomlinson, Jeremy W; Chalder, Susan M; Arlt, Wiebke; Lavery, Gareth G; Bedendo, Oliver; Ray, David W; Laing, Ian; Malunowicz, Ewa; White, Perrin C; Hewison, Martin; Mason, Philip J; Connell, John M; Shackleton, Cedric H L; Stewart, Paul M

    2003-08-01

    In cortisone reductase deficiency (CRD), activation of cortisone to cortisol does not occur, resulting in adrenocorticotropin-mediated androgen excess and a phenotype resembling polycystic ovary syndrome (PCOS; refs. 1,2). This suggests a defect in the gene HSD11B1 encoding 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1), a primary regulator of tissue-specific glucocorticoid bioavailability. We identified intronic mutations in HSD11B1 that resulted in reduced gene transcription in three individuals with CRD. In vivo, 11beta-HSD1 catalyzes the reduction of cortisone to cortisol whereas purified enzyme acts as a dehydrogenase converting cortisol to cortisone. Oxo-reductase activity can be regained using a NADPH-regeneration system and the cytosolic enzyme glucose-6-phosphate dehydrogenase. But the catalytic domain of 11beta-HSD1 faces into the lumen of the endoplasmic reticulum (ER; ref. 6). We hypothesized that endolumenal hexose-6-phosphate dehydrogenase (H6PDH) regenerates NADPH in the ER, thereby influencing directionality of 11beta-HSD1 activity. Mutations in exon 5 of H6PD in individuals with CRD attenuated or abolished H6PDH activity. These individuals have mutations in both HSD11B1 and H6PD in a triallelic digenic model of inheritance, resulting in low 11beta-HSD1 expression and ER NADPH generation with loss of 11beta-HSD1 oxo-reductase activity. CRD defines a new ER-specific redox potential and establishes H6PDH as a potential factor in the pathogenesis of PCOS. PMID:12858176

  15. Participation of free oxygen radicals in damage of porcine erythrocytes

    SciTech Connect

    Jozwiak, J.; Helszer, Z.

    1981-10-01

    Gamma radiation causes disturbances in energy metabolism, decreases in (Na/sup +/-K/sup +/)-ATPase, Mg/sup 2 +/-APTase activity, and increase in the degree of hemolysis in porcine erythrocytes. Our results indicated a contribution of exogenous free radicals in radiation damage to porcine erythrocytes. In the presence of biological and chemical radioprotectors a protective effect with respect to ATPase activity and energy metabolism was observed in the presence of catalase, histidine, glucose, and sulfhydryl compounds. It appears that radiation damage to porcine erythrocytes is due to the action of various radicals formed upon irradiation which react at different rates with various cell constituents.

  16. Comparative study on mannose 6-phosphate residue contents of recombinant lysosomal enzymes.

    PubMed

    Togawa, Tadayasu; Takada, Masaru; Aizawa, Yoshiaki; Tsukimura, Takahiro; Chiba, Yasunori; Sakuraba, Hitoshi

    2014-03-01

    As most recombinant lysosomal enzymes are incorporated into cells via mannose 6-phosphate (M6P) receptors, the M6P content is important for effective enzyme replacement therapy (ERT) for lysosomal diseases. However, there have been no comprehensive reports of the M6P contents of lysosomal enzymes. We developed an M6P assay method comprising three steps, i.e., acid hydrolysis of glycoproteins, derivatization of M6P, and high-performance liquid chromatography, and determined the M6P contents of six recombinant lysosomal enzymes now available for ERT and one in the process of development. The assay is easy, specific, and reproducible. The results of the comparative study revealed that the M6P contents of agalsidase alfa, agalsidase beta, modified α-N-acetylgalactosaminidase, alglucosidase alfa, laronidase, idursulfase, and imiglucerase are 2.1, 2.9, 5.9, 0.7, 2.5, 3.2, and <0.3 mol/mol enzyme, respectively. The results were correlated with those of the biochemical analyses previously performed and that of the binding assay of exposed M6P of the enzymes with the domain 9 of the cation-independent M6P receptor. This assay method is useful for comparison of the M6P contents of recombinant lysosomal enzymes for ERT. PMID:24439675

  17. The phylogenetic utility of nucleotide sequences of sorbitol 6-phosphate dehydrogenase in Prunus (Rosaceae).

    PubMed

    Bortiri, Esteban; Oh, Sang-Hun; Gao, Fang-You; Potter, Dan

    2002-10-01

    Sequences from s6pdh, a gene that encodes sorbitol-6-phosphate dehydrogenase in the Rosaceae, are used to reconstruct the phylogeny of 22 species of Prunus. The s6pdh sequences alone and in combination with previously published sequences of the internal transcribed spacer (ITS) and the cpDNA trnL-trnF spacer are analyzed using parsimony and maximum likelihood methods. Both methods reconstructed the same phylogeny when s6pdh sequences are used alone and in combination with ITS and trnL-trnF, and the topology is in agreement with previous studies that used a larger sample size. The s6pdh sequences have about twice as many informative sites as ITS. A molecular clock is rejected for s6pdh, most likely due to greater rates of evolution in subgenera Padus and Laurocerasus than in the rest of the genus. Phylogenetic reconstruction of Prunus as determined by analysis of the combined data set suggests an early split into two clades. One is composed of subgenera Cerasus, Laurocerasus, and Padus. The second includes subgenera Amygdalus, Emplectocladus, and Prunus. Species of section Microcerasus (formerly in subgenus Cerasus) are nested within subgenus Prunus. The order of branching and relationships among early diverging lineages is weakly supported, as a result of very short branches that may indicate rapid radiation. PMID:21665596

  18. Cloning and partial characterization of the mouse glutamine:fructose-6-phosphate amidotransferase (GFAT) gene promoter.

    PubMed Central

    Sayeski, P P; Wang, D; Su, K; Han, I O; Kudlow, J E

    1997-01-01

    Glutamine:fructose-6-phosphate amidotransferase (GFAT) is the enzyme that is rate limiting in the synthesis of glucosamine and hexosamines. Glucosamine has been proposed to contribute to the glucotoxicity of diabetes. Evidence that the gene encoding GFAT is transcriptionally regulated prompted us to clone and characterize its promoter. The position of the mouse GFAT promoter relative to the translational start site was located by primer extension and found to be 149 bp upstream of the translational start site. A 1.9 kb SacI fragment of the GFAT gene was found to contain the promoter and 88 bp of sequence downstream of the transcriptional start site. This promoter segment could drive expression of a luciferase reporter gene, could confer correct transcriptional initiation to the reporter and could confer the EGF-responsiveness previously observed in the native gene. The mouse GFAT promoter lacks a canonical TATA box and has several GC boxes within a highly GC-rich region. Deletional analysis of the promoter indicated that a proximal element extending to -120 relative to the transcriptional start site could confer reporter expression at a level of 57% of the 1.9 kb construct. Detailed analysis of this proximal region by DNase I footprinting, electrophoretic mobility shift assays and site-directed mutagenesis indicated that Sp1 binds to three elements in this proximal promoter segment and plays a vital role in regulation of transcription from this gene. PMID:9060444

  19. Lrp1/LDL Receptor Play Critical Roles in Mannose 6-Phosphate-Independent Lysosomal Enzyme Targeting.

    PubMed

    Markmann, Sandra; Thelen, Melanie; Cornils, Kerstin; Schweizer, Michaela; Brocke-Ahmadinejad, Nahal; Willnow, Thomas; Heeren, Joerg; Gieselmann, Volkmar; Braulke, Thomas; Kollmann, Katrin

    2015-07-01

    Most lysosomal enzymes require mannose 6-phosphate (M6P) residues for efficient receptor-mediated lysosomal targeting. Although the lack of M6P residues results in missorting and hypersecretion, selected lysosomal enzymes reach normal levels in lysosomes of various cell types, suggesting the existence of M6P-independent transport routes. Here, we quantify the lysosomal proteome in M6P-deficient mouse fibroblasts (PT(ki)) using Stable Isotope Labeling by Amino acids in Cell culture (SILAC)-based comparative mass spectrometry, and find unchanged amounts of 20% of lysosomal enzymes, including cathepsins D and B (Ctsd and Ctsb). Examination of fibroblasts from a new mouse line lacking both M6P and sortilin, a candidate for M6P-independent transport of lysosomal enzymes, revealed that sortilin does not act as cargo receptor for Ctsb and Ctsd. Using fibroblast lines deficient for endocytic lipoprotein receptors, we could demonstrate that both LDL receptor and Lrp1 mediate the internalization of non-phosphorylated Ctsb and Ctsd. Furthermore, the presence of Lrp1 inhibitor increased the secretion of Ctsd from PT(ki) cells. These findings establish Lrp1 and LDL receptors in M6P-independent secretion-recapture targeting mechanism for lysosomal enzymes.

  20. Cloning, expression, purification, crystallization and preliminary X-ray crystallographic analysis of the mannose 6-phosphate isomerase from Salmonella typhimurium

    SciTech Connect

    Gowda, Giri; Sagurthi, Someswar Rao; Savithri, H. S.; Murthy, M. R. N.

    2008-02-01

    The cloning, expression, purification, crystallization and preliminary X-ray crystallographic studies of mannose 6-phosphate isomerase from S. typhimurium are reported. Mannose 6-phosphate isomerase (MPI; EC 5.3.1.8) catalyzes the reversible isomerization of d-mannose 6-phosphate (M6P) and d-fructose 6-phosphate (F6P). In the eukaryotes and prokaryotes investigated to date, the enzyme has been reported to play a crucial role in d-mannose metabolism and supply of the activated mannose donor guanosine diphosphate d-mannose (GDP-d-mannose). In the present study, MPI was cloned from Salmonella typhimurium, overexpressed in Escherichia coli and purified using Ni–NTA affinity column chromatography. Purified MPI crystallized in space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 36.03, b = 92.2, c = 111.01 Å. A data set extending to 1.66 Å resolution was collected with 98.8% completeness using an image-plate detector system mounted on a rotating-anode X-ray generator. The asymmetric unit of the crystal cell was compatible with the presence of a monomer of MPI. A preliminary structure solution of the enzyme has been obtained by molecular replacement using Candida albicans MPI as the phasing model and the program Phaser. Further refinement and model building are in progress.

  1. Structures of mannose-6-phosphate isomerase from Salmonella typhimurium bound to metal atoms and substrate: implications for catalytic mechanism.

    PubMed

    Sagurthi, S R; Gowda, Giri; Savithri, H S; Murthy, M R N

    2009-07-01

    Mannose-6-phosphate isomerase (MPI) catalyzes the interconversion of mannose 6-phosphate and fructose 6-phosphate. X-ray crystal structures of MPI from Salmonella typhimurium in the apo form (with no metal bound) and in the holo form (with bound Zn2+) and two other structures with yttrium bound at an inhibitory site and complexed with Zn2+ and fructose 6-phosphate (F6P) were determined in order to gain insights into the structure and the isomerization mechanism. Isomerization involves acid/base catalysis with proton transfer between the C1 and C2 atoms of the substrate. His99, Lys132, His131 and Asp270 are close to the substrate and are likely to be the residues involved in proton transfer. The interactions observed at the active site suggest that the ring-opening step is probably catalyzed by His99 and Asp270. An active-site loop consisting of residues 130-133 undergoes conformational changes upon substrate binding. Zn2+ binding induces structural order in the loop consisting of residues 50-54. The metal atom appears to play a role in substrate binding and is probably also important for maintaining the architecture of the active site. Isomerization probably follows the previously suggested cis-enediol mechanism.

  2. Metabolism of acetylcholine in human erythrocytes

    SciTech Connect

    Chapman, E.S.

    1990-01-01

    In order to examine the possible role of erythrocyte acetylcholinesterase in the maintenance of membrane phospholipid content and membrane fluidity, experiments were performed to monitor the activity of the enzyme and follow the fate of one of its hydrolytic products, choline. Intact human erythrocytes were incubated with acetylcholine (choline methyl-{sup 14}C). The incubation resulted in the hydrolysis of acetylcholine to acetate and choline; the reaction was catalyzed by membrane acetylcholinesterase. The studies demonstrate the further metabolism of choline. Experiments were carried out to determine rate of hydrolysis of acetylcholine, uptake of choline, identification of intracellular metabolites of choline, and identification of radiolabeled membrane components. Erythrocytes at a 25% hematocrit were incubated in an isoosmotic bicarbonate buffer pH 7.4, containing glucose, adenosine, streptomycin and penicillin with 0.3 {mu}Ci of acetylcholine (choline methyl-{sup 14}C), for 24 hours. Aliquots of the erythrocyte suspension were taken throughout for analysis. Erythrocytes were washed free of excess substrate, lysed, and the hemolysate was extracted for choline and its metabolites. Blank samples containing incubation buffer and radiolabeled acetylcholine only, and erythrocyte hemolysate extracts were analyzed for choline content, the difference between blank samples and hemolysate extracts was the amount of choline originating from acetylcholine and attributable to acetylcholinesterase activity. The conversion of choline to {sup 14}C-betaine is noted after several minutes of incubation; at 30 minutes, more than 80% of {sup 14}C-choline is taken up and after several hours, detectable levels of radiolabeled S-adenosylmethionine were present in the hemolysate extract.

  3. Interactions of HIV-1 and HIV-2 envelope glycoproteins with sulphated polysaccharides and mannose-6-phosphate.

    PubMed

    Mbemba, E; Gluckman, J C; Gattegno, L

    1994-02-01

    Envelope glycoproteins of human immunodeficiency viruses (HIV-1 and HIV-2) can interact with high-mannose glycans and with the mannosyl or N-acetylglucosaminyl core of complex-type oligosaccharidic structures. HIV-1 glycoproteins also specifically bind sulphated polysaccharides such as dextran sulphate (DS) and heparin. Here, we show that the latter property is shared by HIV-2 recombinant gp140 (rgp140) precursor glycoprotein. Binding of rgp140 and of corresponding rgp160 of HIV-1 to heparin- and DS-substituted (sulphated dextran beads; SDB) affinity matrices was inhibited by the soluble specific ligand and also by fetuin, asialofetuin or the anionic simple carbohydrate derivative mannose-6-phosphate (M6P). Interaction of HIV-1 rgp120 subunit with the two affinity matrices was also inhibited by M6P, but only rgp120 binding to heparin-agarose, and not that to SDB, was affected by fetuin and asialofetuin. These results suggest that HIV-1 and HIV-2 envelope glycoproteins presumably display different sulphated polysaccharide and carbohydrate recognition sites. Some of these may be common or in close proximity: with respect to rgp160, for example, the sites may be common on the gp41 moiety and/or in a region of gp120 which would be more accessible when expressed on rgp160 than on processed gp120, while they may be distinct on the cleaved gp120 subunit. Finally, because M6P is a marker of lysosomal enzymes, we verified that HIV-1 and HIV-2 envelope glycoproteins could specifically bind in a M6P-inhibitable manner to a representative lysosomal enzyme, bovine liver beta-glucuronidase coupled to agarose, suggesting that they may possibly interfere with lysosomal enzyme sorting in HIV-infected cells.

  4. Recognition of mannose 6-phosphate ligands by dystrophic rat retinal pigment epithelium

    SciTech Connect

    Tarnowski, B.; Shepherd, V.; McLaughlin, B.

    1986-05-01

    Retinal pigment epithelium (RPE) phagocytize discarded rod outer segments (ROS) during normal eye function. In the dystrophic rat, an animal model for retinitis pigmentosa in humans, ROS phagocytosis is defective. Dystrophic RPE can phagocytize particles other than ROS, suggesting that the defect may be in the RPE phagocytic recognition. They are currently investigating the recognition markers on RPE in dystrophic rats. In studies using ligand-coated latex beads, no uptake of mannose-coated beads was found in dystrophic rat RPE. They found that dystrophic RPE could specifically phagocytize phosphomannan-coated beads. Studies were begun to examine the presence and function of a phosphomannan receptor (PMR) on dystrophic RPE. ..cap alpha..-Mannosidase, isolated from D. discoideum has been shown to be an efficient ligand for the PMR in fibroblasts and macrophages. It is also recognized by the macrophage mannose receptor. Dystrophic rat RPE and retina explants were placed in culture dishes (5-7/well). /sup 125/I-Labelled ..cap alpha..-mannosidase was added to each well in the presence or absence of 10 mM mannose 6-phosphate (M6P) or yeast mannan (lmg/ml). Explants were incubated at 37/sup 0/ for 2 hr., washed and bound /sup 125/I-mannosidase quantitated. Approximately 2-3% of total counts added were bound to the RPE via a M6P-inhibitable recognition process. The binding to RPE was not blocked by mannan. No mannan or M6P-specific binding was found in retina explants. These results support the findings of specific uptake of phosphomannan-coated beads and demonstrate the presence of a specific PMR on dystrophic RPE phagocytic membranes.

  5. A Tale of Two Sugars: Trehalose 6-Phosphate and Sucrose1[OPEN

    PubMed Central

    2016-01-01

    Trehalose 6-phosphate (Tre6P), the intermediate of trehalose biosynthesis, is an essential signal metabolite in plants, linking growth and development to carbon status. The Suc-Tre6P nexus model postulates that Tre6P is both a signal and negative feedback regulator of Suc levels, forming part of a mechanism to maintain Suc levels within an optimal range and functionally comparable to the insulin-glucagon system for regulating blood Glc levels in animals. The target range and sensitivity of the Tre6P-Suc feedback control circuit can be adjusted according to the cell type, developmental stage, and environmental conditions. In source leaves, Tre6P modulates Suc levels by affecting Suc synthesis, whereas in sink organs it regulates Suc consumption. In illuminated leaves, Tre6P influences the partitioning of photoassimilates between Suc, organic acids, and amino acids via posttranslational regulation of phosphoenolpyruvate carboxylase and nitrate reductase. At night, Tre6P regulates the remobilization of leaf starch reserves to Suc, potentially linking starch turnover in source leaves to carbon demand from developing sink organs. Use of Suc for growth in developing tissues is strongly influenced by the antagonistic activities of two protein kinases: SUC-NON-FERMENTING-1-RELATED KINASE1 (SnRK1) and TARGET OF RAPAMYCIN (TOR). The relationship between Tre6P and SnRK1 in developing tissues is complex and not yet fully resolved, involving both direct and indirect mechanisms, and positive and negative effects. No direct connection between Tre6P and TOR has yet been described. The roles of Tre6P in abiotic stress tolerance and stomatal regulation are also discussed. PMID:27482078

  6. Transcriptome analysis of potato leaves expressing the trehalose-6-phosphate synthase 1 gene of yeast.

    PubMed

    Kondrák, Mihály; Marincs, Ferenc; Kalapos, Balázs; Juhász, Zsófia; Bánfalvi, Zsófia

    2011-01-01

    Transgenic lines of the potato cultivar White Lady expressing the trehalose-6-phosphate synthase (TPS1) gene of yeast exhibit improved drought tolerance, but grow slower and have a lower carbon fixation rate and stomatal density than the wild-type. To understand the molecular basis of this phenomenon, we have compared the transcriptomes of wild-type and TPS1-transgenic plants using the POCI microarray containing 42,034 potato unigene probes. We show that 74 and 25 genes were up-, and down-regulated, respectively, in the mature source leaves of TPS1-transgenic plants when compared with the wild-type. The differentially regulated genes were assigned into 16 functional groups. All of the seven genes, which were assigned into carbon fixation and metabolism group, were up-regulated, while about 42% of the assigned genes are involved in transcriptional and post-transcriptional regulation. Expression of genes encoding a 14-3-3 regulatory protein, and four transcription factors were down-regulated in the TPS1-transgenic leaves. To verify the microarray results, we used RNA gel blot analysis to examine the expression of eight genes and found that the RNA gel blot and microarray data correlated in each case. Using the putative Arabidopsis orthologs of the assigned potato sequences we have identified putative transcription binding sites in the promoter region of the differentially regulated genes, and putative protein-protein interactions involving some of the up- and down-regulated genes. We have also demonstrated that starch content is lower, while malate, inositol and maltose contents are higher in the TPS1-transgenic than in the wild-type leaves. Our results suggest that a complex regulatory network, involving transcription factors and other regulatory proteins, underpins the phenotypic alterations we have observed previously in potato when expressing the TPS1 gene of yeast.

  7. The Redox-Sensitive Chloroplast Trehalose-6-Phosphate Phosphatase AtTPPD Regulates Salt Stress Tolerance

    PubMed Central

    Krasensky, Julia; Broyart, Caroline; Rabanal, Fernando A.

    2014-01-01

    Abstract Aims: High salinity stress impairs plant growth and development. Trehalose metabolism has been implicated in sugar signaling, and enhanced trehalose metabolism can positively regulate abiotic stress tolerance. However, the molecular mechanism(s) of the stress-related trehalose pathway and the role of individual trehalose biosynthetic enzymes for stress tolerance remain unclear. Results: Trehalose-6-phosphate phosphatase (TPP) catalyzes the final step of trehalose metabolism. Investigating the subcellular localization of the Arabidopsis thaliana TPP family members, we identified AtTPPD as a chloroplast-localized enzyme. Plants deficient in AtTPPD were hypersensitive, whereas plants overexpressing AtTPPD were more tolerant to high salinity stress. Elevated stress tolerance of AtTPPD overexpressors correlated with high starch levels and increased accumulation of soluble sugars, suggesting a role for AtTPPD in regulating sugar metabolism under salinity conditions. Biochemical analyses indicate that AtTPPD is a target of post-translational redox regulation and can be reversibly inactivated by oxidizing conditions. Two cysteine residues were identified as the redox-sensitive sites. Structural and mutation analyses suggest that the formation of an intramolecular disulfide bridge regulates AtTPPD activity. Innovation: The activity of different AtTPP isoforms, located in the cytosol, nucleus, and chloroplasts, can be redox regulated, suggesting that the trehalose metabolism might relay the redox status of different cellular compartments to regulate diverse biological processes such as stress responses. Conclusion: The evolutionary conservation of the two redox regulatory cysteine residues of TPPs in spermatophytes indicates that redox regulation of TPPs might be a common mechanism enabling plants to rapidly adjust trehalose metabolism to the prevailing environmental and developmental conditions. Antioxid. Redox Signal. 21, 1289–1304. PMID:24800789

  8. Structural flexibility, an essential component of the allosteric activation in Escherichia coli glucosamine-6-phosphate deaminase.

    PubMed

    Rudiño-Piñera, E; Morales-Arrieta, S; Rojas-Trejo, S P; Horjales, E

    2002-01-01

    A new crystallographic structure of the free active-site R conformer of the allosteric enzyme glucosamine-6-phosphate deaminase from Escherichia coli, coupled with previously reported structures of the T and R conformers, generates a detailed description of the heterotropic allosteric transition in which structural flexibility plays a central role. The T conformer's external zone [Horjales et al. (1999), Structure, 7, 527-536] presents higher B values than in the R conformers. The ligand-free enzyme (T conformer) undergoes an allosteric transition to the free active-site R conformer upon binding of the allosteric activator. This structure shows three alternate conformations of the mobile section of the active-site lid (residues 163-182), in comparison to the high B values for the unique conformation of the T conformer. One of these alternate R conformations corresponds to the active-site lid found when the substrate is bound. The disorder associated with the three alternate conformations can be related to the biological regulation of the K(m) of the enzyme for the reaction, which is metabolically required to maintain adequate concentrations of the activator, which holds the enzyme in its R state. Seven alternate conformations for the active-site lid and three for the C-terminus were refined for the T structure using isotropic B factors. Some of these conformers approach that of the R conformer in geometry. Furthermore, the direction of the atomic vibrations obtained with anisotropic B refinement supports the hypothesis of an oscillating rather than a tense T state. The concerted character of the allosteric transition is also analysed in view of the apparent dynamics of the conformers.

  9. Stabilization of mutant 46-kDa mannose 6-phosphate receptors by proteasomal inhibitor lactacystin.

    PubMed

    Breuer, P; Braulke, T

    1998-12-11

    Palmitoylation of cysteine residue 34 within the 67-amino acid cytoplasmic domain of the 46-kDa mannose 6-phosphate receptor (MPR 46), which may be anchored to the lipid bilayer, prevents the receptor from entering lysosomes (Schweizer, A., Kornfeld, S., and Rohrer, J. (1996) J. Cell Biol. 132, 577-584). In the present study, we examined the importance of the spacing between the transmembrane domain and the palmitoylation anchor site in the cytoplasmic domain for stability and trafficking of MPR 46. MPR 46 mutants with deletions of residues 20-23 and 24-29 expressed in baby hamster kidney cells were rapidly degraded with half-lives of less than 10 h. The replacement of residues 24-29 by alanine resulted in prolongation of receptor stability (t(1)/(2) approximately 20 h). Whereas mutant MPR 46 could not be detected in lysosomal fractions and inhibitors of lysosomal proteases failed to prevent degradation, treatment with the proteasome inhibitor lactacystin resulted in increased stability of mutant MPR 46. Pulse-chase experiments at low temperature and the acquirement of endoglucosaminidase H-resistant oligosaccharides indicate that the majority of mutant MPR 46 is degraded after leaving the Golgi compartment. Altered trafficking of mutant MPR 46 may be the result of decreased palmitoylation reaching 40% of wild type receptors. The data suggest that the spacing between the transmembrane domain and the proposed palmitoylation anchor site in the cytoplasmic domain of MPR 46 is important for a post Golgi sorting step preventing receptor degradation by multiple proteolytic systems including the proteasome. PMID:9837896

  10. Mannose-6-phosphate facilitates early peripheral nerve regeneration in thy-1-YFP-H mice.

    PubMed

    Harding, A J; Christmas, C R; Ferguson, M W J; Loescher, A R; Robinson, P P; Boissonade, F M

    2014-10-24

    The formation of scar tissue following nerve injury has been shown to adversely affect nerve regeneration and evidence suggests that mannose-6-phosphate (M6P), a potential scar reducing agent that affects transforming growth factor (TGF)-β activation, may enhance nerve regeneration. In this study we utilized thy-1-YFP-H mice - a transgenic strain expressing yellow fluorescent protein (YFP) within a subset of axons - to enable visual analysis of axons regenerating through a nerve graft. Using this strain of mouse we have developed analysis techniques to visualize and quantify regeneration of individual axons across the injury site following the application of either M6P or vehicle to the site of nerve injury. No significant differences were found in the proportion of axons regenerating through the graft between M6P- and vehicle-treated grafts at any point along the graft length. Maximal sprouting occurred at 1.0mm from the proximal graft ending in both groups. The maximum change in sprouting levels for both treatment groups occurred between the graft start and 0.5-mm interval for both treatment groups. The difference between repair groups was significant at this point with a greater increase seen in the vehicle group than the M6P group. The average length of axons regenerating across the initial graft entry was significantly shorter in M6P- than in vehicle-treated grafts, indicating that they encountered less impedance. Application of M6P appears to reduce the disruption of regenerating axons and may therefore facilitate quicker recovery; this is likely to result from altered scar tissue formation in M6P grafts in the early stages of recovery. This study also establishes the usefulness of our methods of analysis using the thy-1-YFP-H mouse strain to visualize and quantify regeneration at the level of the individual axon.

  11. A Novel Single-Chain Antibody Fragment for Detection of Mannose 6-Phosphate-Containing Proteins

    PubMed Central

    Müller-Loennies, Sven; Galliciotti, Giovanna; Kollmann, Katrin; Glatzel, Markus; Braulke, Thomas

    2010-01-01

    Newly synthesized soluble lysosomal hydrolases require mannose 6-phosphate (Man6P) residues on their oligosaccharides for their transport to lysosomes. The formation of Man6P residues is catalyzed by the GlcNAc-1-phosphotransferase, which is defective in the lysosomal storage disorders mucolipidosis type II (ML II) and ML III. Both hypersecretion and reduced intracellular level of lysosomal enzymes as well as direct sequencing of GlcNAc-1-phosphotransferase genes are important diagnostic markers for ML II and ML III. A high-affinity Man6P-specific single-chain antibody fragment was generated, allowing the rapid indirect demonstration of defective GlcNAc-1-phosphotransferase. In media and extracts of cultured fibroblasts of healthy controls but not of ML II and ML III patients, several Man6P-containing proteins could be detected by anti-Man6P Western blotting. Immunoprecipitation of Man6P-containing proteins from conditioned media or mouse brain extracts followed by arylsulfatase A and cathepsin D Western blotting confirmed the specificity of the antibody fragment for lysosomal proteins. Application of the antibody fragment in immunohistochemistry of human brain slices from nonaffected patients showed strong neuronal immunoreactivity, which was not observed in cortical sections of an ML II patient. Finally, in brain extracts of a novel GlcNAc-1-phosphotransferase knock-in mouse no Man6P-containing proteins were detectable. Thus, the single-chain antibody fragment against Man6P was demonstrated to allow the specific, rapid, and convenient detection of Man6P-containing proteins and facilitates the diagnosis of ML II and ML III. PMID:20472886

  12. Regulation of Glucose Metabolism and Cell Wall Synthesis in Avena Stem Segments by Gibberellic Acid 1

    PubMed Central

    Montague, Michael J.; Ikuma, Hiroshi

    1978-01-01

    Gibberellic acid (GA) stimulated both the elongation of Avena sativa stem segments and increased synthesis of cell wall material. The effects of GA on glucose metabolism, as related to cell wall synthesis, have been investigated in order to find specific events regulated by GA. GA caused a decline in the levels of glucose, glucose 6-phosphate, and fructose 6-phosphate if exogenous sugar was not supplied to the segments, whereas the hormone caused no change in the levels of glucose 6-phosphate, fructose 6-phosphate, UDP-glucose, or the adenylate energy charge if the segments were incubated in 0.1 m glucose. No GA-induced change could be demonstrated in the activities of hexokinase, phosphoglucomutase, UDP-glucose pyrophosphorylase, or polysaccharide synthetases using UDP-glucose, UDP-galactose, UDP-xylose, and UDP-arabinose as substrates. GA stimulated the activity of GDP-glucose-dependent β-glucan synthetase by 2- to 4-fold over the control. When glucan synthetase was assayed using UDP-glucose as substrate, only β-1,3-linked glucan was synthesized in vitro, whereas with GDP-glucose, only β-1,4-linked glucan was synthesized. These results suggest that one part of the mechanism by which GA stimulates cell wall synthesis concurrently with elongation in Avena stem segments may be through a stimulation of cell wall polysaccharide synthetase activity. PMID:16660524

  13. Trehalose-6-phosphate synthesis controls yeast gluconeogenesis downstream and independent of SNF1.

    PubMed

    Deroover, Sofie; Ghillebert, Ruben; Broeckx, Tom; Winderickx, Joris; Rolland, Filip

    2016-06-01

    Trehalose-6-P (T6P), an intermediate of trehalose biosynthesis, was identified as an important regulator of yeast sugar metabolism and signaling. tps1Δ mutants, deficient in T6P synthesis (TPS), are unable to grow on rapidly fermentable medium with uncontrolled influx in glycolysis, depletion of ATP and accumulation of sugar phosphates. However, the exact molecular mechanisms involved are not fully understood. We show that SNF1 deletion restores the tps1Δ growth defect on glucose, suggesting that lack of TPS hampers inactivation of SNF1 or SNF1-regulated processes. In addition to alternative, non-fermentable carbon metabolism, SNF1 controls two major processes: respiration and gluconeogenesis. The tps1Δ defect appears to be specifically associated with deficient inhibition of gluconeogenesis, indicating more downstream effects. Consistently, Snf1 dephosphorylation and inactivation on glucose medium are not affected, as confirmed with an in vivo Snf1 activity reporter. Detailed analysis shows that gluconeogenic Pck1 and Fbp1 expression, protein levels and activity are not repressed upon glucose addition to tps1Δ cells, suggesting a link between the metabolic defect and persistent gluconeogenesis. While SNF1 is essential for induction of gluconeogenesis, T6P/TPS is required for inactivation of gluconeogenesis in the presence of glucose, downstream and independent of SNF1 activity and the Cat8 and Sip4 transcription factors.

  14. Trehalose-6-phosphate synthesis controls yeast gluconeogenesis downstream and independent of SNF1.

    PubMed

    Deroover, Sofie; Ghillebert, Ruben; Broeckx, Tom; Winderickx, Joris; Rolland, Filip

    2016-06-01

    Trehalose-6-P (T6P), an intermediate of trehalose biosynthesis, was identified as an important regulator of yeast sugar metabolism and signaling. tps1Δ mutants, deficient in T6P synthesis (TPS), are unable to grow on rapidly fermentable medium with uncontrolled influx in glycolysis, depletion of ATP and accumulation of sugar phosphates. However, the exact molecular mechanisms involved are not fully understood. We show that SNF1 deletion restores the tps1Δ growth defect on glucose, suggesting that lack of TPS hampers inactivation of SNF1 or SNF1-regulated processes. In addition to alternative, non-fermentable carbon metabolism, SNF1 controls two major processes: respiration and gluconeogenesis. The tps1Δ defect appears to be specifically associated with deficient inhibition of gluconeogenesis, indicating more downstream effects. Consistently, Snf1 dephosphorylation and inactivation on glucose medium are not affected, as confirmed with an in vivo Snf1 activity reporter. Detailed analysis shows that gluconeogenic Pck1 and Fbp1 expression, protein levels and activity are not repressed upon glucose addition to tps1Δ cells, suggesting a link between the metabolic defect and persistent gluconeogenesis. While SNF1 is essential for induction of gluconeogenesis, T6P/TPS is required for inactivation of gluconeogenesis in the presence of glucose, downstream and independent of SNF1 activity and the Cat8 and Sip4 transcription factors. PMID:27189362

  15. Intracellular movement of two mannose 6-phosphate receptors: return to the Golgi apparatus.

    PubMed

    Duncan, J R; Kornfeld, S

    1988-03-01

    We have used Chinese hamster ovary (CHO) cells and a murine lymphoma cell line to study the recycling of the 215-kD and the 46-kD mannose 6-phosphate receptors to various regions of the Golgi to determine the site where the receptors first encounter newly synthesized lysosomal enzymes. For assessing return to the trans-most Golgi compartments containing sialyltransferase (trans-cisternae and trans-Golgi network), the oligosaccharides of receptor molecules on the cell surface were labeled with [3H]galactose at 4 degrees C. Upon warming to 37 degrees C, the [3H]galactose residues on both receptors were substituted with sialic acid with a t1/2 approximately 3 hrs. Other glycoproteins acquired sialic acid at least 8-10 times slower. Return of the receptors to the trans-Golgi cisternae containing galactosyltransferase could not be detected. Return to the cis/middle Golgi cisternae containing alpha-mannosidase I was measured by adding deoxymannojirimycin, a mannosidase I inhibitor, during the initial posttranslational passage of [3H]mannose-labeled glycoproteins through the Golgi, thereby preserving oligosaccharides which would be substrates for alpha-mannosidase I. After removal of the inhibitor, return to the early Golgi with subsequent passage through the Golgi complex was measured by determining the conversion of the oligosaccharides from high mannose to complex-type units. This conversion was very slow for the receptors and other glycoproteins (t1/2 approximately 20 h). Exposure of the receptors and other glycoproteins to the dMM-sensitive alpha-mannosidase without movement through the Golgi apparatus was determined by measuring the loss of mannose residues from these proteins. This loss was also slow. These results indicate that both Man-6-P receptors routinely return to the Golgi compartment which contains sialyltransferase and recycle through other regions of the Golgi region less frequently. We infer that the trans-Golgi network is the major site for lysosomal

  16. Convenient preparative synthesis of ( sup 14 C)trehalose from ( sup 14 C)glucose by intact Escherichia coli cells

    SciTech Connect

    Brand, B.; Boos, W. )

    1989-09-01

    At high osmolarity, Escherichia coli synthesizes trehalose intracellularly, irrespective of the nature of the carbon source. Synthesis proceeds via the transfer of UDP-glucose to glucose 6-phosphate, yielding trehalose 6-phosphate, followed by its dephosphorylation to trehalose. This reaction was exploited to preparatively synthesize ({sup 14}C)trehalose from exogenous ({sup 14}C)glucose by using intact bacteria of a mutant (DF214) that could not metabolize glucose. The total yield of radiochemically pure trehalose from glucose was routinely more than 50%.

  17. Protein preparation and preliminary X-ray crystallographic analysis of a putative glucosamine 6-phosphate deaminase from Streptococcus mutants

    SciTech Connect

    Hu, Guan-Jing; Li, Lan-Fen; Li, Dan; Liu, Cong; Wei, Shi-Cheng; Liang, Yu-He Su, Xiao-Dong

    2007-09-01

    A glucosamine 6-phosphate deaminase homologue from S. mutans was expressed, purified and crystallized. Diffraction data have been collected to 2.4 Å resolution. The SMU.636 protein from Streptococcus mutans is a putative glucosamine 6-phosphate deaminase with 233 residues. The smu.636 gene was PCR-amplified from S. mutans genomic DNA and cloned into the expression vector pET-28a(+). The resultant His-tagged fusion protein was expressed in Escherichia coli and purified to homogeneity in two steps. Crystals of the fusion protein were obtained by the hanging-drop vapour-diffusion method. The crystals diffracted to 2.4 Å resolution and belong to space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 53.83, b = 82.13, c = 134.70 Å.

  18. [Experimental Evaluation of Radioprotective Efficacy of Synthetic Genistein on Criteria of Glutathione System and Lipid Peroxidation in Erythrocytes of Peripheral Blood in Irradiated Rats].

    PubMed

    Grebenyuk, A N; Tarumov, R A; Basharin, V A; Kovtun, V U

    2015-01-01

    The study was aimed to evaluate experimentally the radioprotective effectiveness of synthetic genistein in terms of the glutathione system and lipid peroxidation in erythrocytes of irradiated rats. The animals were exposed to single acute X-ray irradiation at a dose of 6 Gy. Genistein was administered intraperitoneally at 200 mg/kg 1 hour before radiation exposure. The irradiation caused the initiation of lipid peroxidation in the background depletion of reduced glutathione. Decrease by 25% in the number of malondialdehyde in the rats treated with genistein was registered 5 min after irradiation compared with the control. It is established thatl day after irradiation the level of reduced glutathione in the rats treated with genistein was 26% higher. However, intraperitoneal administration of genistein did not cause statistically significant changes in the activity of glutathione reductase, glutathione-S-transferase, or glucose-6-phosphate dehydrogenase during the whole period of observation. The results suggest that the radioprotective effect of synthetic genistein is implemented, along with other mechanisms, by stimulating the glutathione system and reducing the severity of lipid peroxidation. PMID:26863780

  19. Structure of the Trehalose-6-phosphate Phosphatase from Brugia malayi Reveals Key Design Principles for Anthelmintic Drugs

    PubMed Central

    Farelli, Jeremiah D.; Galvin, Brendan D.; Li, Zhiru; Liu, Chunliang; Aono, Miyuki; Garland, Megan; Hallett, Olivia E.; Causey, Thomas B.; Ali-Reynolds, Alana; Saltzberg, Daniel J.; Carlow, Clotilde K. S.; Dunaway-Mariano, Debra; Allen, Karen N.

    2014-01-01

    Parasitic nematodes are responsible for devastating illnesses that plague many of the world's poorest populations indigenous to the tropical areas of developing nations. Among these diseases is lymphatic filariasis, a major cause of permanent and long-term disability. Proteins essential to nematodes that do not have mammalian counterparts represent targets for therapeutic inhibitor discovery. One promising target is trehalose-6-phosphate phosphatase (T6PP) from Brugia malayi. In the model nematode Caenorhabditis elegans, T6PP is essential for survival due to the toxic effect(s) of the accumulation of trehalose 6-phosphate. T6PP has also been shown to be essential in Mycobacterium tuberculosis. We determined the X-ray crystal structure of T6PP from B. malayi. The protein structure revealed a stabilizing N-terminal MIT-like domain and a catalytic C-terminal C2B-type HAD phosphatase fold. Structure-guided mutagenesis, combined with kinetic analyses using a designed competitive inhibitor, trehalose 6-sulfate, identified five residues important for binding and catalysis. This structure-function analysis along with computational mapping provided the basis for the proposed model of the T6PP-trehalose 6-phosphate complex. The model indicates a substrate-binding mode wherein shape complementarity and van der Waals interactions drive recognition. The mode of binding is in sharp contrast to the homolog sucrose-6-phosphate phosphatase where extensive hydrogen-bond interactions are made to the substrate. Together these results suggest that high-affinity inhibitors will be bi-dentate, taking advantage of substrate-like binding to the phosphoryl-binding pocket while simultaneously utilizing non-native binding to the trehalose pocket. The conservation of the key residues that enforce the shape of the substrate pocket in T6PP enzymes suggest that development of broad-range anthelmintic and antibacterial therapeutics employing this platform may be possible. PMID:24992307

  20. Structure of the trehalose-6-phosphate phosphatase from Brugia malayi reveals key design principles for anthelmintic drugs.

    PubMed

    Farelli, Jeremiah D; Galvin, Brendan D; Li, Zhiru; Liu, Chunliang; Aono, Miyuki; Garland, Megan; Hallett, Olivia E; Causey, Thomas B; Ali-Reynolds, Alana; Saltzberg, Daniel J; Carlow, Clotilde K S; Dunaway-Mariano, Debra; Allen, Karen N

    2014-07-01

    Parasitic nematodes are responsible for devastating illnesses that plague many of the world's poorest populations indigenous to the tropical areas of developing nations. Among these diseases is lymphatic filariasis, a major cause of permanent and long-term disability. Proteins essential to nematodes that do not have mammalian counterparts represent targets for therapeutic inhibitor discovery. One promising target is trehalose-6-phosphate phosphatase (T6PP) from Brugia malayi. In the model nematode Caenorhabditis elegans, T6PP is essential for survival due to the toxic effect(s) of the accumulation of trehalose 6-phosphate. T6PP has also been shown to be essential in Mycobacterium tuberculosis. We determined the X-ray crystal structure of T6PP from B. malayi. The protein structure revealed a stabilizing N-terminal MIT-like domain and a catalytic C-terminal C2B-type HAD phosphatase fold. Structure-guided mutagenesis, combined with kinetic analyses using a designed competitive inhibitor, trehalose 6-sulfate, identified five residues important for binding and catalysis. This structure-function analysis along with computational mapping provided the basis for the proposed model of the T6PP-trehalose 6-phosphate complex. The model indicates a substrate-binding mode wherein shape complementarity and van der Waals interactions drive recognition. The mode of binding is in sharp contrast to the homolog sucrose-6-phosphate phosphatase where extensive hydrogen-bond interactions are made to the substrate. Together these results suggest that high-affinity inhibitors will be bi-dentate, taking advantage of substrate-like binding to the phosphoryl-binding pocket while simultaneously utilizing non-native binding to the trehalose pocket. The conservation of the key residues that enforce the shape of the substrate pocket in T6PP enzymes suggest that development of broad-range anthelmintic and antibacterial therapeutics employing this platform may be possible.

  1. The overexpressed human 46-kDa mannose 6-phosphate receptor mediates endocytosis and sorting of. beta. -glucuronidase

    SciTech Connect

    Watanabe, H.; Grubb, J.H.; Sly, W.S. )

    1990-10-01

    The authors studied the function of the human small (46-kDa) mannose 6-phosphate receptor (SMPR) in transfected mouse L cells that do not express the larger insulin-like growth factor II/mannose 6-phosphate receptor. Cells overexpressing human SMPR were studied for enzyme binding to cell surface receptors, for binding to intracellular receptors in permeabilized cells, and for receptor-mediated endocytosis of recombinant human {beta}-glucuronidase. Specific binding to human SMPR in permeabilized cells showed a pH optimum between pH 6.0 and pH 6.5. Binding was significant in the present of EDTA but was enhanced by added divalent cations. Up to 2.3{percent} of the total functional receptor could be detected on the cell surface by enzyme binding. They present experiments showing that at very high levels of overexpression, and at pH 6.5, human SMPR mediated the endocytosis of {beta}-glucuronidase. At pH 7.5, the rate of endocytosis was only 14{percent} the rate seen at pH 6.5. Cells overexpressing human SMPR also showed reduced secretion of newly synthesized {beta}-glucuronidase when compared to cells transfected with vector only, suggesting that overexpressed human SMPR can participate in sorting of newly synthesized {beta}-glucuronidase and partially correct the sorting defect in mouse L cells that do not express the insulin-like growth factor II/mannose 6-phosphate receptor.

  2. Substrate specificity of a galactose 6-phosphate isomerase from Lactococcus lactis that produces d-allose from d-psicose.

    PubMed

    Park, Ha-Young; Park, Chang-Su; Kim, Hye-Jung; Oh, Deok-Kun

    2007-10-15

    We purified recombinant galactose 6-phosphate isomerase (LacAB) from Lactococcus lactis using HiTrap Q HP and Phenyl-Sepharose columns. The purified LacAB had a final specific activity of 1.79units/mg to produce d-allose. The molecular mass of native galactose 6-phosphate isomerase was estimated at 135.5kDa using Sephacryl S-300 gel filtration, and the enzyme exists as a hetero-octamer of LacA and LacB subunits. The activity of galactose 6-phosphate isomerase was maximal at pH 7.0 and 30 degrees C, and enzyme activity was independent of metal ions. When 100g/L of d-psicose was used as the substrate, 25g/L of d-allose and 13g/L of d-altrose were simultaneously produced at pH 7.0 and 30 degrees C after 12h of incubation. The enzyme had broad specificity for various aldoses and ketoses. The interconversion of sugars with the same configuration except at the C2 position was driven by using a large amount of enzyme in extended reactions. The interconversion occurred via two isomerization reactions, i.e., the interconversion of d-allose<-->d-psicose<-->d-altrose, and d-allose to d-psicose reaction was faster than d-altrose to d-psicose reaction.

  3. Developmental differences between cation-independent and cation-dependent mannose-6-phosphate receptors in rat brain at perinatal stages.

    PubMed

    Romano, P S; Carvelli, L; López, A C; Jofré, G; Sartor, T; Sosa, M A

    2005-08-01

    Mannose-6-phosphate receptors (MPRs) play a role in the selective transport of macromolecules bearing mannose-6-phosphate residue to lysosomes. To date, two types of MPRs have been described in most of cells and tissues: the cation-dependent (CD-MPR) and cation-independent mannose-6-phosphate receptor (CI-MPR). In order to elucidate their possible role in the central nervous system, the expression and binding properties of both MPRs were studied in rat brain along perinatal development. It was observed that the expression of CI-MPR decreases progressively from fetuses to adults, while the CD-MPR increases around the 10th day of birth, and maintains these values up to adulthood. Binding assays showed differences in the Bmax and KD values between the ages studied, and they did not correlate with the expression levels of both MPRs. Variations in lysosomal enzyme activities and expression of phosphomannosylated ligands during development correlated more with CD-MPR than with CI-MPR expression. These results suggest that both receptors play a different role in rat brain during perinatal development, being CD-MPR mostly involved in lysosome maturation.

  4. Oxidative Hemolysis of Erythrocytes

    ERIC Educational Resources Information Center

    Wlodek, Lidia; Kusior, Dorota

    2006-01-01

    This exercise for students will allow them to simultaneously observe lipid peroxidation and consequent hemolysis of rat erythrocytes and the effect of sodium azide, a catalase inhibitor, on these processes. It will also demonstrate a protective action of antioxidants, the therapeutically used N-acetylcysteine and albumins present in plasma.

  5. Monitoring the Dynamics of Monomer Exchange Using Electrospray Mass Spectrometry: The Case of the Dimeric Glucosamine-6-Phosphate Synthase

    NASA Astrophysics Data System (ADS)

    Chevreux, Guillaume; Atmanene, Cédric; Lopez, Philippe; Ouazzani, Jamal; Van Dorsselaer, Alain; Badet, Bernard; Badet-Denisot, Marie-Ange; Sanglier-Cianférani, Sarah

    2011-03-01

    Escherichia coli glucosamine-6-phosphate synthase (GlmS) is a dimeric enzyme from the glutamine-dependent amidotransferases family, which catalyses the conversion of D-fructose-6-phosphate (Fru6P) and glutamine (Gln) into D-glucosamine-6-phosphate (GlcN6P) and glutamate, respectively. Extensive X-ray crystallography investigations have been reported, highlighting the importance of the dimeric association to form the sugar active site as well as significant conformational changes of the protein upon substrate and product binding. In the present work, an approach based on time-resolved noncovalent mass spectrometry has been developed to study the dynamics of GlmS subunit exchange. Using 14N versus 15N labeled proteins, the kinetics of GlmS subunit exchange was monitored with the wild-type enzyme in the presence of different substrates and products as well as with the protein bearing a key amino acid mutation specially designed to weaken the dimer interface. Determination of rate constants of subunit exchange revealed important modifications of the protein dynamics: while glutamine, glutamate, and K603A mutation accelerates subunit exchange, Fru6P and GlcN6P totally prevent it. These results are described in light of the available structural information, providing additional useful data for both the characterization of GlmS catalytic process and the design of new GlmS inhibitors. Finally, time-resolved noncovalent MS can be proposed as an additional biophysical technique for real-time monitoring of protein dynamics.

  6. Structural Diversity Within the Mononuclear and Binuclear Active Sites of N-Acetyl-D-Glucosamine-6-Phosphate Deacetylase

    SciTech Connect

    Hall,R.; Brown, S.; Fedorov, A.; Fedorov, E.; Xu, C.; Babbitt, P.; Almo, S.; Raushel, F.

    2007-01-01

    NagA catalyzes the hydrolysis of N-acetyl-D-glucosamine-6-phosphate to D-glucosamine-6-phosphate and acetate. X-ray crystal structures of NagA from Escherichia coli were determined to establish the number and ligation scheme for the binding of zinc to the active site and to elucidate the molecular interactions between the protein and substrate. The three-dimensional structures of the apo-NagA, Zn-NagA, and the D273N mutant enzyme in the presence of a tight-binding N-methylhydroxyphosphinyl-D-glucosamine-6-phosphate inhibitor were determined. The structure of the Zn-NagA confirms that this enzyme binds a single divalent cation at the beta-position in the active site via ligation to Glu-131, His-195, and His-216. A water molecule completes the ligation shell, which is also in position to be hydrogen bonded to Asp-273. In the structure of NagA bound to the tight binding inhibitor that mimics the tetrahedral intermediate, the methyl phosphonate moiety has displaced the hydrolytic water molecule and is directly coordinated to the zinc within the active site. The side chain of Asp-273 is positioned to activate the hydrolytic water molecule via general base catalysis and to deliver this proton to the amino group upon cleavage of the amide bond of the substrate. His-143 is positioned to help polarize the carbonyl group of the substrate in conjunction with Lewis acid catalysis by the bound zinc. The inhibitor is bound in the {alpha}-configuration at the anomeric carbon through a hydrogen bonding interaction of the hydroxyl group at C-1 with the side chain of His-251. The phosphate group of the inhibitor attached to the hydroxyl at C-6 is ion paired with Arg-227 from the adjacent subunit. NagA from Thermotoga maritima was shown to require a single divalent cation for full catalytic activity.

  7. Structures of trehalose-6-phosphate phosphatase from pathogenic fungi reveal the mechanisms of substrate recognition and catalysis.

    PubMed

    Miao, Yi; Tenor, Jennifer L; Toffaletti, Dena L; Washington, Erica J; Liu, Jiuyu; Shadrick, William R; Schumacher, Maria A; Lee, Richard E; Perfect, John R; Brennan, Richard G

    2016-06-28

    Trehalose is a disaccharide essential for the survival and virulence of pathogenic fungi. The biosynthesis of trehalose requires trehalose-6-phosphate synthase, Tps1, and trehalose-6-phosphate phosphatase, Tps2. Here, we report the structures of the N-terminal domain of Tps2 (Tps2NTD) from Candida albicans, a transition-state complex of the Tps2 C-terminal trehalose-6-phosphate phosphatase domain (Tps2PD) bound to BeF3 and trehalose, and catalytically dead Tps2PD(D24N) from Cryptococcus neoformans bound to trehalose-6-phosphate (T6P). The Tps2NTD closely resembles the structure of Tps1 but lacks any catalytic activity. The Tps2PD-BeF3-trehalose and Tps2PD(D24N)-T6P complex structures reveal a "closed" conformation that is effected by extensive interactions between each trehalose hydroxyl group and residues of the cap and core domains of the protein, thereby providing exquisite substrate specificity. Disruption of any of the direct substrate-protein residue interactions leads to significant or complete loss of phosphatase activity. Notably, the Tps2PD-BeF3-trehalose complex structure captures an aspartyl-BeF3 covalent adduct, which closely mimics the proposed aspartyl-phosphate intermediate of the phosphatase catalytic cycle. Structures of substrate-free Tps2PD reveal an "open" conformation whereby the cap and core domains separate and visualize the striking conformational changes effected by substrate binding and product release and the role of two hinge regions centered at approximately residues 102-103 and 184-188. Significantly, tps2Δ, tps2NTDΔ, and tps2D705N strains are unable to grow at elevated temperatures. Combined, these studies provide a deeper understanding of the substrate recognition and catalytic mechanism of Tps2 and provide a structural basis for the future design of novel antifungal compounds against a target found in three major fungal pathogens. PMID:27307435

  8. Structures of trehalose-6-phosphate phosphatase from pathogenic fungi reveal the mechanisms of substrate recognition and catalysis

    PubMed Central

    Miao, Yi; Tenor, Jennifer L.; Toffaletti, Dena L.; Washington, Erica J.; Liu, Jiuyu; Shadrick, William R.; Schumacher, Maria A.; Lee, Richard E.; Perfect, John R.; Brennan, Richard G.

    2016-01-01

    Trehalose is a disaccharide essential for the survival and virulence of pathogenic fungi. The biosynthesis of trehalose requires trehalose-6-phosphate synthase, Tps1, and trehalose-6-phosphate phosphatase, Tps2. Here, we report the structures of the N-terminal domain of Tps2 (Tps2NTD) from Candida albicans, a transition-state complex of the Tps2 C-terminal trehalose-6-phosphate phosphatase domain (Tps2PD) bound to BeF3 and trehalose, and catalytically dead Tps2PD(D24N) from Cryptococcus neoformans bound to trehalose-6-phosphate (T6P). The Tps2NTD closely resembles the structure of Tps1 but lacks any catalytic activity. The Tps2PD–BeF3–trehalose and Tps2PD(D24N)–T6P complex structures reveal a “closed” conformation that is effected by extensive interactions between each trehalose hydroxyl group and residues of the cap and core domains of the protein, thereby providing exquisite substrate specificity. Disruption of any of the direct substrate–protein residue interactions leads to significant or complete loss of phosphatase activity. Notably, the Tps2PD–BeF3–trehalose complex structure captures an aspartyl-BeF3 covalent adduct, which closely mimics the proposed aspartyl-phosphate intermediate of the phosphatase catalytic cycle. Structures of substrate-free Tps2PD reveal an “open” conformation whereby the cap and core domains separate and visualize the striking conformational changes effected by substrate binding and product release and the role of two hinge regions centered at approximately residues 102–103 and 184–188. Significantly, tps2Δ, tps2NTDΔ, and tps2D705N strains are unable to grow at elevated temperatures. Combined, these studies provide a deeper understanding of the substrate recognition and catalytic mechanism of Tps2 and provide a structural basis for the future design of novel antifungal compounds against a target found in three major fungal pathogens. PMID:27307435

  9. Asymmetric phosphorylation through catalytic P(III) phosphoramidite transfer: Enantioselective synthesis of d-myo-inositol-6-phosphate

    PubMed Central

    Jordan, Peter A.; Kayser-Bricker, Katherine J.; Miller, Scott J.

    2010-01-01

    Despite the ubiquitous use of phosphoramidite chemistry in the synthesis of biophosphates, catalytic asymmetric phosphoramidite transfer remains largely unexplored for phosphate ester synthesis. We have discovered that a tetrazole-functionalized peptide, in the presence of 10-Å molecular sieves, functions as an enantioselective catalyst for phosphite transfer. This chemistry in turn has been used as the key step in a streamlined synthesis of myo-inositol-6-phosphate. Mechanistic insights implicate phosphate as a directing group for a highly selective kinetic resolution of a protected inositol monophosphate. This work represents a distinct and efficient method for the selective catalytic phosphorylation of natural products. PMID:20439750

  10. Molecular Docking Studies of Catechin and Its Derivatives as Anti-bacterial Inhibitor for Glucosamine-6-Phosphate Synthase

    NASA Astrophysics Data System (ADS)

    Fikrika, H.; Ambarsari, L.; Sumaryada, T.

    2016-01-01

    Molecular docking simulation of catechin and its derivatives on Glucosamine-6- Phosphate Synthase (GlmS) has been performed in this research. GlmS inhibition by a particular ligand will suppress the production of bacterial cell wall and significantly reduce the population of invading bacteria. In this study, catechin derivatives i.e epicatechin, galloatechin and epigalloatechin were found to have stronger binding affinities as compared to natural ligand of GlmS, Fructose-6-Phosphate (F6P). Those three ligands were docked on the same pocket in GlmS target as F6P, with 70% binding sites similarity. Based on the docking results, gallocatechin turns out to be the most potent ligand for anti-bacterial agent with ΔG= -8.00 kcal/mol. The docking between GlmS and catechin derivatives are characterized by a constant present of a strong hydrogen bond between functional group O3 and Ser-349. This hydrogen bond most likely plays a significant role in the docking mechanism and binding modes selection. The surprising result is catechin itself exhibited a quite strong binding with GlmS (ΔG= -7.80 kcal.mol), but docked on a completely different pocket compared to other ligands. This results suggest that catechin might still have a curing effect but with a completely different pathway and mechanism as compared to its derivatives.

  11. Lysosome sorting of β-glucocerebrosidase by LIMP-2 is targeted by the mannose 6-phosphate receptor

    PubMed Central

    Zhao, Yuguang; Ren, Jingshan; Padilla-Parra, Sergi; Fry, Elizabeth E.; Stuart, David I.

    2014-01-01

    The integral membrane protein LIMP-2 has been a paradigm for mannose 6-phosphate receptor (MPR) independent lysosomal targeting, binding to β-glucocerebrosidase (β-GCase) and directing it to the lysosome, before dissociating in the late-endosomal/lysosomal compartments. Here we report structural results illuminating how LIMP-2 binds and releases β-GCase according to changes in pH, via a histidine trigger, and suggesting that LIMP-2 localizes the ceramide portion of the substrate adjacent to the β-GCase catalytic site. Remarkably, we find that LIMP-2 bears P-Man9GlcNAc2 covalently attached to residue N325, and that it binds MPR, via mannose 6-phosphate, with a similar affinity to that observed between LIMP-2 and β-GCase. The binding sites for β-GCase and the MPR are functionally separate, so that a stable ternary complex can be formed. By fluorescence lifetime imaging microscopy, we also demonstrate that LIMP-2 interacts with MPR in living cells. These results revise the accepted view of LIMP-2–β-GCase lysosomal targeting. PMID:25027712

  12. Synthesis of a truncated Mr 46,000 mannose 6-phosphate receptor that is secreted and retains ligand binding.

    PubMed

    Wendland, M; Hille, A; Nagel, G; Waheed, A; von Figura, K; Pohlmann, R

    1989-05-15

    The Mr 46,000 mannose 6-phosphate receptor is an integral membrane protein with its ligand-binding site in the ectoplasmic domain. By site-directed mutagenesis, a stop codon was introduced in the receptor cDNA at the border between the ectoplasmic and membrane-spanning domain. The truncated receptor was expressed in three different systems, Xenopus oocytes, COS cells and BHK-21 cells. In all three systems the truncated receptor behaved as a soluble protein. In oocytes only small amounts of the truncated receptor were secreted within 48 h after synthesis. Accumulation of endoglucosaminidase H-sensitive forms of the truncated receptor in oocytes suggested that exit from the endoplasmic reticulum was slowed down. In COS and BHK-21 cells, the truncated receptor was secreted and, as for wild-type receptor, most of the N-linked oligosaccharides were processed to complex forms. Both the intracellularly-retained (oocytes) and the secreted (COS and BHK-21 cells) truncated receptors bound to phosphomannan-Sepharose in a mannose-6-phosphate-dependent manner. Using chemical cross-linking, the truncated receptor was shown to be secreted as a homodimer. PMID:2549951

  13. Synthesis of a truncated Mr 46,000 mannose 6-phosphate receptor that is secreted and retains ligand binding.

    PubMed Central

    Wendland, M; Hille, A; Nagel, G; Waheed, A; von Figura, K; Pohlmann, R

    1989-01-01

    The Mr 46,000 mannose 6-phosphate receptor is an integral membrane protein with its ligand-binding site in the ectoplasmic domain. By site-directed mutagenesis, a stop codon was introduced in the receptor cDNA at the border between the ectoplasmic and membrane-spanning domain. The truncated receptor was expressed in three different systems, Xenopus oocytes, COS cells and BHK-21 cells. In all three systems the truncated receptor behaved as a soluble protein. In oocytes only small amounts of the truncated receptor were secreted within 48 h after synthesis. Accumulation of endoglucosaminidase H-sensitive forms of the truncated receptor in oocytes suggested that exit from the endoplasmic reticulum was slowed down. In COS and BHK-21 cells, the truncated receptor was secreted and, as for wild-type receptor, most of the N-linked oligosaccharides were processed to complex forms. Both the intracellularly-retained (oocytes) and the secreted (COS and BHK-21 cells) truncated receptors bound to phosphomannan-Sepharose in a mannose-6-phosphate-dependent manner. Using chemical cross-linking, the truncated receptor was shown to be secreted as a homodimer. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. PMID:2549951

  14. Changes in phosphomannosyl ligands correlate with cation-dependent mannose-6-phosphate receptors in rat liver during perinatal development.

    PubMed

    Romano, Patricia S; Jofré, Guillermo; Carvelli, Lorena; López, Ana C; Sartor, Tirso; Sosa, Miguel A

    2006-06-01

    The co-existence of two mannose-6-phosphate receptors (CD-MPR and CI-MPR) in most cell types is still a dilemma to be resolved. In this study, some parameters were measured to explore lysosomal apparatus evolution in rat liver during perinatal development, and establish a possible involvement of CD- and/or CI-MPR in lysosome maturation. Activity of four acid hydrolases was measured in the whole organ at different ages and it was found that N-acetyl-beta-D-glucosaminidase (NAG), beta-galactosidase, and beta-glucuronidase change during development, reaching a peak at the 10th day after birth. These results correlated with the expression and binding properties of CD-MPR previously reported. We also used a method that recognizes phosphomannosylated ligands by using purified biotinylated CI-MPR as a probe, and found that the highest concentrations of ligands also appear around the 10th day. Binding assays were also carried out, incubating endogenous NAG from 10-day-old and adult rats with membranes from their respective ages, and the results indicated that cation-dependent mannose-6-phosphate receptor (CD-MPR) has more impact on trafficking of the enzyme at the 10th day after birth. We concluded that lysosome maturation in the rat liver occurs around the 10th day after birth, and that the CD-MPR may participate in that event.

  15. Enhanced suicidal erythrocyte death in mice carrying a loss-of-function mutation of the adenomatous polyposis coli gene.

    PubMed

    Qadri, Syed M; Mahmud, Hasan; Lang, Elisabeth; Gu, Shuchen; Bobbala, Diwakar; Zelenak, Christine; Jilani, Kashif; Siegfried, Alexandra; Föller, Michael; Lang, Florian

    2012-05-01

    Loss-of-function mutations in human adenomatous polyposis coli (APC) lead to multiple colonic adenomatous polyps eventually resulting in colonic carcinoma. Similarly, heterozygous mice carrying defective APC (apc(Min/+)) suffer from intestinal tumours. The animals further suffer from anaemia, which in theory could result from accelerated eryptosis, a suicidal erythrocyte death triggered by enhanced cytosolic Ca(2+) activity and characterized by cell membrane scrambling and cell shrinkage. To explore, whether APC-deficiency enhances eryptosis, we estimated cell membrane scrambling from annexin V binding, cell size from forward scatter and cytosolic ATP utilizing luciferin-luciferase in isolated erythrocytes from apc(Min/+) mice and wild-type mice (apc(+/+)). Clearance of circulating erythrocytes was estimated by carboxyfluorescein-diacetate-succinimidyl-ester labelling. As a result, apc(Min/+) mice were anaemic despite reticulocytosis. Cytosolic ATP was significantly lower and annexin V binding significantly higher in apc(Min/+) erythrocytes than in apc(+/+) erythrocytes. Glucose depletion enhanced annexin V binding, an effect significantly more pronounced in apc(Min/+) erythrocytes than in apc(+/+) erythrocytes. Extracellular Ca(2+) removal or inhibition of Ca(2+) entry with amiloride (1 mM) blunted the increase but did not abrogate the genotype differences of annexin V binding following glucose depletion. Stimulation of Ca(2+) -entry by treatment with Ca(2+) -ionophore ionomycin (10 μM) increased annexin V binding, an effect again significantly more pronounced in apc(Min/+) erythrocytes than in apc(+/+) erythrocytes. Following retrieval and injection into the circulation of the same mice, apc(Min/+) erythrocytes were more rapidly cleared from circulating blood than apc(+/+) erythrocytes. Most labelled erythrocytes were trapped in the spleen, which was significantly enlarged in apc(Min/+) mice. The observations point to accelerated eryptosis and subsequent

  16. A Cationic-Independent Mannose 6-Phosphate Receptor Inhibitor (PXS64) Ameliorates Kidney Fibrosis by Inhibiting Activation of Transforming Growth Factor-β1

    PubMed Central

    Zhang, Jie; Wong, Muh Geot; Wong, May; Gross, Simon; Chen, Jason; Pollock, Carol; Saad, Sonia

    2015-01-01

    The activity of transforming growth factor-β1 (TGF-β1) is regulated by its conversion from the latent to the active form. We have previously shown that the conversion is at least in part mediated by the cationic-independent mannose 6-phosphate receptor (CI-M6PR), as the CI-M6PR inhibitor, PXS-25 has anti-fibrotic properties in human kidney tubular (HK-2) cells under high glucose conditions. However, its clinical use is limited by low bioavailability. Our aim was to determine the effects of PXS64, a pro-drug of PXS25, in in vitro and in vivo models of renal fibrosis. HK-2 cells were exposed to latent TGFβ1+/- PXS64 for 48 hours. The mRNA and protein levels of pro-fibrotic and pro-inflammatory markers were determined. A 7 day unilateral ureteric obstruction (UUO) model was used and the following experimental groups were studied: (i) Sham operated, (ii) UUO, (iii) UUO + telmisartan (iv) UUO + PSX64. HK-2 cells exposed to PXS64 reduced TGFβ mediated effects on collagen IV, fibronectin, macrophage chemotactic protein-1 (MCP-1) and phospho-smad2 protein expression, consistent with inhibition of the conversion of latent to active TGF-β1. PXS 64 treated UUO mice had a lower tubulointerstitial fibrosis index, collagen IV and fibronectin protein and mRNA expression when compared to untreated UUO mice. In addition, these animals had lower MCP-1 mRNA expression, reduced inflammarory cell infiltrate, as indicated by fewer CD45, F4/80 positive cells, and reduced phospho-Smad2 protein expression when compared to untreated UUO animals. Our data demonstrates that PSX64 is an effective anti-fibrotic agent by inhibiting the activation of latent TGF-β1. PMID:25658916

  17. Trehalose metabolism in the blue crab Callinectes sapidus: isolation of multiple structural cDNA isoforms of trehalose-6-phosphate synthase and their expression in muscles.

    PubMed

    Shi, Q; Chung, J Sook

    2014-02-15

    Adult blue crab Callinectes sapidus exhibit behavioral and ecological dimorphisms: females migrating from the low salinity water to the high salinity area vs. males remaining in the same areas. The flesh basal muscle of the swimming paddle shows a dimorphic color pattern in that levator (Lev) and depressor (Dep) of females tend to be much darker than those of males, while both genders have the same light colored remoter (Rem) and promoter (Pro). The full-length cDNA sequence of four structural isoforms of trehalose-6-phosphate synthase (TPS) is isolated from chela muscles of an adult female, C. sapidus. Two isoforms of the C. sapidus TPS encode functional domains of TPS and trehalose-6-phosphorylase (TPP) in tandem as a fused gene product of Escherichia coli Ost A and Ost B. The other two isoforms contain only a single TPS domain. In both males and females, the darker (Lev+Dep) muscles exhibit greater amounts of trehalose, TPS and trehalase activities than the light colored (Rem+Pro). The fact that adult females show higher levels of trehalase activity in the basal muscles and of glucose in Lev+Dep than those of adult males suggests that there may be a metabolic dimorphism. Moreover, the involvement of trehalose in energy metabolism that was examined under the condition of strenuous swimming activity mimicked in adult females demonstrates the intrinsic trehalose metabolism in Lev+Dep, which subsequently results in hemolymphatic hyperglycemia and hyperlactemia. Our data support that trehalose serves as an additional carbohydrate source of hemolymphatic hyperglycemia in this species. Behavioral and ecological dimorphisms of C. sapidus adults may be supported by a functional dimorphism in energy metabolism.

  18. Glucose cycling in islets from healthy and diabetic rats

    SciTech Connect

    Khan, A.; Chandramouli, V.; Ostenson, C.G.; Loew, H.L.; Landau, B.R.; Efendic, S. )

    1990-04-01

    Pancreatic islets from healthy (control) and neonatally streptozocin-induced diabetic (STZ-D) rats, a model for non-insulin-dependent diabetes mellitus, were incubated with {sup 3}H{sub 2}O and 5.5 or 16.7 mM glucose. At 5.5 mM glucose, no detectable ({sup 3}H)glucose was formed. At 16.7 mM, 2.2 patom.islet-1.h-1 of {sup 3}H was incorporated into glucose by the control islets and 5.4 patom.islet-1.h-1 by STZ-D islets. About 75% of the {sup 3}H was bound to carbon-2 of the glucose. Glucose utilization was 35.3 pmol.islet-1.h-1 by the control and 19.0 pmol.islet-1.h-1 by the STZ-D islets. Therefore, 4.5% of the glucose-6-phosphate formed by the control islets and 15.7% by the STZ-D islets was dephosphorylated. This presumably occurred in the beta-cells of the islets catalyzed by glucose-6-phosphatase. An increased glucose cycling, i.e., glucose----glucose-6-phosphate----glucose, in islets of STZ-D rats may contribute to the decreased insulin secretion found in these animals.

  19. Plasma lipids profile and erythrocytes system in patients with coronary heart disease

    NASA Astrophysics Data System (ADS)

    Malinova, Lidia I.; Simonenko, Georgy V.; Tuchin, Valery V.; Denisova, Tatyana P.

    2006-08-01

    Erythrocytes system study can provide a framework for detailed exploration of blood cell-cell and cell-vessel wall interactions, one of the key patterns in blood and vascular pathophysiology. Our objective was to explore erythrocytes system in patients with stable angina pectoris II f.c. (Canadian classification). The participants (N = 56, age 40 - 55 years) without obesity, glucose tolerance violations, lipid lowering drugs treating, heart failure of II and more functional classes (NYHA), coronary episode at least 6 months before study were involved in the study. Blood samples were incubated with glucose solutions of increasing concentrations (from 2.5% to 20% with 2.5% step) during 60 mm (36° C). In prepared blood smears erythrocyte's sizes were studied. Plasma total cholesterol, triglyceride and glucose levels were also measured. Received data were approximated by polynomials of high degree, with after going first and second derivations. Erythrocytes system "behavior" was studied by means of phase pattern constructing. By lipids levels all the patient were divided into five groups: 1) patients with normal lipids levels, 2) patients with borderline total cholesterol level, 3) patients with isolated hypercholesterolemia, 4) patients with isolated hypertriglyceridemia and 5) patients with combined hyperlipidemia. Erythrocytes size lowering process was of set of "stages", which characteristics differ significantly (p > 0.05) in all five groups. Their rate and acceleration characteristics allow us to detect type of lipid profile in patients. Erythrocyte system disturbing by glucose concentration increase show to be most resistant in group of patients with isolated hypercholesterolemia.

  20. [Effects of La3+ on calcium binding to erythrocyte cytoskeleton].

    PubMed

    Kravtsov, G M; Postnov, Iu V

    1992-02-01

    When the whole erythrocytes were exposed to LaCl3, A--23187, ionomycin, orthovanadate and saponin, there was Ca2+ binding only following La3+ treatment of the cells. The binding was evident at a wide range (0.1 microM--1.OmM) of La3+ concentrations. Iodoacetamide-induced (incubation for 3 hours, 37 degrees C) decrease in erythrocyte ATP levels was found to result in a 3-fold reduction in Ca2+ binding to the cytoskeleton. La(3+)-induced Ca2+ binding enhanced the incorporation of 14C-glucose and/or its metabolites into the red cell skeleton. Thus, the detected new type of Ca2+ binding to the cytoskeleton of human and rat erythrocytes is likely to be due to the cumulative process: direct binding of La3+ to the outer surface of a membrane and the metal-induced trigger of nucleotide--dependent intracellular process.

  1. Studies on the role of goat heart galectin-1 as an erythrocyte membrane perturbing agent.

    PubMed

    Ashraf, Ghulam Md; Perveen, Asma; Zaidi, Syed Kashif; Tabrez, Shams; Kamal, Mohammad A; Banu, Naheed

    2015-01-01

    Galectins are β-galactoside binding lectins with a potential hemolytic role on erythrocyte membrane integrity and permeability. In the present study, goat heart galectin-1 (GHG-1) was purified and investigated for its hemolytic actions on erythrocyte membrane. When exposed to various saccharides, lactose and sucrose provided maximum protection against hemolysis, while glucose and galactose provided lesser protection against hemolysis. GHG-1 agglutinated erythrocytes were found to be significantly hemolyzed in comparison with unagglutinated erythrocytes. A concentration dependent rise in the hemolysis of trypsinized rabbit erythrocytes was observed in the presence of GHG-1. Similarly, a temperature dependent gradual increase in percent hemolysis was observed in GHG-1 agglutinated erythrocytes as compared to negligible hemolysis in unagglutinated cells. The hemolysis of GHG-1 treated erythrocytes showed a sharp rise with the increasing pH up to 7.5 which became constant till pH 9.5. The extent of erythrocyte hemolysis increased with the increase in the incubation period, with maximum hemolysis after 5 h of incubation. The results of this study establish the ability of galectins as a potential hemolytic agent of erythrocyte membrane, which in turn opens an interesting avenue in the field of proteomics and glycobiology.

  2. Inhibition of glucose metabolism by n-hexadecane in Cladosporium (Amorphotheca) resinae.

    PubMed Central

    Siporin, C; Cooney, J J

    1976-01-01

    When Cladosporium resinae is provided with n-hexadecane and glucose, n-hexadecane is used preferentially. Studies using [14C]glucose indicated that n-hexadecane did not inhibit glucose uptake but did retard oxidation of glucose to CO2 and assimilation of glucose carbon into trichloroacetic acid-insoluble material. Glucose could be recovered quantitatively from hydrocarbon-grown cells that had been transferred to glucose. Four enzymes that may be involved in glucose metabolism, hexokinase, glucose-6-phosphate dehydrogenase, glucose-phosphate isomerase, and succinate dehydrogenase, were not detected in cells grown on hexadecane but were present in cells grown on glucose. Addition of hexadecane to extracts of glucose-grown cells resulted in immediate loss of activity for each of the four enzymes, but two other enzymes did not directly involved in glucose metabolism, adenosine triphosphatase and alanine-ketoacid aminotransferase, were not inhibited by hexadecane in vitro. Cells grown on hexadecane and transferred to glucose metabolize intracellular hexadecane; after 1 day, activity of hexokinase, glucose-6-phosphate dehydrogenase, glucosephosphate isomerase, and succinate dehydrogenase could be detected and 22% of the intracellular hydrocarbon had been metabolized. Hexadecane-grown cells transferred to glucose plus cycloheximide showed the same level of activity of all the four enzymes as cells transferred to glucose alone. Thus, intracellular n-hexadecane or a metabolite of hexadecane can inthesis of those enzymes is not inhibited. PMID:135754

  3. Control of Pyrophosphated-Fructose-6-Phosphate 1-Phosphotransferase Activity in the Cotyledons of Citrullus lanatus1

    PubMed Central

    Botha, Anna-Maria; Botha, Frederik C.

    1990-01-01

    After initiation of radicle elongation, the pyrophosphate:d-fructose-6-phosphate 1-phosphotransferase (PFP) activity sharply increases in the cotyledons of Citrullus lanatus. Removal of the radicle early during incubation prevents the increase in PFP activity in the cotyledons evident in the control. Removal of the radicle at any stage after germination results in a decrease in PFP activity in the cotyledons. Application of kinetin (0.5 micromolar) or 2-chlorophosphonic acid (0.1 micromolar) to isolated cotyledons replaces the effect of the radicle. Gibberellic acid (0.09 micromolar GA3) also partially mimics the presence of the radicle. Anaerobic conditions, as well as cycloheximide application (0.18 micromolar) to intact embryos or to kinetin and ethrel treated isolated cotyledons prevent the increase in PFP activity evident in the control. PMID:16667523

  4. Isolation of the GFA1 gene encoding glucosamine-6-phosphate synthase of Sporothrix schenckii and its expression in Saccharomyces cerevisiae.

    PubMed

    Sánchez-López, Juan Francisco; González-Ibarra, Joaquín; Álvarez-Vargas, Aurelio; Milewski, Slawomir; Villagómez-Castro, Julio César; Cano-Canchola, Carmen; López-Romero, Everardo

    2015-06-01

    Glucosamine-6-phosphate synthase (GlcN-6-P synthase) is an essential enzyme involved in cell wall biogenesis that has been proposed as a strategic target for antifungal chemotherapy. Here we describe the cloning and functional characterization of Sporothrix schenckii GFA1 gene which was isolated from a genomic library of the fungus. The gene encodes a predicted protein of 708 amino acids that is homologous to GlcN-6-P synthases from other sources. The recombinant enzyme restored glucosamine prototrophy of the Saccharomyces cerevisiae gfa1 null mutant. Purification and biochemical analysis of the recombinant enzyme revealed some differences from the wild type enzyme, such as improved stability and less sensitivity to UDP-GlcNAc. The sensitivity of the recombinant enzyme to the selective inhibitor FMDP [N(3)-(4-methoxyfumaroyl)-l-2,3-diaminopropanoic acid] and other properties were similar to those previously reported for the wild type enzyme.

  5. Beneficial Effect of Sugar Osmolytes on the Refolding of Guanidine Hydrochloride-Denatured Trehalose-6-phosphate Hydrolase from Bacillus licheniformis

    PubMed Central

    Chen, Jiau-Hua; Chi, Meng-Chun; Lin, Min-Guan; Lin, Long-Liu; Wang, Tzu-Fan

    2015-01-01

    The influence of three sugar osmolytes on the refolding of guanidine hydrochloride- (GdnHCl-) denatured trehalose-6-phosphate hydrolase of Bacillus licheniformis (BlTreA) was studied by circular dichroism (CD) spectra, fluorescence emission spectra, and the recovery of enzymatic activity. These experimental results clearly indicated that sorbitol, sucrose, and trehalose at a concentration of 0.75 M improved the refolding yields of GdnHCl-denatured  BlTreA, probably due to the fact that these sugars favored the formation of tertiary architectures. Far-UV CD measurements demonstrated the ability of sugar osmolytes to shift the secondary structure of GdnHCl-denatured enzyme towards near-native conformations. ANS fluorescence intensity measurements revealed a reduction of exposed hydrophobic surfaces upon the treatment of denatured enzyme with sugar osmolytes. These observations suggest that sugar osmolytes possibly play a chaperone role in the refolding of chemically denatured BlTreA. PMID:25667926

  6. Trehalose 6-phosphate phosphatase is required for development, virulence and mycotoxin biosynthesis apart from trehalose biosynthesis in Fusarium graminearum.

    PubMed

    Song, Xiu-Shi; Li, He-Ping; Zhang, Jing-Bo; Song, Bo; Huang, Tao; Du, Xiao-Min; Gong, An-Dong; Liu, Yi-Ke; Feng, Yan-Ni; Agboola, Rebecca S; Liao, Yu-Cai

    2014-02-01

    Trehalose 6-phosphate synthase (TPS1) and trehalose 6-phosphate phosphatase (TPS2) are required for trehalose biosynthesis in yeast and filamentous fungi, including Fusarium graminearum. Three null mutants Δtps1, Δtps2 and Δtps1-Δtps2, each carrying either a single deletion of TPS1 or TPS2 or a double deletion of TPS1-TPS2, were generated from a toxigenic F. graminearum strain and were not able to synthesize trehalose. In contrast to its reported function in yeasts and filamentous fungi, TPS1 appeared dispensable for development and virulence. However, deletion of TPS2 abolished sporulation and sexual reproduction; it also altered cell polarity and ultrastructure of the cell wall in association with reduced chitin biosynthesis. The cell polarity alteration was exhibited as reduced apical growth and increased lateral growth and branching with increased hyphal and cell wall widths. Moreover, the TPS2-deficient strain displayed abnormal septum development and nucleus distribution in its conidia and vegetative hyphae. The Δtps2 mutant also had 62% lower mycelial growth on potato dextrose agar and 99% lower virulence on wheat compared with the wild-type. The Δtps1, Δtps2 and Δtps1-Δtps2 mutants synthesized over 3.08-, 7.09- and 2.47-fold less mycotoxins, respectively, on rice culture compared with the wild-type. Comparative transcriptome analysis revealed that the Δtps1, Δtps2 and Δtps1-Δtps2 mutants had 486, 1885 and 146 genotype-specific genes, respectively, with significantly changed expression profiles compared with the wild-type. Further dissection of this pathway will provide new insights into regulation of fungal development, virulence and trichothecene biosynthesis.

  7. In vivo survival of (14C)sucrose-loaded porcine carrier erythrocytes

    SciTech Connect

    DeLoach, J.R.

    1983-06-01

    Porcine carrier erythrocyte survival was measured in adult pigs. (14C)Sucrose-loaded erythrocytes had a biphasic survival curve, with as much as 50% of the cells removed from circulation in the first 24 hours. The remaining cells had a 35-day half-life. Encapsulation values were measured for porcine erythrocytes and entrapment of (14C)sucrose was greater than 45%. Addition of inosine and glucose to the dialyzed cells and to the final wash buffer before reinjection of autologous cells did not improve their survival.

  8. Isolation of streptomycin-nonproducing mutants deficient in biosynthesis of the streptidine moiety or linkage between streptidine 6-phosphate and dihydrostreptose.

    PubMed

    Ohnuki, T; Imanaka, T; Aiba, S

    1985-03-01

    Eight streptidine idiotrophic mutants (SD20, SD81, SD141, SD189, SD245, SD261, SD263, and SD274) which required streptidine to produce streptomycin were derived from Streptomyces griseus ATCC 10137 by UV mutagenesis. By both the characterization of intermediates accumulated by the idiotrophs and the assay of enzymes involved in streptidine biosynthesis, the biochemical lesions of the mutants were deduced as follows: SD20 and SD263, transamination; SD81, SD261, and SD274, phosphorylation; SD141, transamidination; SD189, dehydrogenation; SD245, linkage between streptidine 6-phosphate and dihydrostreptose. An accumulation of streptidine 6-phosphate was found in SD245 to impair its aminotransferase activity. This finding suggests that aminotransferase activity might have been negatively controlled by the end product, streptidine 6-phosphate, of the streptidine biosynthetic pathway.

  9. The Structure of MurNAc 6-Phosphate Hydrolase (MurQ) from Haemophilus influenzae with Bound Inhibitor

    PubMed Central

    Hadi, Timin; Hazra, Saugata; Tanner, Martin E.; Blanchard, John S.

    2014-01-01

    The breakdown and recycling of peptidoglycan, an essential polymeric cell structure, occurs in a number of bacterial species. A key enzyme in the recycling pathway of one of the components of the peptidoglycan layer, N-acetylmuramic acid (MurNAc), is MurNAc 6-phosphate hydrolase (MurQ). This enzyme catalyzes the cofactor-independent cleavage of a relatively non-labile ether bond and presents an interesting target for mechanistic studies. Open-chain product and substrate analogs were synthesized and tested as competitive inhibitors (Kis values of 1.1 +/− 0.3 mM and 0.23 +/− 0.02 mM, respectively) of the MurNAc 6P hydrolase from Escherichia coli (MurQ-EC). To identify the roles of active site residues important for catalysis, the substrate analog was co-crystallized with the MurNAc 6P hydrolase from Haemophilus influenzae (MurQ-HI) that was amenable to crystallographic studies. The co-crystal structure of MurQ-HI with the substrate analog showed that Glu89 was located in close proximity to both the carbon at the C2 position and the oxygen at the C3 position of the bound inhibitor, and that no other potential acid/base residue that could act as an active site acid/base was located in the vicinity. The conserved residues Glu120 and Lys239 were found within hydrogen-bonding distance of the C5 hydroxyl group and C6 phosphate group, suggesting that they play a role in substrate binding and ring-opening. Combining these results with previous biochemical data, a one base mechanism of action where Glu89 functions to both deprotonate at the C2 position and assist in the departure of the lactyl ether at the C3 position is proposed. This same residue would serve to deprotonate the incoming water and reprotonate the enolate in the second half of the catalytic cycle. PMID:24251551

  10. Two mechanisms for toxic effects of hydroxylamines in human erythrocytes: involvement of free radicals and risk of potentiation.

    PubMed

    Evelo, C T; Spooren, A A; Bisschops, R A; Baars, L G; Neis, J M

    1998-09-01

    The toxic potency of three industrially used hydroxylamines was studied in human blood cells in vitro. The parent compound hydroxylamine and the O-ethyl derivative gave very similar results. Both compounds induced a high degree of methemoglobin formation and glutathione depletion. Cytotoxicity was visible as Heinz body formation and hemolysis. High levels of lipid peroxidation occurred, in this respect O-ethyl hydroxylamine was more active than hydroxylamine. In contrast H2O2 induced lipid peroxidation was lowered after O-ethyl hydroxylamine or hydroxylamine treatment, this is explained by the ferrohemoglobin dependence of H2O2 induced radical species formation. Glutathione S-transferase (GST) and NADPH methemoglobin reductase (NADPH-HbR) activities were also impaired, probably as a result of the radical stress occurring. The riboflavin availability was decreased. Other enzyme activities glutathione reductase (GR), glucose 6-phosphate dehydrogenase (G6PDH), glucose phosphate isomerase and NADH methemoglobin reductase, were not or only slightly impaired by hydroxylamine or O-ethyl hydroxylamine treatment. A different scheme of reactivity was found for N,O-dimethyl hydroxylamine. This compound gave much less methemoglobin formation and no hemolysis or Heinz body formation at concentrations up to and including 7 mM. Lipid peroxidase induction was not detectable, but could be induced by subsequent H2O2 treatment. GST and NADPH-HbR activities and riboflavin availability were not decreased. On the other hand GR and G6PDH activities were inhibited. These results combined with literature data indicate the existence of two different routes of hematotoxicity induced by hydroxylamines. Hydroxylamine as well as O-alkylated derivatives primarily induce methemoglobin, a process involving radical formation. The radical stress occurring is probably responsible for most other effects. N-alkylated species like N,O-dimethyl hydroxylamine primarily lead to inhibition of the protective

  11. How do sugars regulate plant growth and development? New insight into the role of trehalose-6-phosphate.

    PubMed

    O'Hara, Liam E; Paul, Matthew J; Wingler, Astrid

    2013-03-01

    Plant growth and development are tightly controlled in response to environmental conditions that influence the availability of photosynthetic carbon in the form of sucrose. Trehalose-6-phosphate (T6P), the precursor of trehalose in the biosynthetic pathway, is an important signaling metabolite that is involved in the regulation of plant growth and development in response to carbon availability. In addition to the plant's own pathway for trehalose synthesis, formation of T6P or trehalose by pathogens can result in the reprogramming of plant metabolism and development. Developmental processes that are regulated by T6P range from embryo development to leaf senescence. Some of these processes are regulated in interaction with phytohormones, such as auxin. A key interacting factor of T6P signaling in response to the environment is the protein kinase sucrose non-fermenting related kinase-1 (SnRK1), whose catalytic activity is inhibited by T6P. SnRK1 is most likely involved in the adjustment of metabolism and growth in response to starvation. The transcription factor bZIP11 has recently been identified as a new player in the T6P/SnRK1 regulatory pathway. By inhibiting SnRK1, T6P promotes biosynthetic reactions. This regulation has important consequences for crop production, for example, in the developing wheat grain and during the growth of potato tubers.

  12. Glycan structure determinants for cation-independent mannose 6-phosphate receptor binding and cellular uptake of a recombinant protein.

    PubMed

    Zhou, Qun; Avila, Luis Z; Konowicz, Paul A; Harrahy, John; Finn, Patrick; Kim, Jennifer; Reardon, Michael R; Kyazike, Josephine; Brunyak, Elizabeth; Zheng, Xiaoyang; Patten, Scott M Van; Miller, Robert J; Pan, Clark Q

    2013-12-18

    The cation-independent mannose 6-phosphate receptor (CI-MPR) plays a critical role in intracellular transport of lysosomal enzymes as well as the uptake of recombinant proteins. To define the minimal glycan structure determinants necessary for receptor binding and cellular uptake, we synthesized a series of glycans containing mono-, di-, tri-, tetra-, and hexamannoses terminated with either one or two phosphates for conjugating to a model protein, recombinant human acid α-glucosidase. A high affinity interaction with the CI-MPR can be achieved for the enzyme conjugated to a dimannose glycan with a single phosphate. However, tightest binding to a CI-MPR affinity column was observed with a hexamannose structure containing two phosphates. Moreover, maximal cellular uptake and a 5-fold improvement in in vivo potency were achieved when the bisphosphorylated hexamannose glycan is conjugated to the protein by a β linker. Nevertheless, even a monophosphorylated dimannose glycan conjugate showed stronger binding to the receptor affinity column, higher cellular uptake, and significantly greater in vivo efficacy compared to the unconjugated protein which contains a low level of high affinity glycan structure. These results demonstrate that the phosphorylated dimannose moiety appears to be the minimal structure determinant for enhanced CI-MPR binding and that the orientation of the glycan is critical for maximum receptor interaction. In summary, we have improved the understanding of the mechanism of CI-MPR binding and developed a simple alternative for CI-MPR targeting.

  13. Requirement of the Human GARP Complex for Mannose 6-phosphate-receptor-dependent Sorting of Cathepsin D to Lysosomes

    PubMed Central

    Pérez-Victoria, F. Javier; Mardones, Gonzalo A.

    2008-01-01

    The biosynthetic sorting of acid hydrolases to lysosomes relies on transmembrane, mannose 6-phosphate receptors (MPRs) that cycle between the TGN and endosomes. Herein we report that maintenance of this cycling requires the function of the mammalian Golgi-associated retrograde protein (GARP) complex. Depletion of any of the three GARP subunits, Vps52, Vps53, or Vps54, by RNAi impairs sorting of the precursor of the acid hydrolase, cathepsin D, to lysosomes and leads to its secretion into the culture medium. As a consequence, lysosomes become swollen, likely due to a buildup of undegraded materials. Missorting of cathepsin D in GARP-depleted cells results from accumulation of recycling MPRs in a population of light, small vesicles downstream of endosomes. These vesicles might correspond to intermediates in retrograde transport from endosomes to the TGN. Depletion of GARP subunits also blocks the retrograde transport of the TGN protein, TGN46, and the B subunit of Shiga toxin. These observations indicate that the mammalian GARP complex plays a general role in the delivery of retrograde cargo into the TGN. We also report that a Vps54 mutant protein in the Wobbler mouse strain is active in retrograde transport, thus explaining the viability of these mutant mice. PMID:18367545

  14. Disruption in Candida albicans of the TPS2 gene encoding trehalose-6-phosphate phosphatase affects cell integrity and decreases infectivity.

    PubMed

    Zaragoza, Oscar; de Virgilio, Claudio; Pontón, José; Gancedo, Carlos

    2002-05-01

    The gene CaTPS2 encoding trehalose-6-phosphate (T6P) phosphatase from Candida albicans has been cloned and disrupted in this organism. The Catps2/Catps2 mutant did not accumulate trehalose but accumulated high levels of T6P. Disruption of the two copies of the CaTPS2 gene did not abolish growth even at 42 degrees C, but decreased the growth rate. In the stationary phase, the Catps2/Catps2 mutant aggregated, more than 50% of its cells became permeable to propidium iodide and a large amount of protein was found in the culture medium. Aggregation occurred only at pH values higher than 7 and was avoided by osmoprotectants; it was never observed during the exponential phase of growth. The mutant formed colonies with a smooth border on Spider medium. Mice inoculated with 1.5 x 10(6) c.f.u. of wild-type cells died after 8 days, while 80% of those inoculated with the same number of c.f.u. of the Catps2/Catps2 mutant survived for at least 1 month. Reintroduction of the wild-type CaTPS2 gene in the Catps2/Catps2 mutant abolished the phenotypes described. It is hypothesized that the accumulation of T6P interferes with the assembly of a normal cell wall.

  15. Identification of three internalization sequences in the cytoplasmic tail of the 46 kDa mannose 6-phosphate receptor.

    PubMed Central

    Denzer, K; Weber, B; Hille-Rehfeld, A; Figura, K V; Pohlmann, R

    1997-01-01

    The cytoplasmic tail of the human 46 kDa mannose 6-phosphate receptor (MPR 46) is necessary for rapid internalization of the receptor and sufficient to mediate internalization of a resident plasma membrane protein. To localize the internalization sequences within the 67 amino acids of the cytoplasmic tail, the tail was progressively shortened from its C-terminus, internal deletions of between four and eight amino acids were introduced into the tail, and individual residues were substituted by alanine, glycine or serine. Three sequences were identified that contribute to the internalization of MPR 46. The first is located within the 23 juxtamembrane cytoplasmic residues of the tail. It contains four essential residues within a heptapeptide and does not resemble known internalization signals. The second sequence contains as a critical residue Tyr-45. The third region is located within the C-terminal seven residues and contains a di-leucine pair as essential residues. The first and third sequences were shown to function as autonomous internalization sequences. Substitution of critically important residues within a single internalization sequence was tolerated, with no or only a moderate decrease in the internalization rate. When essential residues from two or all three internalization sequences were substituted, however, the internalization rate was decreased by more than 60% and 90% respectively. This indicates that the autonomous internalization signals in the cytoplasmic tail of MPR 46 function in an additive manner, but are partly redundant. PMID:9291124

  16. Expression and binding properties of the two mannose-6-phosphate receptors differ during perinatal development in rat liver.

    PubMed

    Romano, Patricia S; López, Ana C; Mariani, María L; Sartor, Tirso; Belmonte, Silvia A; Sosa, Miguel A

    2002-07-26

    Mammalian tissues express both cation-dependent (CD-MPR) and cation-independent (CI-MPR) mannose-6-phosphate receptors, which mediate the targeting of acid hydrolases to lysosomes. The coexistence of the two receptors in all cell types and tissues is still poorly understood. To determine whether these receptors might play a role in maturation, we studied their expression and binding properties in rat liver during perinatal development. CI-MPR expression decreases progressively from 18-day fetuses to adults, whereas the CD-MPR showed a transient decrease in newborn and at the 5th day after birth. Immunostaining of the tissues showed that both receptors localize to hepatocytes at all the ages and, additionally, the CD-MPR was reactive in megakaryocytes at early stages. Binding assays showed differences in the B(max) and K(D) values between the ages studied. These results demonstrate that both receptors change differentially during perinatal development, suggesting that they play distinct roles during organ maturation.

  17. Novel mode of inhibition by D-tagatose 6-phosphate through a Heyns rearrangement in the active site of transaldolase B variants.

    PubMed

    Stellmacher, Lena; Sandalova, Tatyana; Schneider, Sarah; Schneider, Gunter; Sprenger, Georg A; Samland, Anne K

    2016-04-01

    Transaldolase B (TalB) and D-fructose-6-phosphate aldolase A (FSAA) from Escherichia coli are C-C bond-forming enzymes. Using kinetic inhibition studies and mass spectrometry, it is shown that enzyme variants of FSAA and TalB that exhibit D-fructose-6-phosphate aldolase activity are inhibited covalently and irreversibly by D-tagatose 6-phosphate (D-T6P), whereas no inhibition was observed for wild-type transaldolase B from E. coli. The crystal structure of the variant TalB(F178Y) with bound sugar phosphate was solved to a resolution of 1.46 Å and revealed a novel mode of covalent inhibition. The sugar is bound covalently via its C2 atom to the ℇ-NH2 group of the active-site residue Lys132. It is neither bound in the open-chain form nor as the closed-ring form of D-T6P, but has been converted to β-D-galactofuranose 6-phosphate (D-G6P), a five-membered ring structure. The furanose ring of the covalent adduct is formed via a Heyns rearrangement and subsequent hemiacetal formation. This reaction is facilitated by Tyr178, which is proposed to act as acid-base catalyst. The crystal structure of the inhibitor complex is compared with the structure of the Schiff-base intermediate of TalB(E96Q) formed with the substrate D-fructose 6-phosphate determined to a resolution of 2.20 Å. This comparison highlights the differences in stereochemistry at the C4 atom of the ligand as an essential determinant for the formation of the inhibitor adduct in the active site of the enzyme. PMID:27050126

  18. Novel mode of inhibition by D-tagatose 6-phosphate through a Heyns rearrangement in the active site of transaldolase B variants.

    PubMed

    Stellmacher, Lena; Sandalova, Tatyana; Schneider, Sarah; Schneider, Gunter; Sprenger, Georg A; Samland, Anne K

    2016-04-01

    Transaldolase B (TalB) and D-fructose-6-phosphate aldolase A (FSAA) from Escherichia coli are C-C bond-forming enzymes. Using kinetic inhibition studies and mass spectrometry, it is shown that enzyme variants of FSAA and TalB that exhibit D-fructose-6-phosphate aldolase activity are inhibited covalently and irreversibly by D-tagatose 6-phosphate (D-T6P), whereas no inhibition was observed for wild-type transaldolase B from E. coli. The crystal structure of the variant TalB(F178Y) with bound sugar phosphate was solved to a resolution of 1.46 Å and revealed a novel mode of covalent inhibition. The sugar is bound covalently via its C2 atom to the ℇ-NH2 group of the active-site residue Lys132. It is neither bound in the open-chain form nor as the closed-ring form of D-T6P, but has been converted to β-D-galactofuranose 6-phosphate (D-G6P), a five-membered ring structure. The furanose ring of the covalent adduct is formed via a Heyns rearrangement and subsequent hemiacetal formation. This reaction is facilitated by Tyr178, which is proposed to act as acid-base catalyst. The crystal structure of the inhibitor complex is compared with the structure of the Schiff-base intermediate of TalB(E96Q) formed with the substrate D-fructose 6-phosphate determined to a resolution of 2.20 Å. This comparison highlights the differences in stereochemistry at the C4 atom of the ligand as an essential determinant for the formation of the inhibitor adduct in the active site of the enzyme.

  19. Genome-Wide Identification and Evolution Analysis of Trehalose-6-Phosphate Synthase Gene Family in Nelumbo nucifera

    PubMed Central

    Jin, Qijiang; Hu, Xin; Li, Xin; Wang, Bei; Wang, Yanjie; Jiang, Hongwei; Mattson, Neil; Xu, Yingchun

    2016-01-01

    Trehalose-6-phosphate synthase (TPS) plays a key role in plant carbohydrate metabolism and the perception of carbohydrate availability. In the present work, the publicly available Nelumbo nucifera (lotus) genome sequence database was analyzed which led to identification of nine lotus TPS genes (NnTPS). It was found that at least two introns are included in the coding sequences of NnTPS genes. When the motif compositions were analyzed we found that NnTPS generally shared the similar motifs, implying that they have similar functions. The dN/dS ratios were always less than 1 for different domains and regions outside domains, suggesting purifying selection on the lotus TPS gene family. The regions outside TPS domain evolved relatively faster than NnTPS domains. A phylogenetic tree was constructed using all predicted coding sequences of lotus TPS genes, together with those from Arabidopsis, poplar, soybean, and rice. The result indicated that those TPS genes could be clearly divided into two main subfamilies (I-II), where each subfamily could be further divided into 2 (I) and 5 (II) subgroups. Analyses of divergence and adaptive evolution show that purifying selection may have been the main force driving evolution of plant TPS genes. Some of the critical sites that contributed to divergence may have been under positive selection. Transcriptome data analysis revealed that most NnTPS genes were predominantly expressed in sink tissues. Expression pattern of NnTPS genes under copper and submergence stress indicated that NNU_014679 and NNU_022788 might play important roles in lotus energy metabolism and participate in stress response. Our results can facilitate further functional studies of TPS genes in lotus. PMID:27746792

  20. Reduction of Tendon Adhesions following Administration of Adaprev, a Hypertonic Solution of Mannose-6-Phosphate: Mechanism of Action Studies

    PubMed Central

    Wong, Jason K. F.; Metcalfe, Anthony D.; Wong, Richard; Bush, Jim; Platt, Chris; Garcon, Arnaud; Goldspink, Nick; McGrouther, Duncan A.; Ferguson, Mark W. J.

    2014-01-01

    Repaired tendons may be complicated by progressive fibrosis, causing adhesion formation or tendon softening leading to tendon rupture and subsequent reduced range of motion. There are few therapies available which improve the gliding of damaged tendons in the hand. We investigate the role of Mannose 6-phosphate (M6P) in a 600 mM hypertonic solution (Adaprev) on tendon adhesion formation in vivo using a mouse model of severed tendon in conjunction with analysis of collagen synthesis, cellular proliferation and receptors involved in TGF beta signalling. Cytotoxicity was assessed by measuring tissue residency, mechanical strength and cell viability of tendons after treatment with Adaprev. To elicit potential modes of action, in vitro and ex vivo studies were performed investigating phosphorylation of p38, cell migration and proliferation. Adaprev treatment significantly (p<0.05) reduced the development of adhesions and improved collagen organisation without reducing overall collagen synthesis following tendon injury in vivo. The bioavailability of Adaprev saw a 40% reduction at the site of administration over 45 minutes and tendon fibroblasts tolerated up to 120 minutes of exposure without significant loss of cell viability or tensile strength. These favourable effects were independent of CI-MPR and TGF-β signalling and possibly highlight a novel mechanism of action related to cellular stress demonstrated by phosphorylation of p38. The effect of treatment reduced tendon fibroblast migration and transiently halted tendon fibroblast proliferation in vitro and ex vivo. Our studies demonstrate that the primary mode of action for Adaprev is potentially via a physical, non-chemical, hyperosmotic effect. PMID:25383548

  1. Trehalose-6-phosphate and SNF1-related protein kinase 1 are involved in the first-fruit inhibition of cucumber.

    PubMed

    Zhang, ZhiPing; Deng, Yukun; Song, Xingxing; Miao, Minmin

    2015-04-01

    In cucumber (Cucumis sativus L.), the preexisting fruits inhibit the growth of subsequent fruits. To study the mechanism underlying this phenomenon, we examined the sink activity, the level of free sugars, and the activity of SNF1-related protein kinase 1 (SnRK1) in the peduncles of two types of fruits. In the two-fruit cucumber plants, the growth rate and sink activity [evaluated by alkaline alpha-galactosidase (CsAGA) activity in the peduncle] of the first fruit were greater than those of the second fruit. The (14)C-labeling experiment revealed that assimilates produced by the leaves closer to the second fruit tended to move to the first fruit. Sucrose and trehalose-6-phosphate (T6P) levels in the peduncle of the first fruit were higher than those in the peduncle of the second fruit. The SnRK1 activity was lower in the peduncle of the first fruit than in that of the second fruit at 0-8 days after anthesis. The growth rate and sink activity of the second fruit were enhanced after the removal of the first fruit or after treatment with 6-benzyl aminopurine, as determined by comparison with an increase in the sucrose and T6P levels and a decrease in the SnRK1 activity in its peduncle. The SnRK1 activity was inhibited by T6P in an in vitro kinase assay, and the mRNA level of CsAGA1 in cucumber calli was up-regulated by exogenous trehalose treatment, confirming that the SnRK1 activity and CsAGA1 expression can be regulated by T6P levels. Our results suggest that the T6P- and SnRK1-mediated signaling functions are involved in the regulation of first-fruit inhibition in cucumber plants.

  2. Membrane proteins in senescent erythrocytes.

    PubMed Central

    Suzuki, T; Dale, G L

    1989-01-01

    The examination of erythrocyte senescence has been facilitated by recent advances in techniques for the isolation of aged red cells. One of these methods, which uses biotinylated rabbit erythrocytes, has been used to examine the state of membrane proteins in effete cells. These aged red cells were found to have normal ratios of alpha-spectrin and beta-spectrin as well as normal levels of ankyrin. The observation concerning ankyrin is particularly important due to the sensitivity of this protein to proteolysis and the postulated action of proteinases in the aging process. The senescent erythrocytes were also found to have an altered ratio of bands 4.1a and 4.1b without any apparent change in the total level of 4.1. In addition, the analysis of the aged cell membranes did not show any large-molecular-mass aggregated protein at the origin of the SDS/polyacrylamide gels, indicating a lack of transglutaminase activity in the senescence process for rabbit erythrocytes. These results indicate that aging of the rabbit erythrocyte is not accompanied by gross proteolytic degradation or transglutaminase-catalysed cross-linking of membrane components. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. Fig. 5. PMID:2522000

  3. FT-IR spectrometry utilization for determining changes in erythrocyte susceptibility to oxidative stress

    NASA Astrophysics Data System (ADS)

    Petibois, Cyril; Deleris, Gdrard Y. R.

    2004-07-01

    We tested the hypothesis that FT-IR spectrometry was useful for determining oxidative stress damage on erythrocytes. Endurance-trained subjects performed a standardized endurance-training session at 75% of maximal oxygen consumption each week over 19 consecutive weeks. Capillary blood samples were taken before and after test-sessions and plasma and erythrocytes were separately analyzed using Fourier-transform infrared spectrometry. Exercise-induced change in plasma concentrations and erythrocyte IR absorptivities (vC-Hn of fatty acyl moieties, vC=O and δN-H of proteins, vP=O of phospholipids, vCOO- of amino-acids, and vC-O of lactate) were monitored and compared to training level. First training weeks induced normalization of plasma concentration changes during exercise (unchanged for glucose, moderately increased for lactate, high increases for triglycerides, glycerol, and fatty acids) while erythrocyte phospholipids alteration remained elevated (P < 0.05). Further, training reduced the exercise-induced erythrocyte lactate content increase (vC-O; P < 0.05) and phospholipids alteration (vC-Hn and vP=O; P < 0.05) during exercise. These changes paralleled the lowering of exercise-induced hemoconcentration (P < 0.05) and plasma lactate concentration increase during exercise (P < 0.05). These correlated changes between plasma and erythrocyte parameters suggest that hemoconcentration and lactate acidosis (plasmatic and intracellular) are important factors contributing to reduce erythrocyte susceptibility to oxidative stress during chronic endurance training.

  4. Studies on metatherian sex chromosomes. I. Inheritance and inactivation of sex-linked allelic genes determining glucose-6-phosphate dehydrogenase variation in kangaroos.

    PubMed

    Johnston, P G; Sharman, G B

    1975-12-01

    Wallaroos (Macropus robustus robustus), which have the G6PD-F electrophoretic phenotype, crossed with euros (M.r.erubescens), of G6PD-S phenotype, produced F1 animals which had only the maternal G6PD type regardless of the direction of the cross. When F1 hybrids were backcrossed to wallaroos or euros, backcross progeny of either perental phenotype resulted. Sex-linked inheritance of allelic G6PD genes is shown to occur in wallaroos, euros and red kangaroos (M. rufus). Dose compensation for X chromosomes at the G6PD locus in kangaroow is achieved by inactivation of the allele of male parental origin.

  5. Molecular Heterogeneity of Glucose-6-Phosphate Dehydrogenase Deficiency in Burkina Faso: G-6-PD Betica Selma and Santamaria in People with Symptomatic Malaria in Ouagadougou

    PubMed Central

    Ouattara, Abdoul Karim; Yameogo, Pouiré; Diarra, Birama; Obiri-Yeboah, Dorcas; Yonli, Albert; Compaore, Tegwindé Rebeca; Soubeiga, Serge Théophile; Djigma, Florencia Wenkuuni; Simpore, Jacques

    2016-01-01

    The G-6-PD deficiency has an important polymorphism with genotypic variants such as 202A/376G, 376G/542T and 376G/968T known in West African populations. It would confer protection against severe forms of malaria although there are differences between the various associations in different studies. In this study we genotyped six (06) variants of the G-6-PD gene in people with symptomatic malaria in urban areas in Burkina Faso. One hundred and eighty-two (182) patients who tested positive using rapid detection test and microscopy were included in this study. A regular PCR with the GENESPARK G6PD African kit was run followed by electrophoresis, allowing initially to genotype six SNPs (G202A, A376G, A542T, G680T, C563T and T968C). Women carrying the mutations 202A and/or 376G were further typed by real-time PCR using TaqMan probes rs1050828 and rs1050829. In the study population the G-6-PD deficiency prevalence was 9.9%. In addition of G-6-PD A- (202A/376G) variant, 376G/542T and 376G/968T variants were also detected. Hemoglobin electrophoresis revealed that 22.5% (41/182) of the individuals had HbAC compared with2.2% with HbAS and one individual had double heterozygous HbSC. There was no correlation between the G-6-PD deficiency or haemoglobinopathies and symptomatic malaria infections in this study. Our study confirms that the G-6-PD deficiency does not confer protection against Plasmodium falciparum infections. As opposed to previous genotyping studies carried out in Burkina Faso, this study shows for the first time the presence of the variant A- (376G/968C) and warrants further investigation at the national level and in specific ethnic groups. PMID:27413522

  6. Molecular Heterogeneity of Glucose-6-Phosphate Dehydrogenase Deficiency in Burkina Faso: G-6-PD Betica Selma and Santamaria in People with Symptomatic Malaria in Ouagadougou.

    PubMed

    Ouattara, Abdoul Karim; Yameogo, Pouiré; Diarra, Birama; Obiri-Yeboah, Dorcas; Yonli, Albert; Compaore, Tegwindé Rebeca; Soubeiga, Serge Théophile; Djigma, Florencia Wenkuuni; Simpore, Jacques

    2016-01-01

    The G-6-PD deficiency has an important polymorphism with genotypic variants such as 202A/376G, 376G/542T and 376G/968T known in West African populations. It would confer protection against severe forms of malaria although there are differences between the various associations in different studies. In this study we genotyped six (06) variants of the G-6-PD gene in people with symptomatic malaria in urban areas in Burkina Faso. One hundred and eighty-two (182) patients who tested positive using rapid detection test and microscopy were included in this study. A regular PCR with the GENESPARK G6PD African kit was run followed by electrophoresis, allowing initially to genotype six SNPs (G202A, A376G, A542T, G680T, C563T and T968C). Women carrying the mutations 202A and/or 376G were further typed by real-time PCR using TaqMan probes rs1050828 and rs1050829. In the study population the G-6-PD deficiency prevalence was 9.9%. In addition of G-6-PD A- (202A/376G) variant, 376G/542T and 376G/968T variants were also detected. Hemoglobin electrophoresis revealed that 22.5% (41/182) of the individuals had HbAC compared with2.2% with HbAS and one individual had double heterozygous HbSC. There was no correlation between the G-6-PD deficiency or haemoglobinopathies and symptomatic malaria infections in this study. Our study confirms that the G-6-PD deficiency does not confer protection against Plasmodium falciparum infections. As opposed to previous genotyping studies carried out in Burkina Faso, this study shows for the first time the presence of the variant A- (376G/968C) and warrants further investigation at the national level and in specific ethnic groups. PMID:27413522

  7. Population study of 1311 C/T polymorphism of Glucose 6 Phosphate Dehydrogenase gene in Pakistan – an analysis of 715 X-chromosomes

    PubMed Central

    Moiz, Bushra; Nasir, Amna; Moatter, Tariq; Naqvi, Zulfiqar Ali; Khurshid, Mohammad

    2009-01-01

    Background Nucleotide 1311 polymorphism at exon 11 of G6PD gene is widely prevalent in various populations of the world. The aim of the study was to evaluate 1311 polymorphism in subjects carrying G6PD Mediterranean gene and in general population living in Pakistan. Results Patients already known to be G6PD deficient were tested for 563C-T (G6PD Mediterranean) and 1311 C-T mutation through RFLP based PCR and gene sequencing. A control group not known to be G6PD deficient was tested for 1311C/T only. C-T transition at nt 1311 was detected in 60/234 X-chromosomes with 563 C-T mutation (gene frequency of 0.26) while in 130 of normal 402 X-chromosomes (gene frequency of 0.32). Conclusion We conclude that 1311 T is a frequent polymorphism both in general populations and in subjects with G6PD Mediterranean gene in Pakistan. The prevalence is higher compared to most of the populations of the world. The present study will help in understanding genetic basis of G6PD deficiency in Pakistani population and in developing ancestral links of its various ethnic groups. PMID:19640310

  8. Glucose-6-Phosphate Dehydrogenase Deficiency and Sickle Cell Trait among Prospective Blood Donors: A Cross-Sectional Study in Berekum, Ghana

    PubMed Central

    Simpong, David Larbi; Takyi, Godfred; Ephraim, Richard K. D.

    2016-01-01

    Background. Blood transfusion is a therapeutic procedure usually undertaken in patients with severe anaemia. In Ghana, severe anaemia is mostly due to malaria caused by severe Plasmodium falciparum infection, road traffic accidents, and haemoglobinopathy-induced acute haemolysis. Method. This cross-sectional study evaluated coinheritance of sickle cell haemoglobin variant and G6PD enzymopathy among individuals that donated blood at the Holy Trinity Hospital, Berekum, in the Brong-Ahafo Region, Ghana. Demographic data and other pertinent information were captured using questionnaire. Sickle cell haemoglobin variants were determined using cellulose acetate electrophoresis (pH 8.6). Qualitative G6PD status and quantitative G6PD enzyme activity were determined using methaemoglobin reduction and Trinity Biotech G6PD test kit, respectively. Results. Prevalence of sickle cell trait (SCT) and G6PD enzymopathy coinheritance was 7%. In addition, 19.5% of the donors had 10%–60% of normal G6PD enzyme activity suggesting that these donor units are prone to stressor-induced acute haemolysis when given to recipients. Mild G6PD activity (p = 0.03, OR: 2.410 (CI: 1.049–5.534)), commercial (p = 0.020, OR: 5.609 (CI: 1.309–24.035)), and voluntary (p = 0.034, OR: 2.404 (CI: 1.071–5.397)) donors were significantly associated with SCT. Conclusion. Screening for red cell pathologies must be incorporated into existing protocols for populations with high incidence of haemoglobinopathies to protect high-risk recipients. PMID:27703480

  9. Serine Arginine Splicing Factor 3 Is Involved in Enhanced Splicing of Glucose-6-phosphate Dehydrogenase RNA in Response to Nutrients and Hormones in Liver*

    PubMed Central

    Walsh, Callee M.; Suchanek, Amanda L.; Cyphert, Travis J.; Kohan, Alison B.; Szeszel-Fedorowicz, Wioletta; Salati, Lisa M.

    2013-01-01

    Expression of G6PD is controlled by changes in the degree of splicing of the G6PD mRNA in response to nutrients in the diet. This regulation involves an exonic splicing enhancer (ESE) in exon 12 of the mRNA. Using the G6PD model, we demonstrate that nutrients and hormones control the activity of serine-arginine-rich (SR) proteins, a family of splicing co-activators, and thereby regulate the splicing of G6PD mRNA. In primary rat hepatocyte cultures, insulin increased the amount of phosphorylated SR proteins, and this effect was counteracted by arachidonic acid. The results of RNA affinity analysis with nuclear extracts from intact liver demonstrated that the SR splicing factor proteins SRSF3 and SRSF4 bound to the G6PD ESE. Consequently, siRNA-mediated depletion of SRSF3, but not SRSF4, in liver cells inhibited accumulation of both mRNA expressed from a minigene containing exon 12 and the endogenous G6PD mRNA. Consistent with the functional role of SRSF3 in regulating splicing, SRSF3 was observed to bind to the ESE in both intact cells and in animals using RNA immunoprecipitation analysis. Furthermore, refeeding significantly increased the binding of SRSF3 coincident with increased splicing and expression of G6PD. Together, these data establish that nutritional regulation of SRSF3 activity is involved in the differential splicing of the G6PD transcript in response to nutrients. Nutritional regulation of other SR proteins presents a regulatory mechanism that could cause widespread changes in mRNA splicing. Nutrients are therefore novel regulators of mRNA splicing. PMID:23233666

  10. Glucose 6-phosphate dehydrogenase variants: Gd (+) Alexandra associated with neonatal jaundice and Gd (-) Camperdown in a young man with lamellar cataracts.

    PubMed

    Harley, J D; Agar, N S; Yoshida, A

    1978-02-01

    Two male subjects are described, with unusual clinical presentations and with hitherto undescribed G6PD variants. The first, of Italian extraction, suffered from severe neonatal jaundice following maternal ingestion of fresh broad beans (Vicia fava) both prenatally and postnatally: the expression of the enzymatic defect was much more severe in the neonatal period than on retesting in adolescence, when biochemical characterization showed unique features which justify designation as a new variant Gd(+) Alexandra. The second patient, a boy of Maltese extraction who was found to have bilateral lamellar cataracts at the age of 4 years, was identified as G6PD deficient only as a result of a survey of children of Mediterranean origin with unexplained cataract formation; he has approximately 15% of normal enzyme activity, with another unique combination of biochemical characteristics which has led to its designation as Gd(-) Camperdown. Although this association may be coincidental, it prompts further attention to the possibility that under certain circumstances G6PD deficiency may favor cataract formation. The two cases illustrate the value of characterization of the mutant enzyme whenever unexpected clinical or laboratory results are obtained. PMID:23402

  11. The effects of salt stress cause a diversion of basal metabolism in barley roots: possible different roles for glucose-6-phosphate dehydrogenase isoforms.

    PubMed

    Cardi, Manuela; Castiglia, Daniela; Ferrara, Myriam; Guerriero, Gea; Chiurazzi, Maurizio; Esposito, Sergio

    2015-01-01

    In this study the effects of salt stress and nitrogen assimilation have been investigated in roots of hydroponically-grown barley plants exposed to 150 mM NaCl, in presence or absence of ammonium as the sole nitrogen source. Salt stress determines a diversion of root metabolism towards the synthesis of osmolytes, such as glycine betaine and proline, and increased levels of reduced glutathione. The metabolic changes triggered by salt stress result in a decrease in both activities and protein abundance of key enzymes, namely GOGAT and PEP carboxylase, and in a slight increase in HSP70. These variations would enhance the requirement for reductants supplied by the OPPP, consistently with the observed increase in total G6PDH activity. The involvement and occurrence of the different G6PDH isoforms have been investigated, and the kinetic properties of partially purified cytosolic and plastidial G6PDHs determined. Bioinformatic analyses examining co-expression profiles of G6PDHs in Arabidopsis and barley corroborate the data presented. Moreover, the gene coding for the root P2-G6PDH isoform was fully sequenced; the biochemical properties of the corresponding protein were examined experimentally. The results are discussed in the light of the possible distinct roles and regulation of the different G6PDH isoforms during salt stress in barley roots.

  12. Over-estimation of glucose-6-phosphatase activity in brain in vivo. Apparent difference in rates of (2-TH)glucose and (U- UC)glucose utilization is due to contamination of precursor pool with UC-labeled products and incomplete recovery of UC-labeled metabolites

    SciTech Connect

    Dienel, G.A.; Nelson, T.; Cruz, N.F.; Jay, T.; Crane, A.M.; Sokoloff, L.

    1988-12-25

    Significant dephosphorylation of glucose 6-phosphate due to glucose-6-phosphatase activity in rat brain in vivo was recently reported. The evidence was an apparent more rapid TH than UC loss from the glucose pool and faster (2-TH)glucose than (U- UC)glucose utilization following pulse labeling of the brain with (2-TH,U- UC)glucose. Radiochemical purity of the glucose and quantitative recovery of the labeled products of glucose metabolism isolated from the brain were obviously essential requirements of their study, but no evidence for purity and recovery was provided. When we repeated these experiments with the described isolation procedures, we replicated the results, but found that: 1) the precursor glucose pool contained detritiated, UC-labeled contaminants arising from glucose metabolism, particularly 2-pyrrolidone-5-carboxylic acid derived from ( UC)glutamine; 2) ( UC)glucose metabolite were not quantitatively recovered; 3) the procedure used to isolate the glucose itself produced detritiated, UC-labeled derivatives of (2-TH,U-14C)glucose. These deficiencies in the isolation procedures could fully account for the observations that were interpreted as evidence of significant glucose 6-phosphate dephosphorylation by glucose-6-phosphatase activity. When glucose was isolated by more rigorous procedures and its purity verified in the present studies, no evidence for such activity in rat brain was found.

  13. Structural Basis for Substrate Specificity in Phosphate Binding (beta/alpha)8-Barrels: D-Allulose 6-Phosphate 3-Epimerase from Escherichia coli K-12

    SciTech Connect

    Chan,K.; Fedorov, A.; Almo, S.; Gerlt, J.

    2008-01-01

    Enzymes that share the ({beta}/{alpha})8-barrel fold catalyze a diverse range of reactions. Many utilize phosphorylated substrates and share a conserved C-terminal ({beta}/a)2-quarter barrel subdomain that provides a binding motif for the dianionic phosphate group. We recently reported functional and structural studies of d-ribulose 5-phosphate 3-epimerase (RPE) from Streptococcus pyogenes that catalyzes the equilibration of the pentulose 5-phosphates d-ribulose 5-phosphate and d-xylulose 5-phosphate in the pentose phosphate pathway [J. Akana, A. A. Fedorov, E. Fedorov, W. R. P. Novack, P. C. Babbitt, S. C. Almo, and J. A. Gerlt (2006) Biochemistry 45, 2493-2503]. We now report functional and structural studies of d-allulose 6-phosphate 3-epimerase (ALSE) from Escherichia coli K-12 that catalyzes the equilibration of the hexulose 6-phosphates d-allulose 6-phosphate and d-fructose 6-phosphate in a catabolic pathway for d-allose. ALSE and RPE prefer their physiological substrates but are promiscuous for each other's substrate. The active sites (RPE complexed with d-xylitol 5-phosphate and ALSE complexed with d-glucitol 6-phosphate) are superimposable (as expected from their 39% sequence identity), with the exception of the phosphate binding motif. The loop following the eighth {beta}-strand in ALSE is one residue longer than the homologous loop in RPE, so the binding site for the hexulose 6-phosphate substrate/product in ALSE is elongated relative to that for the pentulose 5-phosphate substrate/product in RPE. We constructed three single-residue deletion mutants of the loop in ALSE, ?T196, ?S197 and ?G198, to investigate the structural bases for the differing substrate specificities; for each, the promiscuity is altered so that d-ribulose 5-phosphate is the preferred substrate. The changes in kcat/Km are dominated by changes in kcat, suggesting that substrate discrimination results from differential transition state stabilization. In both ALSE and RPE, the phosphate

  14. Abnormalities of the erythrocyte membrane.

    PubMed

    Gallagher, Patrick G

    2013-12-01

    Primary abnormalities of the erythrocyte membrane are characterized by clinical, laboratory, and genetic heterogeneity. Among this group, hereditary spherocytosis patients are more likely to experience symptomatic anemia. Treatment of hereditary spherocytosis with splenectomy is curative in most patients. Growing recognition of the long-term risks of splenectomy has led to re-evaluation of the role of splenectomy. Management guidelines acknowledge these considerations and recommend discussion between health care providers, patient, and family. The hereditary elliptocytosis syndromes are the most common primary disorders of erythrocyte membrane proteins. However, most elliptocytosis patients are asymptomatic and do not require therapy.

  15. Current status of erythrocyte substitutes.

    PubMed Central

    Biro, G. P.

    1983-01-01

    During the last two decades the search for alternatives to whole blood transfusions has led to promising developments in the field of erythrocyte substitutes. Hemoglobin solutions free of fragments of erythrocyte stroma and fluorocarbon emulsions are not blood-type-specific and appear likely to satisfy some proportion of our blood requirements. Both must be modified before becoming clinically useful. The oxygen affinity of the hemoglobin solution must be reduced and its intravascular persistence improved. Fluorocarbons cannot yet contribute significantly to the oxygen supply unless the patient breathes hyperbaric oxygen. Recent advances are leading to solutions for these problems. PMID:6344974

  16. A Demonstration of Erythrocyte Membrane Asymmetry.

    ERIC Educational Resources Information Center

    Pederson, Philip; And Others

    1985-01-01

    A three-period experiment was developed to help students visualize asymmetric distribution of proteins within membranes. It includes: (1) isolating erythrocyte membranes; (2) differential labeling of intact erythrocytes and isolated erythrocyte membranes with an impermeable fluorescent dye; and (3) separating proteins by polyacrylamide gel…

  17. Mice Lacking Mannose 6-Phosphate Uncovering Enzyme Activity Have a Milder Phenotype than Mice Deficient for N-Acetylglucosamine-1-Phosphotransferase Activity

    PubMed Central

    Boonen, Marielle; Vogel, Peter; Platt, Kenneth A.; Dahms, Nancy

    2009-01-01

    The mannose 6-phosphate (Man-6-P) lysosomal targeting signal on acid hydrolases is synthesized by the sequential action of uridine 5′-diphosphate-N-acetylglucosamine: lysosomal enzyme N-acetylglucosamine-1-phosphotransferase (GlcNAc-1-phosphotransferase) and GlcNAc-1-phosphodiester α-N-acetylglucosaminidase (“uncovering enzyme” or UCE). Mutations in the two genes that encode GlcNAc-1-phosphotransferase give rise to lysosomal storage diseases (mucolipidosis type II and III), whereas no pathological conditions have been associated with the loss of UCE activity. To analyze the consequences of UCE deficiency, the UCE gene was inactivated via insertional mutagenesis in mice. The UCE −/− mice were viable, grew normally and lacked detectable histologic abnormalities. However, the plasma levels of six acid hydrolases were elevated 1.6- to 5.4-fold over wild-type levels. These values underestimate the degree of hydrolase hypersecretion as these enzymes were rapidly cleared from the plasma by the mannose receptor. The secreted hydrolases contained GlcNAc-P-Man diesters, exhibited a decreased affinity for the cation-independent mannose 6-phosphate receptor and failed to bind to the cation-dependent mannose 6-phosphate receptor. These data demonstrate that UCE accounts for all the uncovering activity in the Golgi. We propose that in the absence of UCE, the weak binding of the acid hydrolases to the cation-independent mannose 6-phosphate receptor allows sufficient sorting to lysosomes to prevent the tissue abnormalities seen with GlcNAc-1-phosphotranferase deficiency. PMID:19710420

  18. L-ribose production from L-arabinose by using purified L-arabinose isomerase and mannose-6-phosphate isomerase from Geobacillus thermodenitrificans.

    PubMed

    Yeom, Soo-Jin; Kim, Nam-Hee; Park, Chang-Su; Oh, Deok-Kun

    2009-11-01

    Two enzymes, L-arabinose isomerase and mannose-6-phosphate isomerase, from Geobacillus thermodenitrificans produced 118 g/liter L-ribose from 500 g/liter L-arabinose at pH 7.0, 70 degrees C, and 1 mM Co(2+) for 3 h, with a conversion yield of 23.6% and a volumetric productivity of 39.3 g liter(-1) h(-1).

  19. Against All Odds: Trehalose-6-Phosphate Synthase and Trehalase Genes in the Bdelloid Rotifer Adineta vaga Were Acquired by Horizontal Gene Transfer and Are Upregulated during Desiccation

    PubMed Central

    Hespeels, Boris; Li, Xiang; Flot, Jean-François; Pigneur, Lise-Marie; Malaisse, Jeremy; Da Silva, Corinne; Van Doninck, Karine

    2015-01-01

    The disaccharide sugar trehalose is essential for desiccation resistance in most metazoans that survive dryness; however, neither trehalose nor the enzymes involved in its metabolism have ever been detected in bdelloid rotifers despite their extreme resistance to desiccation. Here we screened the genome of the bdelloid rotifer Adineta vaga for genes involved in trehalose metabolism. We discovered a total of four putative trehalose-6-phosphate synthase (TPS) and seven putative trehalase (TRE) gene copies in the genome of this ameiotic organism; however, no trehalose-6-phosphate phosphatase (TPP) gene or domain was detected. The four TPS copies of A. vaga appear more closely related to plant and fungi proteins, as well as to some protists, whereas the seven TRE copies fall in bacterial clades. Therefore, A. vaga likely acquired its trehalose biosynthesis and hydrolysis genes by horizontal gene transfers. Nearly all residues important for substrate binding in the predicted TPS domains are highly conserved, supporting the hypothesis that several copies of the genes might be functional. Besides, RNAseq library screening showed that trehalase genes were highly expressed compared to TPS genes, explaining probably why trehalose had not been detected in previous studies of bdelloids. A strong overexpression of their TPS genes was observed when bdelloids enter desiccation, suggesting a possible signaling role of trehalose-6-phosphate or trehalose in this process. PMID:26161530

  20. Targeted disruption of the M(r) 46,000 mannose 6-phosphate receptor gene in mice results in misrouting of lysosomal proteins.

    PubMed Central

    Köster, A; Saftig, P; Matzner, U; von Figura, K; Peters, C; Pohlmann, R

    1993-01-01

    Lysosomal enzymes containing mannose 6-phosphate recognition markers are sorted to lysosomes by mannose 6-phosphate receptors (MPRs). The physiological importance of this targeting mechanism is illustrated by I-cell disease, a fatal lysosomal storage disorder caused by the absence of mannose 6-phosphate residues in lysosomal enzymes. Most mammalian cells express two MPRs. Although the binding specificities, subcellular distribution and expression pattern of the two receptors can be differentiated, their coexpression is not understood. The larger of the two receptors with an M(r) of approximately 300,000 (MPR300), which also binds IGFII, appears to have a dominant role in lysosomal enzyme targeting, while the function of the smaller receptor with an M(r) of 46,000 (MPR46) is less clear. To investigate the in vivo function of the MPR46, we generated MPR46-deficient mice using gene targeting in embryonic stem cells. Reduced intracellular retention of newly synthesized lysosomal proteins in cells from MPR46 -/- mice demonstrated an essential sorting function of MPR46. The phenotype of MPR46 -/- mice was normal, indicating mechanisms that compensate the MPR46 deficiency in vivo. Images PMID:8262064

  1. Evaluation of synthase and hemisynthase activities of glucosamine-6-phosphate synthase by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

    PubMed

    Gaucher-Wieczorek, Florence; Guérineau, Vincent; Touboul, David; Thétiot-Laurent, Sophie; Pelissier, Franck; Badet-Denisot, Marie-Ange; Badet, Bernard; Durand, Philippe

    2014-08-01

    Glucosamine-6-phosphate synthase (GlmS, EC 2.6.1.16) catalyzes the first and rate-limiting step in the hexosamine biosynthetic pathway, leading to the synthesis of uridine-5'-diphospho-N-acetyl-D-glucosamine, the major building block for the edification of peptidoglycan in bacteria, chitin in fungi, and glycoproteins in mammals. This bisubstrate enzyme converts D-fructose-6-phosphate (Fru-6P) and L-glutamine (Gln) into D-glucosamine-6-phosphate (GlcN-6P) and L-glutamate (Glu), respectively. We previously demonstrated that matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) allows determination of the kinetic parameters of the synthase activity. We propose here to refine the experimental protocol to quantify Glu and GlcN-6P, allowing determination of both hemisynthase and synthase parameters from a single assay kinetic experiment, while avoiding interferences encountered in other assays. It is the first time that MALDI-MS is used to survey the activity of a bisubstrate enzyme.

  2. Against All Odds: Trehalose-6-Phosphate Synthase and Trehalase Genes in the Bdelloid Rotifer Adineta vaga Were Acquired by Horizontal Gene Transfer and Are Upregulated during Desiccation.

    PubMed

    Hespeels, Boris; Li, Xiang; Flot, Jean-François; Pigneur, Lise-Marie; Malaisse, Jeremy; Da Silva, Corinne; Van Doninck, Karine

    2015-01-01

    The disaccharide sugar trehalose is essential for desiccation resistance in most metazoans that survive dryness; however, neither trehalose nor the enzymes involved in its metabolism have ever been detected in bdelloid rotifers despite their extreme resistance to desiccation. Here we screened the genome of the bdelloid rotifer Adineta vaga for genes involved in trehalose metabolism. We discovered a total of four putative trehalose-6-phosphate synthase (TPS) and seven putative trehalase (TRE) gene copies in the genome of this ameiotic organism; however, no trehalose-6-phosphate phosphatase (TPP) gene or domain was detected. The four TPS copies of A. vaga appear more closely related to plant and fungi proteins, as well as to some protists, whereas the seven TRE copies fall in bacterial clades. Therefore, A. vaga likely acquired its trehalose biosynthesis and hydrolysis genes by horizontal gene transfers. Nearly all residues important for substrate binding in the predicted TPS domains are highly conserved, supporting the hypothesis that several copies of the genes might be functional. Besides, RNAseq library screening showed that trehalase genes were highly expressed compared to TPS genes, explaining probably why trehalose had not been detected in previous studies of bdelloids. A strong overexpression of their TPS genes was observed when bdelloids enter desiccation, suggesting a possible signaling role of trehalose-6-phosphate or trehalose in this process.

  3. Recognition of human urine alpha-N-acetylglucosaminidase by rat hepatocytes. Involvement of receptors specific for galactose, mannose 6-phosphate and mannose.

    PubMed

    Ullrich, K; Basner, R; Gieselmann, V; Von Figura, K

    1979-05-15

    Adsorptive endocytosis of alpha-N-acetylglucosaminidase from human urine by isolated rat hepatocytes is inhibited by glycoproteins, polysaccharides and sugars that are known to bind to cell-surface receptors specific for either terminal galactose/N-acetylgalactosamine residues, terminal mannose residues or mannose 6-phosphate residues. Recognition of alpha-N-acetylglucosaminidase by a cell-surface receptor specific for terminal galactose/N-acetylgalactosamine residues is supported by the observations (a) that neuraminidase pretreatment of the enzyme enhances endocytosis, (b) that beta-galactosidase treatment decreases endocytosis and (c) that neuraminidase pretreatment of hepatocytes decreases alpha-N-acetylglucosaminidase endocytosis. Recognition of alpha-N-acetylglucosaminidase via receptors recognizing mannose 6-phosphate residues is lost after treatment of the enzyme with alkaline phosphatase and endoglucosaminidase H. The effect of endoglucosaminidase H supports the view that the mannose 6-phosphate residues reside in N-glycosidically linked oligosaccharide side chains of the high-mannose type. The weak inhibition of endocytosis produced by compounds known to interact with cell-surface receptors specific for mannose residues suggests that this recognition system plays only a minor role in the endocytosis of lysosomal alpha-N-acetylglucosaminidase by hepatocytes. PMID:114170

  4. Recognition of human urine alpha-N-acetylglucosaminidase by rat hepatocytes. Involvement of receptors specific for galactose, mannose 6-phosphate and mannose.

    PubMed Central

    Ullrich, K; Basner, R; Gieselmann, V; Von Figura, K

    1979-01-01

    Adsorptive endocytosis of alpha-N-acetylglucosaminidase from human urine by isolated rat hepatocytes is inhibited by glycoproteins, polysaccharides and sugars that are known to bind to cell-surface receptors specific for either terminal galactose/N-acetylgalactosamine residues, terminal mannose residues or mannose 6-phosphate residues. Recognition of alpha-N-acetylglucosaminidase by a cell-surface receptor specific for terminal galactose/N-acetylgalactosamine residues is supported by the observations (a) that neuraminidase pretreatment of the enzyme enhances endocytosis, (b) that beta-galactosidase treatment decreases endocytosis and (c) that neuraminidase pretreatment of hepatocytes decreases alpha-N-acetylglucosaminidase endocytosis. Recognition of alpha-N-acetylglucosaminidase via receptors recognizing mannose 6-phosphate residues is lost after treatment of the enzyme with alkaline phosphatase and endoglucosaminidase H. The effect of endoglucosaminidase H supports the view that the mannose 6-phosphate residues reside in N-glycosidically linked oligosaccharide side chains of the high-mannose type. The weak inhibition of endocytosis produced by compounds known to interact with cell-surface receptors specific for mannose residues suggests that this recognition system plays only a minor role in the endocytosis of lysosomal alpha-N-acetylglucosaminidase by hepatocytes. PMID:114170

  5. Role of Endogenous Factors in Response of Erythrocyte Membrane in Patients with Cardiovascular Diseases under Conditions of Ischemic Exposure.

    PubMed

    Pivovarov, Yu I; Kuznetsova, E E; Koryakina, L B; Gorokhova, V G; Kuril'skaya, T E

    2015-05-01

    We studied specific features of erythrocyte membrane response to short-term occlusion of the brachial artery in patients with cardiovascular pathology. Under ischemic conditions, processes of sorption were primarily intensified in patients with effort angina and processes of hemoglobin binding with erythrocyte membrane predominated in patients with essential hypertension. These changes in the cell membrane were related to modulation of aggregation properties of erythrocytes (in patients with angina) and plasminogen activity (in patients with essential hypertension). They can also be associated with changes in glucose levels (effort angina) and uric acid (essential hypertension) whose effects can be significantly modified by other endogenous factors.

  6. Glucose Catabolism in Micrococcus sodonensis1

    PubMed Central

    Perry, Jerome J.; Evans, James B.

    1967-01-01

    The inability of Micrococcus sodonensis to grow on glucose as the sole source of carbon and energy was investigated. Estimation of pathways of glucose catabolism indicated that both the glycolytic and hexose monophosphate pathways are present in this organism. Comparative studies with Escherichia coli demonstrated that key enzymes for glucose catabolism were present in M. sodonensis in quantities equivalent to those of E. coli. The glucose-6-phosphate and 6-phosphogluconate dehydrogenases of M. sodonensis were nicotinamide adenine dinucleotide phosphate (NADP) specific, and glyceraldehyde-3-phosphate dehydrogenase was nicotinamide adenine dinucleotide specific. Transhydrogenase and reduced NADP oxidase were absent. Growth of the organism in the presence of glucose did not result in a repressed ability to oxidize tricarboxylic acid cycle intermediates, but these cells did have a decreased capacity for glucose degradation. The addition of substrates rich in growth-promoting substances, e.g., yeast extract, did not provide requisite nutrients for growth on glucose. Studies with 32P suggest that M. sodonensis is incapable of synthesizing energy-rich phosphate compounds during the catabolism of glucose. PMID:4381630

  7. Identification of the phorbol ester receptor in human and avian erythrocytes

    SciTech Connect

    Kramer, C.M.; Sando, J.J.; Speizer, L.A.

    1986-05-01

    The ability of phorbol esters to inhibit the uptake of a fluorescent glucose analogue in goose but not human erythrocytes is consistent with earlier reports that the human red blood cell lacks the phorbol ester receptor. However, they have located specific phorbol 12,13-dibutyrate binding sites in both human and goose erythrocytes. Human and goose red blood cells contain 2 classes of phorbol ester receptors with similar affinities, however the human erythrocyte contains 1/3 as many phorbol ester receptors as does the goose red blood cell. An additional contrast in the binding of phorbol esters to human and goose red blood cells is the temperature-induced enhancement of binding to goose, but not human erythrocytes. Equilibrium phorbol ester binding to goose red blood cells at 37/sup 0/C is enhanced 3.3 +/- 0.4 times that amount bound at 4/sup 0/C. Equilibrium binding of phorbol esters to human erythrocytes is identical at both temperatures. In vivo and in vitro phosphorylation profiles of C-kinase substrates also differ between the human and goose erythrocyte.

  8. Erythrocyte Catabolism by Macrophages In Vitro THE EFFECT OF HYDROCORTISONE ON ERYTHROPHAGOCYTOSIS AND ON THE INDUCTION OF HEME OXYGENASE

    PubMed Central

    Gemsa, Diethard; Woo, C. H.; Fudenberg, H. Hugh; Schmid, Rudi

    1973-01-01

    Phagocytosis of erythrocytes was studied in vitro in an incubation system consisting of rat peritoneal macrophages and antibody-coated 59Fe-labeled erythrocytes. The system was characterized in terms of the rate and magnitude of erythrophagocytosis, determined by the interiorization of the 59Fe label. On incubation of 150 × 106 macrophages with 75 × 106 antibodycoated erythrocytes, erythrophagocytosis began within a few minutes and was essentially completed after 2 h when 50% of the offered red cells had been ingested by the macrophages. Heme oxygenase (HO) activity, which is very low in native macrophages, increased 4- to 10- fold in response to the ingested erythrocytes; this enzyme stimulation occurred with a delay of 3 h in relation to erythrophagocytosis. Actinomycin D or puromycin prevented the increase of HO activity without affecting erythrophagocytosis, which suggests that the enzyme stimulation was due to substrate-mediated enzyme induction. Hydrocortisone (HC) (0.1 mg/ml medium) dissociated erythrophagocytosis from HO induction, leaving the former unimpaired but completely suppressing the latter. The suppressive effect of HC on the enzyme induction was completely prevented by 5 mg glucose and 0.02 U insulin/ml of the medium. In macrophages engaged in erythrophagocytosis. HC also lowered glucose removal from the medium and reduced formation of 14CO2 from [1-14C]glucose. These results suggest that induction of HO in macrophages by the hemoglobin of ingested erythrocytes requires intact transport or metabolism of glucose. Glucose utilization appears to be impaired by HC, but is restored by additional glucose and insulin. The findings suggest that plasma steroid concentrations in the pharmacological range could reduce bilirubin formation in phagocytic cells in vivo without affecting the sequestration and degradation of erythrocytes. This provides a possible explanation for the observation that in patients with hepatogenous jaundice, steroids often lower

  9. Proteomic Profiling of Nonenzymatically Glycated Proteins in Human Plasma and Erythrocyte Membrane

    SciTech Connect

    Zhang, Qibin; Tang, Ning; Schepmoes, Athena A.; Phillips, Lawrence S.; Smith, Richard D.; Metz, Thomas O.

    2008-05-01

    Non-enzymatic glycation of peptides and proteins by D-glucose has important implications in the pathogenesis of diabetes mellitus, particularly in the development of diabetic complications. In this report, a thorough proteomic profiling of glycated proteins was attempted by using phenylboronate affinity chromatography to enrich glycated proteins and glycated, tryptic peptides from human plasma and erythrocyte membranes. Enriched peptides were subsequently analyzed by liquid chromatography coupled with electron transfer dissociation tandem mass spectrometry, and 76 and 31 proteins were confidently identified as glycated from human plasma and erythrocyte membrane, respectively. It was observed that most of the glycated proteins can be identified in samples from individuals with normal glucose tolerance, although samples from individuals with impaired glucose tolerance and type 2 diabetes mellitus have slightly higher numbers of glycated proteins and more glycation sites identified.

  10. Effect of pretreatment with pentobarbital on the extent of (/sup 14/C) incorporation from (U-/sup 14/C)glucose into various rat brain glycolytic intermediates: relevance to regulation at hexokinase and phosphofructokinase

    SciTech Connect

    Miller, L.P.; Mayer, S.; Braun, L.D.; Geiger, P.; Oldendorf, W.H.

    1988-04-01

    In the present investigation we monitored the incorporation of (14C) from (U-14C)glucose into various rat brain glycolytic intermediates of conscious and pentobarbital-anesthetized animals. Labeled glucose was delivered to brain by single bolus intracarotid injection and brain tissue was subsequently prepared at 15, 30, and 45 sec by freeze-blowing. Glycolytic intermediates were then separated by column chromatography. Our results showed a gradual decrease with time of 14C-labeled glucose which gave a calculated rate for glucose metabolism of 0.86 mumol/min/g and 0.56 mumol/min/g in conscious and anesthetized animals, respectively. Compared to the results obtained using conscious animals the administration of pentobarbital not only resulted in a significant attenuation of the rate of glucose metabolism but also caused a similar reduction in the amount of 14C incorporated into several glycolytic intermediates. These intermediates included: glucose 6-phosphate, fructose 6-phosphate, fructose 1,6 diphosphate, dihydroxyacetone phosphate and post glycolytic compounds. In addition, pretreatment with pentobarbital resulted in a 75% increase in the endogenous concentration of glucose, 10% increase in glucose 6-phosphate, no change in fructose 6-phosphate and 42% decrease in lactate compared to levels in brains obtained from conscious animals. These results are discussed in relation to control of glycolysis through coupled regulation at hexokinase-phosphofructokinase.

  11. [Glucose-6-phosphatase from nuclear envelope in rat liver].

    PubMed

    González-Mujica, Freddy

    2008-06-01

    Nuclear envelope (NE) and microsomal glucosa-6-phosphatase (G-6-Pase) activities were compared. Intact microsomes were unable to hydrolyze mannose-6-phosphate (M-6-P), on the other hand, intact NE hydrolyzes this substrate. Galactose-6-phosphate showed to be a good substrate for both NE and microsomal enzymes, with similar latency to that obtained with M-6-P using microsomes. In consequence, this substrate was used to measure the NE integrity. The kinetic parameters (Kii and Kis) of the intact NE G-6-Pase for the phlorizin inhibition using glucose-6-phosphate (G-6-P) and M-6-P as substrates, were very similar. The NE T1 transporter was more sensitive to amiloride than the microsomal T1. The microsomal system was more sensitive to N-ethylmalemide (NEM) than the NE and the latter was insensitive to anion transport inhibitors DIDS and SITS, which strongly affect the microsomal enzyme. The above results allowed to postulate the presence of a hexose-6-phosphate transporter in the NE which is able to carry G-6-P and M-6-P, and perhaps other hexose-6-phosphate which could be different from that present in microsomes or, if it is the same, its activity could by modified by the membrane system where it is included. The higher PPi hydrolysis activity of the intact NE G-6-Pase in comparison to the intact microsomal, suggests differences between the Pi/PPi transport (T2) of both systems. The lower sensitivity of the NE G-6-Pase to NEM suggests that the catalytic subunit of this system has some differences with the microsomal isoform. PMID:18717264

  12. Global N-linked Glycosylation is Not Significantly Impaired in Myoblasts in Congenital Myasthenic Syndromes Caused by Defective Glutamine-Fructose-6-Phosphate Transaminase 1 (GFPT1)

    PubMed Central

    Chen, Qiushi; Müller, Juliane S.; Pang, Poh-Choo; Laval, Steve H.; Haslam, Stuart M.; Lochmüller, Hanns; Dell, Anne

    2015-01-01

    Glutamine-fructose-6-phosphate transaminase 1 (GFPT1) is the first enzyme of the hexosamine biosynthetic pathway. It transfers an amino group from glutamine to fructose-6-phosphate to yield glucosamine-6-phosphate, thus providing the precursor for uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) synthesis. UDP-GlcNAc is an essential substrate for all mammalian glycosylation biosynthetic pathways and N-glycan branching is especially sensitive to alterations in the concentration of this sugar nucleotide. It has been reported that GFPT1 mutations lead to a distinct sub-class of congenital myasthenic syndromes (CMS) termed “limb-girdle CMS with tubular aggregates”. CMS are hereditary neuromuscular transmission disorders in which neuromuscular junctions are impaired. To investigate whether alterations in protein glycosylation at the neuromuscular junction might be involved in this impairment, we have employed mass spectrometric strategies to study the N-glycomes of myoblasts and myotubes derived from two healthy controls, three GFPT1 patients, and four patients with other muscular diseases, namely CMS caused by mutations in DOK7, myopathy caused by mutations in MTND5, limb girdle muscular dystrophy type 2A (LGMD2A), and Pompe disease. A comparison of the relative abundances of bi-, tri-, and tetra-antennary N-glycans in each of the cell preparations revealed that all samples exhibited broadly similar levels of branching. Moreover, although some differences were observed in the relative abundances of some of the N-glycan constituents, these variations were modest and were not confined to the GFPT1 samples. Therefore, GFPT1 mutations in CMS patients do not appear to compromise global N-glycosylation in muscle cells. PMID:26501342

  13. Can erythrocytes release biologically active NO?

    PubMed

    Benz, Peter M; Fleming, Ingrid

    2016-01-01

    Under physiological conditions, endothelial cells and the endothelial nitric oxide (NO) synthase (eNOS) are the main source of NO in the cardiovascular system. However, several other cell types have also been implicated in the NO-dependent regulation of cell function, including erythrocytes. NO derived from red blood cells has been proposed to regulate erythrocyte membrane fluidity, inhibit platelet activation and induce vasodilation in hypoxic areas, but these proposals are highly controversial. In the current issue of Cell Communication and Signaling, an elegant study by Gambaryan et al., assayed NO production by erythrocytes by monitoring the activation of the platelet intracellular NO receptor, soluble guanylyl cyclase, and its downstream kinase protein kinase G. After systematically testing different combinations of erythrocyte/platelet suspensions, the authors found no evidence for platelet soluble guanylyl cyclase/protein kinase G activation by erythrocytes and conclude that erythrocytes do not release biologically active NO to inhibit platelet activation. PMID:27639852

  14. Optical tweezer for probing erythrocyte membrane deformability

    NASA Astrophysics Data System (ADS)

    Khan, Manas; Soni, Harsh; Sood, A. K.

    2009-12-01

    We report that the average rotation speed of optically trapped crenated erythrocytes is direct signature of their membrane deformability. When placed in hypertonic buffer, discocytic erythrocytes are subjected to crenation. The deformation of cells brings in chirality and asymmetry in shape that makes them rotate under the scattering force of a linearly polarized optical trap. A change in the deformability of the erythrocytes, due to any internal or environmental factor, affects the rotation speed of the trapped crenated cells. Here we show how the increment in erythrocyte membrane rigidity with adsorption of Ca++ ions can be exhibited through this approach.

  15. Characterization, Localization, Essentiality, and High-Resolution Crystal Structure of Glucosamine 6-Phosphate N-Acetyltransferase from Trypanosoma brucei ▿ ‡ §

    PubMed Central

    Mariño, Karina; Güther, M. Lucia Sampaio; Wernimont, Amy K.; Qiu, Wei; Hui, Raymond; Ferguson, Michael A. J.

    2011-01-01

    A gene predicted to encode Trypanosoma brucei glucosamine 6-phosphate N-acetyltransferase (TbGNA1; EC 2.3.1.4) was cloned and expressed in Escherichia coli. The recombinant protein was enzymatically active, and its high-resolution crystal structure was obtained at 1.86 Å. Endogenous TbGNA1 protein was localized to the peroxisome-like microbody, the glycosome. A bloodstream-form T. brucei GNA1 conditional null mutant was constructed and shown to be unable to sustain growth in vitro under nonpermissive conditions, demonstrating that there are no metabolic or nutritional routes to UDP-GlcNAc other than via GlcNAc-6-phosphate. Analysis of the protein glycosylation phenotype of the TbGNA1 mutant under nonpermissive conditions revealed that poly-N-acetyllactosamine structures were greatly reduced in the parasite and that the glycosylation profile of the principal parasite surface coat component, the variant surface glycoprotein (VSG), was modified. The significance of results and the potential of TbGNA1 as a novel drug target for African sleeping sickness are discussed. PMID:21531872

  16. RNA interference of a trehalose-6-phosphate synthase gene reveals its roles during larval-pupal metamorphosis in Bactrocera minax (Diptera: Tephritidae).

    PubMed

    Xiong, Ke-Cai; Wang, Jia; Li, Jia-Hao; Deng, Yu-Qing; Pu, Po; Fan, Huan; Liu, Ying-Hong

    2016-01-01

    Trehalose is the major blood sugar in insects, which plays a crucial role as an instant source of energy and the starting substrate for chitin biosynthesis. In insects, trehalose is synthesized by catalysis of an important enzyme, trehalose-6-phosphate synthase (TPS). In the present study, a trehalose-6-phosphate synthase gene from Bactrocera minax (BmTPS) was cloned and characterized. BmTPS contained an open reading frame of 2445 nucleotides encoding a protein of 814 amino acids with a predicted molecular weight of 92.05kDa. BmTPS was detectable in all developmental stages of Bactrocera minax and expressed higher in the final- (third-) instar larvae. Tissue-specific expression patterns of BmTPS showed that it was mainly expressed in the fat body. The 20-hydroxyecdysone (20E) induced the expression of BmTPS and three genes in the chitin biosynthesis pathway. Moreover, injection of double-stranded RNA into third-instar larvae successfully silenced the transcription of BmTPS in B. minax, and thereby decreased the activity of TPS and trehalose content. Additionally, silencing of BmTPS inhibited the expression of three key genes in the chitin biosynthesis pathway and exhibited 52% death and abnormal phenotypes. The findings demonstrate that BmTPS is indispensable for larval-pupal metamorphosis. Besides, the establishment of RNAi experimental system in B. minax would lay a solid foundation for further investigation of molecular biology and physiology of this pest. PMID:27405007

  17. Substrate specificity of a mannose-6-phosphate isomerase from Bacillus subtilis and its application in the production of L-ribose.

    PubMed

    Yeom, Soo-Jin; Ji, Jung-Hwan; Kim, Nam-Hee; Park, Chang-Su; Oh, Deok-Kun

    2009-07-01

    The uncharacterized gene previously proposed as a mannose-6-phosphate isomerase from Bacillus subtilis was cloned and expressed in Escherichia coli. The maximal activity of the recombinant enzyme was observed at pH 7.5 and 40 degrees C in the presence of 0.5 mM Co(2+). The isomerization activity was specific for aldose substrates possessing hydroxyl groups oriented in the same direction at the C-2 and C-3 positions, such as the d and l forms of ribose, lyxose, talose, mannose, and allose. The enzyme exhibited the highest activity for l-ribulose among all pentoses and hexoses. Thus, L-ribose, as a potential starting material for many L-nucleoside-based pharmaceutical compounds, was produced at 213 g/liter from 300-g/liter L-ribulose by mannose-6-phosphate isomerase at 40 degrees C for 3 h, with a conversion yield of 71% and a volumetric productivity of 71 g liter(-1) h(-1).

  18. Production of L-ribose from L-ribulose by a triple-site variant of mannose-6-phosphate isomerase from Geobacillus thermodenitrificans.

    PubMed

    Lim, Yu-Ri; Yeom, Soo-Jin; Oh, Deok-Kun

    2012-06-01

    A triple-site variant (W17Q N90A L129F) of mannose-6-phosphate isomerase from Geobacillus thermodenitrificans was obtained by combining variants with residue substitutions at different positions after random and site-directed mutagenesis. The specific activity and catalytic efficiency (k(cat)/K(m)) for L-ribulose isomerization of this variant were 3.1- and 7.1-fold higher, respectively, than those of the wild-type enzyme at pH 7.0 and 70°C in the presence of 1 mM Co(2+). The triple-site variant produced 213 g/liter l-ribose from 300 g/liter L-ribulose for 60 min, with a volumetric productivity of 213 g liter(-1) h(-1), which was 4.5-fold higher than that of the wild-type enzyme. The k(cat)/K(m) and productivity of the triple-site variant were approximately 2-fold higher than those of the Thermus thermophilus R142N variant of mannose-6-phosphate isomerase, which exhibited the highest values previously reported.

  19. Induction of Suicidal Erythrocyte Death by Cantharidin

    PubMed Central

    Alzoubi, Kousi; Egler, Jasmin; Briglia, Marilena; Fazio, Antonella; Faggio, Caterina; Lang, Florian

    2015-01-01

    The natural phosphoprotein phosphatase inhibitor cantharidin, primarily used for topical treatment of warts, has later been shown to trigger tumor cell apoptosis and is thus considered for the treatment of malignancy. Similar to apoptosis of tumor cells, erythrocytes may undergo eryptosis, a suicidal cell death characterized by cell shrinkage and translocation of cell membrane phosphatidylserine to the erythrocyte surface. Signaling of eryptosis includes increase of cytosolic Ca2+-activity ([Ca2+]i), ceramide, oxidative stress and dysregulation of several kinases. Phosphatidylserine abundance at the erythrocyte surface was quantified utilizing annexin-V-binding, cell volume from forward scatter, [Ca2+]i from Fluo3-fluorescence, ceramide from antibody binding, and reactive oxidant species (ROS) from 2′,7′-dichlorodihydrofluorescein diacetate (DCFDA) fluorescence. A 48 h treatment of human erythrocytes with cantharidin significantly increased the percentage of annexin-V-binding cells (≥10 μg/mL), significantly decreased forward scatter (≥25 μg/mL), significantly increased [Ca2+]i (≥25 μg/mL), but did not significantly modify ceramide abundance or ROS. The up-regulation of annexin-V-binding following cantharidin treatment was not significantly blunted by removal of extracellular Ca2+ but was abolished by kinase inhibitor staurosporine (1 μM) and slightly decreased by p38 inhibitor skepinone (2 μM). Exposure of erythrocytes to cantharidin triggers suicidal erythrocyte death with erythrocyte shrinkage and erythrocyte membrane scrambling, an effect sensitive to kinase inhibitors staurosporine and skepinone. PMID:26226001

  20. Induction of Suicidal Erythrocyte Death by Cantharidin.

    PubMed

    Alzoubi, Kousi; Egler, Jasmin; Briglia, Marilena; Fazio, Antonella; Faggio, Caterina; Lang, Florian

    2015-08-01

    The natural phosphoprotein phosphatase inhibitor cantharidin, primarily used for topical treatment of warts, has later been shown to trigger tumor cell apoptosis and is thus considered for the treatment of malignancy. Similar to apoptosis of tumor cells, erythrocytes may undergo eryptosis, a suicidal cell death characterized by cell shrinkage and translocation of cell membrane phosphatidylserine to the erythrocyte surface. Signaling of eryptosis includes increase of cytosolic Ca2+-activity ([Ca2+]i), ceramide, oxidative stress and dysregulation of several kinases. Phosphatidylserine abundance at the erythrocyte surface was quantified utilizing annexin-V-binding, cell volume from forward scatter, [Ca2+]i from Fluo3-fluorescence, ceramide from antibody binding, and reactive oxidant species (ROS) from 2',7'-dichlorodihydrofluorescein diacetate (DCFDA) fluorescence. A 48 h treatment of human erythrocytes with cantharidin significantly increased the percentage of annexin-V-binding cells (≥10 mg/mL), significantly decreased forward scatter (≥25 mg/mL), significantly increased [Ca2+]i (≥25 mg/mL), but did not significantly modify ceramide abundance or ROS. The up-regulation of annexin-V-binding following cantharidin treatment was not significantly blunted by removal of extracellular Ca2+ but was abolished by kinase inhibitor staurosporine (1 mM) and slightly decreased by p38 inhibitor skepinone (2 mM). Exposure of erythrocytes to cantharidin triggers suicidal erythrocyte death with erythrocyte shrinkage and erythrocyte membrane scrambling, an effect sensitive to kinase inhibitors staurosporine and skepinone. PMID:26226001

  1. α-Thalassemia does not seem to influence erythrocyte deformability in sickle cell trait carriers.

    PubMed

    Vayá, Amparo; Collado, Susana; Alis, Rafael; Vera, Belen; Romagnoli, Marco; Barragán, Eva

    2014-01-01

    Studies dealing with rheological red blood cell (RBC) behavior in sickle cell trait carriers are scarce. Moreover, the association with α-thalassemia (α-thal), which also modifies erythrocyte behavior, has not always been taken into account. We analyzed erythrocyte deformability by means of a shear stress diffractometer, along with hematological and biochemical parameters (glucose and plasma lipids), given their possible influence on erythrocyte deformability, in 14 sickle cell trait carriers and 23 healthy controls. Nine patients were also α-thal carriers and five were not. Among the thalassemia carriers, eight were heterozygous and one was homozygous. When compared with controls, sickle cell trait carriers showed no differences for any of the biochemical parameters analyzed (p > 0.05), but significantly lower hemoglobin (Hb) (p = 0.003), mean corpuscular volume (MCV) and mean corpuscular Hb (MCH) (p < 0.001) levels, although no differences in erythrocyte deformability were observed at any of the shear stresses tested (p > 0.05). When comparing sickle cell trait carriers, with and without α-thal, no differences in erythrocyte deformability were observed (p > 0.05), in spite of the former showing lower MCV and MCH (p < 0.05) levels. Carriers of α-thal had lower Hb S [β6(A3)Glu → Val; HBB: c.20A > T] levels (p = 0.013) than non carriers. The existence of a compensating mechanism seems reasonable because, despite presenting lower erythrocyte indices, which could worsen erythrocyte deformability, this rheological property improves when the percentage of Hb S is lower. PMID:24601859

  2. An overview of muscle glucose uptake during exercise. Sites of regulation.

    PubMed

    Wasserman, D H; Halseth, A E

    1998-01-01

    The uptake of blood glucose by skeletal muscle is a complex process. In order to be metabolized, glucose must travel the path from blood to interstitium to intracellular space and then be phosphorylated to glucose 6-phosphate (G6P). Movement of glucose from blood to interstitium is determined by skeletal muscle blood flow, capillary recruitment and the endothelial permeability to glucose. The influx of glucose from the interstitium to intracellular space is determined by the number of glucose transporters in the sarcolemma and the glucose gradient across the sarcolemma. The capacity to phosphorylate glucose is determined by the amount of skeletal muscle hexokinase II, hexokinase II compartmentalization within the cell, and the concentration of the hexokinase II inhibitor G6P. Any change in glucose uptake occurs due to an alteration in one or more of these steps. Based on the low calculated intracellular glucose levels and the higher affinity of glucose for phosphorylation relative to transport, glucose transport is generally considered rate-determining for basal muscle glucose uptake. Exercise increases both the movement of glucose from blood to sarcolemma and the permeability of the sarcolemma to glucose. Whether the ability to phosphorylate glucose is increased in the working muscle remains to be clearly shown. It is possible that the accelerated glucose delivery and transport rates during exercise bias regulation so that muscle glucose phosphorylation exerts more control on muscle glucose uptake. Conditions that alter glucose uptake during exercise, such as increased NEFA concentrations, decreased oxygen availability and adrenergic stimulation, must work by altering one or more of the three steps involved in glucose uptake. This review describes the regulation of glucose uptake during exercise at each of these sites under a number of conditions, as well as describing muscle glucose uptake in the post-exercise state.

  3. Erythrocyte membrane phosphatidylserine exposure in obesity.

    PubMed

    Solá, Eva; Vayá, Amparo; Martínez, Marcial; Moscardó, Antonio; Corella, Dolores; Santaolaria, Maria-Luisa; España, Francisco; Hernández-Mijares, Antonio

    2009-02-01

    It has been suggested that increased erythrocyte membrane phosphatidylserine (PS) exposure could contribute to hypercoagulability and hemorheological disturbances in obesity. The aim of our study was to evaluate PS exposure in obese patients and in a control group and to correlate this with hemorheological properties, i.e., erythrocyte aggregability (EA) and deformability, and to evaluate the effect of weight loss on these parameters. An anthropometric and analytical evaluation was performed at baseline and after 3 months on a diet (very low-calorie diet for 4 weeks and low-calorie diet for 2 months) on 49 severe or morbid obese patients (37 women, 12 men) and 55 healthy volunteers (39 women, 16 men). PS exposure on erythrocyte membrane was performed by flow cytometry. Erythrocyte aggregation was measured using the Myrenne MA(1) and the Sefam aggregometer. Erythrocyte deformability was determined in a stress diffractometer. Prothrombin fragment F1+2 (F1+2) was determined as a marker of the hypercoagulable state, and plasma malondialdehyde (MDA) as an indicator of oxidative stress. Obese patients had a higher EA index, higher PS exposure on erythrocyte membranes and higher levels of MDA and F1+2. The differences in erythrocyte aggregation and F1+2 between obese patients and the control group were maintained after adjusting for PS exposure. After 3 months of diet, a significant reduction in PS exposure on erythrocyte membrane was observed. Obese patients show increased PS exposure on erythrocyte membrane, with no effect on rheological properties. Increased PS exposure could contribute to hypercoagulability in these patients. Weight loss obtained with diet treatment reduces PS exposure on erythrocyte membrane.

  4. The Tertiary Origin of the Allosteric Activation of E. coli Glucosamine-6-Phosphate Deaminase Studied by Sol-Gel Nanoencapsulation of Its T Conformer

    PubMed Central

    Zonszein, Sergio; Álvarez-Añorve, Laura I.; Vázquez-Núñez, Roberto J.; Calcagno, Mario L.

    2014-01-01

    The role of tertiary conformational changes associated to ligand binding was explored using the allosteric enzyme glucosamine-6-phosphate (GlcN6P) deaminase from Escherichia coli (EcGNPDA) as an experimental model. This is an enzyme of amino sugar catabolism that deaminates GlcN6P, giving fructose 6-phosphate and ammonia, and is allosterically activated by N-acetylglucosamine 6-phosphate (GlcNAc6P). We resorted to the nanoencapsulation of this enzyme in wet silica sol-gels for studying the role of intrasubunit local mobility in its allosteric activation under the suppression of quaternary transition. The gel-trapped enzyme lost its characteristic homotropic cooperativity while keeping its catalytic properties and the allosteric activation by GlcNAc6P. The nanoencapsulation keeps the enzyme in the T quaternary conformation, making possible the study of its allosteric activation under a condition that is not possible to attain in a soluble phase. The involved local transition was slowed down by nanoencapsulation, thus easing the fluorometric analysis of its relaxation kinetics, which revealed an induced-fit mechanism. The absence of cooperativity produced allosterically activated transitory states displaying velocity against substrate concentration curves with apparent negative cooperativity, due to the simultaneous presence of subunits with different substrate affinities. Reaction kinetics experiments performed at different tertiary conformational relaxation times also reveal the sequential nature of the allosteric activation. We assumed as a minimal model the existence of two tertiary states, t and r, of low and high affinity, respectively, for the substrate and the activator. By fitting the velocity-substrate curves as a linear combination of two hyperbolic functions with Kt and Kr as KM values, we obtained comparable values to those reported for the quaternary conformers in solution fitted to MWC model. These results are discussed in the background of the known

  5. Accumulation of d-Glucose from Pentoses by Metabolically Engineered Escherichia coli

    PubMed Central

    Xia, Tian; Han, Qi; Costanzo, William V.; Zhu, Yixuan; Urbauer, Jeffrey L.

    2015-01-01

    Escherichia coli that is unable to metabolize d-glucose (with knockouts in ptsG, manZ, and glk) accumulates a small amount of d-glucose (yield of about 0.01 g/g) during growth on the pentoses d-xylose or l-arabinose as a sole carbon source. Additional knockouts in the zwf and pfkA genes, encoding, respectively, d-glucose-6-phosphate 1-dehydrogenase and 6-phosphofructokinase I (E. coli MEC143), increased accumulation to greater than 1 g/liter d-glucose and 100 mg/liter d-mannose from 5 g/liter d-xylose or l-arabinose. Knockouts of other genes associated with interconversions of d-glucose-phosphates demonstrate that d-glucose is formed primarily by the dephosphorylation of d-glucose-6-phosphate. Under controlled batch conditions with 20 g/liter d-xylose, MEC143 generated 4.4 g/liter d-glucose and 0.6 g/liter d-mannose. The results establish a direct link between pentoses and hexoses and provide a novel strategy to increase carbon backbone length from five to six carbons by directing flux through the pentose phosphate pathway. PMID:25746993

  6. Glucose phosphorylation is not rate limiting for accumulation of glycogen from glucose in perfused livers from fasted rats

    SciTech Connect

    Youn, J.H.; Ader, M.; Bergman, R.N.

    1989-01-05

    Incorporation of Glc and Fru into glycogen was measured in perfused livers from 24-h fasted rats using (6-3H)Glc and (U-14C)Fru. For the initial 20 min, livers were perfused with low Glc (2 mM) to deplete hepatic glycogen and were perfused for the following 30 min with various combinations of Glc and Fru. With constant Fru (2 mM), increasing perfusate Glc increased the relative contribution of Glc carbons to glycogen (7.2 +/- 0.4, 34.9 +/- 2.8, and 59.1 +/- 2.7% at 2, 10, and 20 mM Glc, respectively; n = 5 for each). During perfusion with substrate levels seen during refeeding (10 mM Glc, 1.8 mumol/g/min gluconeogenic flux from 2 mM Fru), Fru provided 54.7 +/- 2.7% of the carbons for glycogen, while Glc provided only 34.9 +/- 2.8%, consistent with in vivo estimations. However, the estimated rate of Glc phosphorylation was at least 1.10 +/- 0.11 mumol/g/min, which exceeded by at least 4-fold the glycogen accumulation rate (0.28 +/- 0.04 mumol of glucose/g/min). The total rate of glucose 6-phosphate supply via Glc phosphorylation and gluconeogenesis (2.9 mumol/g/min) exceeded reported in vivo rates of glycogen accumulation during refeeding. Thus, in perfused livers of 24-h fasted rats there is an apparent redundancy in glucose 6-phosphate supply. These results suggest that the rate-limiting step for hepatic glycogen accumulation during refeeding is located between glucose 6-phosphate and glycogen, rather than at the step of Glc phosphorylation or in the gluconeogenic pathway.

  7. Pathways of hepatic glycogen formation in humans following ingestion of a glucose load in the fed state

    SciTech Connect

    Magnusson, I.; Chandramouli, V.; Schumann, W.C.; Kumaran, K.; Wahren, J.; Landau, B.R.

    1989-06-01

    The relative contributions of the direct and the indirect pathways to hepatic glycogen formation following a glucose load given to humans four hours after a substantial breakfast have been examined. Glucose loads labeled with (6-(/sup 14/)C)glucose were given to six healthy volunteers along with diflunisal (1 g) or acetaminophen (1.5 g), drugs excreted in urine as glucuronides. Distribution of /sup 14/C in the glucose unit of the glucuronide was taken as a measure of the extent to which glucose was deposited directly in liver glycogen (ie, glucose----glucose-6-phosphate----glycogen) rather than indirectly (ie, glucose----C3-compound----glucose-6-phosphate----glycogen). The maximum contribution to glycogen formation by the direct pathway was estimated to be 77% +/- 4%, which is somewhat higher than previous estimates in humans fasted overnight (65% +/- 1%, P less than 0.05). Thus, the indirect pathway of liver glycogen formation following a glucose load is operative in both the overnight fasted and the fed state, although its contribution may be somewhat less in the fed state.

  8. Competitive inhibitors of type B ribose 5-phosphate isomerases: design, synthesis and kinetic evaluation of new D-allose and D-allulose 6-phosphate derivatives.

    PubMed

    Mariano, Sandrine; Roos, Annette K; Mowbray, Sherry L; Salmon, Laurent

    2009-05-12

    This study reports syntheses of d-allose 6-phosphate (All6P), D-allulose (or D-psicose) 6-phosphate (Allu6P), and seven D-ribose 5-phosphate isomerase (Rpi) inhibitors. The inhibitors were designed as analogues of the 6-carbon high-energy intermediate postulated for the All6P to Allu6P isomerization reaction (Allpi activity) catalyzed by type B Rpi from Escherichiacoli (EcRpiB). 5-Phospho-D-ribonate, easily obtained through oxidative cleavage of either All6P or Allu6P, led to the original synthon 5-dihydrogenophospho-D-ribono-1,4-lactone from which the other inhibitors could be synthesized through nucleophilic addition in one step. Kinetic evaluation on Allpi activity of EcRpiB shows that two of these compounds, 5-phospho-D-ribonohydroxamic acid and N-(5-phospho-D-ribonoyl)-methylamine, indeed behave as new efficient inhibitors of EcRpiB; further, 5-phospho-D-ribonohydroxamic acid was demonstrated to have competitive inhibition. Kinetic evaluation on Rpi activity of both EcRpiB and RpiB from Mycobacterium tuberculosis (MtRpiB) shows that several of the designed 6-carbon high-energy intermediate analogues are new competitive inhibitors of both RpiBs. One of them, 5-phospho-D-ribonate, not only appears as the strongest competitive inhibitor of a Rpi ever reported in the literature, with a K(i) value of 9 microM for MtRpiB, but also displays specific inhibition of MtRpiB versus EcRpiB.

  9. Interchangeable but Essential Functions of SNX1 and SNX2 in the Association of Retromer with Endosomes and the Trafficking of Mannose 6-Phosphate Receptors▿ †

    PubMed Central

    Rojas, Raul; Kametaka, Satoshi; Haft, Carol R.; Bonifacino, Juan S.

    2007-01-01

    The retromer is a cytosolic/peripheral membrane protein complex that mediates the retrieval of the cation-independent mannose 6-phosphate receptor from endosomes to the trans-Golgi network (TGN) in mammalian cells. Previous studies showed that the mammalian retromer comprises three proteins, named Vps26, Vps29, and Vps35, plus the sorting nexin, SNX1. There is conflicting evidence, however, as to whether a homologous sorting nexin, SNX2, is truly a component of the retromer. In addition, the nature of the subunit interactions and assembly of the mammalian retromer complex are poorly understood. We have addressed these issues by performing biochemical and functional analyses of endogenous retromers in the human cell line HeLa. We found that the mammalian retromer complex consists of two autonomously assembling subcomplexes, namely, a Vps26-Vps29-Vps35 obligate heterotrimer and a SNX1/2 alternative heterodimer or homodimer. The association of Vps26-Vps29-Vps35 with endosomes requires the presence of either SNX1 or SNX2, whereas SNX1/2 can be recruited to endosomes independently of Vps26-Vps29-Vps35. We also found that the presence of either SNX1 or SNX2 is essential for the retrieval of the cation-independent mannose 6-phosphate receptor to the TGN. These observations indicate that the mammalian retromer complex assembles by sequential association of SNX1/2 and Vps26-Vps29-Vps35 subcomplexes on endosomal membranes and that SNX1 and SNX2 play interchangeable but essential roles in retromer structure and function. PMID:17101778

  10. Enhanced freeze tolerance of baker's yeast by overexpressed trehalose-6-phosphate synthase gene (TPS1) and deleted trehalase genes in frozen dough.

    PubMed

    Tan, Haigang; Dong, Jian; Wang, Guanglu; Xu, Haiyan; Zhang, Cuiying; Xiao, Dongguang

    2014-08-01

    Several recombinant strains with overexpressed trehalose-6-phosphate synthase gene (TPS1) and/or deleted trehalase genes were obtained to elucidate the relationships between TPS1, trehalase genes, content of intracellular trehalose and freeze tolerance of baker's yeast, as well as improve the fermentation properties of lean dough after freezing. In this study, strain TL301(TPS1) overexpressing TPS1 showed 62.92 % higher trehalose-6-phosphate synthase (Tps1) activity and enhanced the content of intracellular trehalose than the parental strain. Deleting ATH1 exerted a significant effect on trehalase activities and the degradation amount of intracellular trehalose during the first 30 min of prefermentation. This finding indicates that acid trehalase (Ath1) plays a role in intracellular trehalose degradation. NTH2 encodes a functional neutral trehalase (Nth2) that was significantly involved in intracellular trehalose degradation in the absence of the NTH1 and/or ATH1 gene. The survival ratio, freeze-tolerance ratio and relative fermentation ability of strain TL301(TPS1) were approximately twice as high as those of the parental strain (BY6-9α). The increase in freeze tolerance of strain TL301(TPS1) was accompanied by relatively low trehalase activity, high Tps1 activity and high residual content of intracellular trehalose. Our results suggest that overexpressing TPS1 and deleting trehalase genes are sufficient to improve the freeze tolerance of baker's yeast in frozen dough. The present study provides guidance for the commercial baking industry as well as the research on the intracellular trehalose mobilization and freeze tolerance of baker's yeast. PMID:24951963

  11. Bovine heart fructose-6-phosphate 2-kinase/fructose-2,6-bisphosphatase: complete amino acid sequence and localization of phosphorylation sites.

    PubMed Central

    Sakata, J; Uyeda, K

    1990-01-01

    We have shown previously that bovine heart fructose-6-phosphate 2-kinase/fructose-2,6-bisphosphatase (EC 2.7.1.105/3.1.3.46) is phosphorylated by cAMP-dependent protein kinase and protein kinase C; phosphorylation results in activation of kinase. This activation of heart enzyme is in contrast to results with the liver isozyme, in which phosphorylation by cAMP-dependent protein kinase inhibits the kinase activity. As an initial step toward understanding this difference between the isozymes we have determined the DNA sequence of the heart enzyme and analyzed the amino acid sequence with special emphasis on the location of the phosphorylation site. We isolated and sequenced two overlapping cDNA fragments, which together could encode the complete amino acid sequence of bovine heart fructose-6-phosphate 2-kinase/fructose-2,6-bisphosphatase, a protein of 530 amino acids, with a calculated molecular weight of 60,679. Since the deduced protein contained amino acid sequences identical to the sequences of four known tryptic peptides from this enzyme we concluded that the deduced protein sequence did represent bovine heart enzyme. In addition, a cDNA fragment hybridized to a 4-kilobase mRNA from bovine heart. The phosphorylation sites of the heart enzyme were located near the C terminus, whereas the phosphorylation site of the liver isozyme is known to be located near the N terminus. These opposite locations of the phosphorylation sites may explain the contrasting effect of the covalent modification on the enzymes' activities. Images PMID:2164212

  12. Increased mannosylphosphorylation of N-glycans by heterologous expression of YlMPO1 in glyco-engineered Saccharomyces cerevisiae for mannose-6-phosphate modification.

    PubMed

    Gil, Jin Young; Park, Jeong-Nam; Lee, Kyung Jin; Kang, Ji-Yeon; Kim, Yeong Hun; Kim, Seonghun; Kim, Sang-Yoon; Kwon, Ohsuk; Lim, Yong Taik; Kang, Hyun Ah; Oh, Doo-Byoung

    2015-07-20

    Mannosylphosphorylated N-glycans found in yeasts can be converted to those containing mannose-6-phosphate, which is a key factor for lysosomal targeting. In the traditional yeast Saccharomyces cerevisiae, both ScMNN4 and ScMNN6 genes are required for efficient mannosylphosphorylation. ScMnn4 protein has been known to be a positive regulator of ScMnn6p, a real enzyme for mannosylphosphorylation. On the other hand, YlMpo1p, a ScMnn4p homologue, mediates mannosylphosphorylation in Yarrowia lypolytica without the involvement of ScMnn6p homologues. In this study, we show that heterologous expression of YlMpo1p can perform and enhance mannosylphosphorylation in S. cerevisiae in the absence of ScMnn4p and ScMnn6p. Moreover, the level of mannosylphosphorylation of N-glycans enhanced by YlMpo1p overexpression is much higher than that with ScMnn4p overexpression, and this is highlighted further in Scmnn4- and Scmnn6-disrupted mutants. When YlMpo1p overexpression is applied to glyco-engineered S. cerevisiae in which the synthesis of immunogenic glycans is abolished, a great increase of bi-mannosylphosphorylated glycan is observed. Through an in vitro process involving the uncapping of the outer mannose residue, this bi-mannosylphosphorylated structure is changed to a bi-phosphorylated structure with high affinity for mannose-6-phosphate receptor. The superior ability of YlMpo1p to increase bi-mannosylphosphorylated glycan in yeast shows promise for the production of therapeutic enzymes with improved lysosomal targeting capability.

  13. Characterization of a mannose-6-phosphate isomerase from Thermus thermophilus and increased L-ribose production by its R142N mutant.

    PubMed

    Yeom, Soo-Jin; Seo, Eun-Sun; Kim, Bi-Na; Kim, Yeong-Su; Oh, Deok-Kun

    2011-02-01

    An uncharacterized gene from Thermus thermophilus, thought to encode a mannose-6-phosphate isomerase, was cloned and expressed in Escherichia coli. The maximal activity of the recombinant enzyme for L-ribulose isomerization was observed at pH 7.0 and 75°C in the presence of 0.5 mM Cu(2+). Among all of the pentoses and hexoses evaluated, the enzyme exhibited the highest activity for the conversion of L-ribulose to L-ribose, a potential starting material for many L-nucleoside-based pharmaceutical compounds. The active-site residues, predicted according to a homology-based model, were separately replaced with Ala. The residue at position 142 was correlated with an increase in L-ribulose isomerization activity. The R142N mutant showed the highest activity among mutants modified with Ala, Glu, Tyr, Lys, Asn, or Gln. The specific activity and catalytic efficiency (k(cat)/K(m)) for L-ribulose using the R142N mutant were 1.4- and 1.6-fold higher than those of the wild-type enzyme, respectively. The k(cat)/K(m) of the R142N mutant was 3.8-fold higher than that of Geobacillus thermodenitrificans mannose-6-phosphate isomerase, which exhibited the highest activity to date for the previously reported k(cat)/K(m). The R142N mutant enzyme produced 213 g/liter L-ribose from 300 g/liter L-ribulose for 2 h, with a volumetric productivity of 107 g liter(-1) h(-1), which was 1.5-fold higher than that of the wild-type enzyme.

  14. Characterization of mammalian glucose transport proteins using photoaffinity labeling techniques

    SciTech Connect

    Wadzinski, B.E.

    1989-01-01

    A carrier-free radioiodinated phenylazide derivative of forskolin, 3-iodo-4-azidophenethylamido-7-O-succinyl-deacetyl-forskolin (({sup 125}I)IAPS-forskolin), has been shown to be a highly selective photoaffinity probe for the human erythrocyte glucose transported and the glucose transport proteins found in several mammalian tissues and cultured cells where the glucose transport protein is present at a low concentration. The photoincorporation of ({sup 125}I)IAPS-forskolin into these glucose transporters was blocked by D- (but not L-) glucose, cytochalasin B, and forskolin. In addition to labeling the mammalian glucose transport proteins, ({sup 125}I)IAPS-forskolin also labeled the L-arabinose transporter from E. coli. In muscle and adipose tissues, glucose transport is markedly increased in response to insulin. ({sup 125}I)IAPS-forskolin was shown to selectivity tag the glucose transporter in membranes derived from these cells. In addition, the covalent derivatization of the transport protein in subcellular fractions of the adipocyte has provided a means to study the hormonal regulation of glucose transport. ({sup 125}I)IAPS-forskolin has also been used to label the purified human erythrocyte glucose transporter. The site of insertion has therefore been localized by analysis of the radiolabeled peptides which were produced following chemical and proteolytic digestion of the labeled transport protein.

  15. Triggering of Programmed Erythrocyte Death by Alantolactone

    PubMed Central

    Alzoubi, Kousi; Calabrò, Salvatrice; Egler, Jasmin; Faggio, Caterina; Lang, Florian

    2014-01-01

    The sesquiterpene alantolactone counteracts malignancy, an effect at least in part due to stimulation of suicidal death or apoptosis of tumor cells. Signaling of alantolactone induced apoptosis involves altered gene expression and mitochondrial depolarization. Erythrocytes lack mitochondria and nuclei but may enter suicidal death or eryptosis, which is characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine exposure at the erythrocyte surface. Cellular mechanisms involved in triggering of eryptosis include increase of cytosolic Ca2+-activity ([Ca2+]i) and oxidative stress. The present study explored, whether alantolactone stimulates eryptosis. To this end, erythrocyte volume was estimated from forward scatter, phosphatidylserine-exposure at the erythrocyte surface from FITC-annexin-V-binding, [Ca2+]i from Fluo3-fluorescence, ceramide abundance from binding of fluorescent antibodies, and oxidative stress from 2',7'-dichlorodihydrofluorescein-diacetate (DCFDA) fluorescence. As a result, a 48 h exposure of human erythrocytes to alantolactone (≥20 μM) significantly decreased erythrocyte forward scatter and increased the percentage of annexin-V-binding cells. Alantolactone significantly increased Fluo3 fluorescence (60 μM), ceramide abundance (60 μM) and DCFDA fluorescence (≥40 μM). The effect of alantolactone (60 μM) on annexin-V-binding was not significantly modified by removal of extracellular Ca2+. In conclusion, alantolactone stimulates suicidal erythrocyte death or eryptosis, an effect paralleled by increase of [Ca2+]i, ceramide abundance and oxidative stress. PMID:25533522

  16. Erythrocyte rouleau formation under polarized electromagnetic fields

    NASA Astrophysics Data System (ADS)

    Sebastián, José Luis; San Martín, Sagrario Muñoz; Sancho, Miguel; Miranda, José Miguel; Álvarez, Gabriel

    2005-09-01

    We study the influence of an external electromagnetic field of 1.8GHz in the formation or disaggregation of long rouleau of identical erythrocyte cells. In particular we calculate the variation of the transmembrane potential of an individual erythrocyte illuminated by the external field due to the presence of the neighboring erythrocytes in the rouleau, and compare the total electric energy of isolated cells with the total electric energy of the rouleau. We show that the polarization of the external electromagnetic field plays a fundamental role in the total energy variation of the cell system, and consequently in the formation or disaggregation of rouleau.

  17. Inhibition of hepatic glucose 6-phosphatase system by the green tea flavanol epigallocatechin gallate.

    PubMed

    Csala, Miklós; Margittai, Eva; Senesi, Silvia; Gamberucci, Alessandra; Bánhegyi, Gábor; Mandl, József; Benedetti, Angelo

    2007-04-17

    Effect of 5-100 microM epigallocatechin gallate (EGCG) on hepatic glucose 6-phosphatase (G6Pase) system was investigated. EGCG inhibited G6Pase in intact but not in permeabilized rat liver microsomes, suggesting the interference with the transport. However, EGCG did not hinder microsomal glucose 6-phosphate (G6P) uptake. Instead, it increased the accumulation of radioactivity after the addition of [(14)C]G6P, presumably due to a slower release of [(14)C]glucose, the product of luminal hydrolysis. Indeed, EGCG was found to inhibit microsomal glucose efflux. Since G6Pase activity is depressed by glucose in a concentration-dependent manner, we concluded that EGCG inhibits G6Pase through an elevated luminal glucose level.

  18. The Molecular Basis of Erythrocyte Shape

    NASA Astrophysics Data System (ADS)

    Elgsaeter, Arnljot; Stokke, Bjorn T.; Mikkelsen, Arne; Branton, Daniel

    1986-12-01

    Recent discoveries about the molecular organization and physical properties of the mammalian erythrocyte membrane and its associated structural proteins can now be used to explain, and may eventually be used to predict, the shape of the erythrocyte. Such explanations are possible because the relatively few structural proteins of the erythrocyte are regularly distributed over the entire cytoplasmic surface of the cell membrane and because the well-understood topological associations of these proteins seem to be stable in comparison with the time required for the cell to change shape. These simplifications make the erythrocyte the first nonmuscle cell for which it will be possible to extend our knowledge of molecular interactions to the next hierarchical level of organization that deals with shape and shape transformations.

  19. The Erythrocyte Ghost Is a Perfect Osmometer

    PubMed Central

    Kwant, W. O.; Seeman, Philip

    1970-01-01

    The osmotic swelling of intact erythrocytes in hypotonic solutions was measured using microhematocrit tubes, Van Allen tubes, and a calibrated Coulter counter. In agreement with earlier workers the intact cells did not behave as perfect osmometers, the cells swelling less than predicted by the Boyle-van't Hoff law. Erythrocyte ghosts were prepared from fresh intact erythrocytes by one-step hemolysis in 0.25% NaCl at an extremely dilute concentration of cells and the membranes were sealed at 37°. The ghosts were mixed with NaCl solutions of different osmolarities and the MCV (mean cell volume) of the shrunken cells immediately monitored by a calibrated Coulter counter. It was found that the MCV values of the shrunken ghosts were accurately predicted by the Boyle-van't Hoff law. These results indicate that these erythrocyte ghosts behaved as perfect osmometers. PMID:5413078

  20. Transposon-encoded sucrose metabolism in Lactococcus lactis. Purification of sucrose-6-phosphate hydrolase and genetic linkage to N5-(L-1-carboxyethyl)-L-ornithine synthase in strain K1.

    PubMed

    Thompson, J; Nguyen, N Y; Sackett, D L; Donkersloot, J A

    1991-08-01

    Sucrose-6-phosphate hydrolase from Lactococcus lactis subsp. lactis K1-23 (formerly Streptococcus lactis K1-23) has been purified 600-fold to electrophoretic homogeneity. Purification of the enzyme was achieved by DEAE-Sephacel, phosphocellulose P-11, and gel exclusion (Ultrogel AcA 54) chromatography. The purified enzyme (specific activity 31 units/mg) catalyzed the hydrolysis of both 6-O-phosphoryl-alpha-D-glucopyranosyl-1,2-beta-D-fructofuranoside (sucrose 6-phosphate) and sucrose (Km = 0.1 and 100 mM, respectively). Ultracentrifugal analysis of sucrose-6-phosphate hydrolase indicated an Mr = 52,200. The purified enzyme migrated as a single protein during sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Mr = 52,000). However, four distinct polypeptides were detected by analytical electrofocusing, and all four species hydrolyzed sucrose and sucrose 6-phosphate. The amino acid composition of sucrose-6-phosphate hydrolase, and the sequence of the first 12 amino acids from the NH2 terminus, have been determined. Hybridization studies with oligonucleotide probes show that the genes for sucrose-6-phosphate hydrolase (scrB), Enzyme IIScr of the phosphoenolypyruvate-dependent sucrose:phosphotransferase system (scrA), and N5-(carboxyethyl)ornithine synthase (ceo) are encoded by the same approximately 20-kilobase EcoRI fragment. This fragment is part of a large transposon Tn5306 that also encodes the nisin precursor gene, spaN, and IS904. In L. lactis ATCC 11454, spaN, IS904, scrA, and scrB (but not ceo) are encoded on a related transposon, Tn5307.

  1. Induction of Suicidal Erythrocyte Death by Nelfinavir

    PubMed Central

    Bissinger, Rosi; Waibel, Sabrina; Lang, Florian

    2015-01-01

    The HIV protease inhibitor, nelfinavir, primarily used for the treatment of HIV infections, has later been shown to be effective in various infectious diseases including malaria. Nelfinavir may trigger mitochondria-independent cell death. Erythrocytes may undergo eryptosis, a mitochondria-independent suicidal cell death characterized by cell shrinkage and phosphatidylserine translocation to the erythrocyte surface. Triggers of eryptosis include oxidative stress and increase of cytosolic Ca2+-activity ([Ca2+]i). During malaria, accelerated death of infected erythrocytes may decrease parasitemia and thus favorably influence the clinical course of the disease. In the present study, phosphatidylserine abundance at the cell surface was estimated from annexin V binding, cell volume from forward scatter, reactive oxidant species (ROS) from 2',7'-dichlorodihydrofluorescein diacetate (DCFDA) fluorescence, and [Ca2+]i from Fluo3-fluorescence. A 48 h treatment of human erythrocytes with nelfinavir significantly increased the percentage of annexin-V-binding cells (≥5µg/mL), significantly decreased forward scatter (≥2.5µg/mL), significantly increased ROS abundance (10 µg/mL), and significantly increased [Ca2+]i (≥5 µg/mL). The up-regulation of annexin-V-binding following nelfinavir treatment was significantly blunted, but not abolished by either addition of the antioxidant N-acetylcysteine (1 mM) or removal of extracellular Ca2+. In conclusion, exposure of erythrocytes to nelfinavir induces oxidative stress and Ca2+ entry, thus leading to suicidal erythrocyte death characterized by erythrocyte shrinkage and erythrocyte membrane scrambling. PMID:26008229

  2. Triggering of Erythrocyte Death by Triparanol

    PubMed Central

    Officioso, Arbace; Manna, Caterina; Alzoubi, Kousi; Lang, Florian

    2015-01-01

    The cholesterol synthesis inhibitor Triparanol has been shown to trigger apoptosis in several malignancies. Similar to the apoptosis of nucleated cells, erythrocytes may enter eryptosis, the suicidal death characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Triggers of eryptosis include oxidative stress which may activate erythrocytic Ca2+ permeable unselective cation channels with subsequent Ca2+ entry and increase of cytosolic Ca2+ activity ([Ca2+]i). The present study explored whether and how Triparanol induces eryptosis. To this end, phosphatidylserine exposure at the cell surface was estimated from annexin-V-binding, cell volume from forward scatter, hemolysis from hemoglobin release, [Ca2+]i from Fluo3-fluorescence, and ROS formation from 2’,7’-dichlorodihydrofluorescein diacetate (DCFDA) dependent fluorescence. As a result, a 48 h exposure of human erythrocytes to Triparanol (20 µM) significantly increased DCFDA fluorescence and significantly increased Fluo3-fluorescence. Triparanol (15 µM) significantly increased the percentage of annexin-V-binding cells, and significantly decreased the forward scatter. The effect of Triparanol on annexin-V-binding was significantly blunted, but not abolished by removal of extracellular Ca2+. In conclusion, Triparanol leads to eryptosis, the suicidal erythrocyte death characterized by cell shrinkage and phospholipid scrambling of the erythrocyte cell membrane. Triparanol is at least in part effective by stimulating ROS formation and Ca2+ entry. PMID:26305256

  3. [Kinetics of Cu crossing human erythrocyte membrane].

    PubMed

    Dun, Zhu Ci Ren

    2014-12-01

    This study was aimed to investigate various factors influencing the proceduction of Cu(II) crossing human erythrocyte membrane, including concentration of Cu²⁺, pH value of the medium, temperature and time of incubation, and to derive kinetic equation of Cu(II) crossing human erythrocyte membrane. Suspension red blood cells were incubated by Cu²⁺, then content of Cu²⁺ crossed human erythrocyte membrane was determined by atomic absorption spectrometry under various conditions after digestion. The results showed that content of Cu²⁺ crossed human erythrocyte membrane increased with the increase of extracellular Cu²⁺ and enhancement of incubation temperature, and the content of Cu²⁺ crossed human erythrocyte membrane showed a increasing tendency when pH reached to 6.2-7.4, and to maximum at pH 7.4, then gradually decreased at range of pH 7.4-9.2. It is concluded that the Cu²⁺ crossing human erythrocyte has been confirmed to be the first order kinetics characteristics within 120 min, and the linear equation is 10³ × Y = 0.0497t +6.5992.

  4. The glucosidic pathways and glucose production by frog muscle.

    PubMed

    Fournier, P A; Petrof, E O; Guderley, H

    1992-04-25

    Resting muscle is generally perceived as a glucose-utilizing organ; however, we show that resting well-oxygenated frog muscle recovering from strenuous exercise can release significant amounts of glucose. The metabolic pathway responsible for this process does not involve glucose-6-phosphatase because this enzyme is undetectable in frog muscle. The participation of amylo-1,6-glucosidase in the production of glucose is also ruled out since neither marked net phosphorolytic breakdown of glycogen nor considerable cycling between glycogen and glucose 6-phosphate occur. The glucosidic pathways of glycogen breakdown are the likely source of glucose as they are the only metabolic avenues with sufficient capacity to account for the rate at which glucose is released from post-exercised muscle. This rate of glucose production is high enough to be of physiological importance. Our results clearly indicate that to measure lactate glycogenesis in muscle, the simultaneous hydrolysis of muscle glycogen by the glucosidic pathways must be taken into account to prevent marked underestimation of the rate of glycogen synthesis. The glucosidic pathways seem the predominant avenues of glycogen breakdown in post-exercised resting frog muscle and are active enough to account for the rate of glycogen breakdown in resting muscle, suggesting that these rather than the phosphorolytic pathways are the chief routes of glycogen breakdown in resting muscle. PMID:1569076

  5. Novel Stable Isotope Analyses Demonstrate Significant Rates of Glucose Cycling in Mouse Pancreatic Islets

    PubMed Central

    Pound, Lynley D.; Trenary, Irina; O’Brien, Richard M.

    2015-01-01

    A polymorphism located in the G6PC2 gene, which encodes an islet-specific glucose-6-phosphatase catalytic subunit, is the most important common determinant of variations in fasting blood glucose (FBG) levels in humans. Studies of G6pc2 knockout (KO) mice suggest that G6pc2 represents a negative regulator of basal glucose-stimulated insulin secretion (GSIS) that acts by hydrolyzing glucose-6-phosphate (G6P), thereby reducing glycolytic flux. However, this conclusion conflicts with the very low estimates for the rate of glucose cycling in pancreatic islets, as assessed using radioisotopes. We have reassessed the rate of glucose cycling in pancreatic islets using a novel stable isotope method. The data show much higher levels of glucose cycling than previously reported. In 5 mmol/L glucose, islets from C57BL/6J chow-fed mice cycled ∼16% of net glucose uptake. The cycling rate was further increased at 11 mmol/L glucose. Similar cycling rates were observed using islets from high fat–fed mice. Importantly, glucose cycling was abolished in G6pc2 KO mouse islets, confirming that G6pc2 opposes the action of the glucose sensor glucokinase by hydrolyzing G6P. The demonstration of high rates of glucose cycling in pancreatic islets explains why G6pc2 deletion enhances GSIS and why variants in G6PC2 affect FBG in humans. PMID:25552595

  6. Detection of erythrocytes influenced by aging and type 2 diabetes using atomic force microscope

    SciTech Connect

    Jin, Hua; Xing, Xiaobo; Zhao, Hongxia; Chen, Yong; Huang, Xun; Ma, Shuyuan; Ye, Hongyan; Cai, Jiye

    2010-01-22

    The pathophysiological changes of erythrocytes are detected at the molecular scale, which is important to reveal the onset of diseases. Type 2 diabetes is an age-related metabolic disorder with high prevalence in elderly (or old) people. Up to now, there are no treatments to cure diabetes. Therefore, early detection and the ability to monitor the progression of type 2 diabetes are very important for developing effective therapies. Type 2 diabetes is associated with high blood glucose in the context of insulin resistance and relative insulin deficiency. These abnormalities may disturb the architecture and functions of erythrocytes at molecular scale. In this study, the aging- and diabetes-induced changes in morphological and biomechanical properties of erythrocytes are clearly characterized at nanometer scale using atomic force microscope (AFM). The structural information and mechanical properties of the cell surface membranes of erythrocytes are very important indicators for determining the healthy, diseased or aging status. So, AFM may potentially be developed into a powerful tool in diagnosing diseases.

  7. A Role of Rab29 in the Integrity of the Trans-Golgi Network and Retrograde Trafficking of Mannose-6-Phosphate Receptor

    PubMed Central

    Wang, Shicong; Ma, Zexu; Xu, Xiaohui; Wang, Zhen; Sun, Lixiang; Zhou, Yunhe; Lin, Xiaosi; Hong, Wanjin; Wang, Tuanlao

    2014-01-01

    Rab29 (also referred as Rab7L1) is a novel Rab protein, and is recently demonstrated to regulate phagocytosis and traffic from the Golgi to the lysosome. However, its roles in membrane trafficking have not been investigated extensively. Our results in this study revealed that Rab29 is associated with the trans-Golgi network (TGN), and is essential for maintaining the integrity of the TGN, because inhibition of the activity of Rab29 or depletion of Rab29 resulted in fragmentation of the TGN marked by TGN46. Expression of the dominant negative form Rab29T21N or shRNA-Rab29 also altered the distribution of mannose-6-phosphate receptor (M6PR), and interrupted the retrograde trafficking of M6PR through monitoring the endocytosis of CD8-tagged calcium dependent M6PR (cdM6PR) or calcium independent M6PR (ciM6PR), but without significant effects on the anterograde trafficking of vesicular stomatitis virus G protein (VSV-G). Our results suggest that Rab29 is essential for the integrity of the TGN and participates in the retrograde trafficking of M6PRs. PMID:24788816

  8. Insulin-like growth factors (IGFs) stimulate the release of alpha 1-antichymotrypsin and soluble IGF-II/mannose 6-phosphate receptor from MCF7 breast cancer cells.

    PubMed

    Confort, C; Rochefort, H; Vignon, F

    1995-09-01

    The growth of hormone-responsive MCF7 human breast cancer cells is controlled by steroid hormones and growth factors. By metabolic labeling of cells grown in steroid- and growth factor-stripped serum conditions, we show that insulin-like growth factors (IGF-I and IGF-II) increase by approximately 5-fold the release of several proteins including cathepsin D, alpha 1-antichymotrypsin, and soluble forms of the multifunctional IGF-II/mannose 6-phosphate (M6P) receptor. Two soluble forms of IGF-II/M6P receptors were detected, one major (approximately 260 kilodaltons) and one minor (approximately 85 kilodaltons) that probably represents a proteolytic fragment of the larger soluble molecule. IGFs increased receptor release in a dose-dependent fashion with 50-60% of newly synthesized receptor released at 5-10 nM IGFs. The release of IGF-II/M6P receptors correlated with the levels of secreted cathepsin D in different human breast cancer cells or in rats stable transfectants that are constitutively expressing variable levels of human cathepsin D. IGFs had a stronger effect on IGF-II/M6P receptor release, whereas estradiol treatment preferentially enhanced the release of protease and antiprotease. We thus demonstrate that in human breast cancer cells, IGFs not only act as strong mitogens but also regulate release of alpha 1-antichymotrypsin, IGF-II/M6P-soluble receptor, and cathepsin D; three proteins that potentially regulate cell proliferation and/or invasion.

  9. Extending the Mannose 6-Phosphate Glycoproteome by High Resolution/Accuracy Mass Spectrometry Analysis of Control and Acid Phosphatase 5-Deficient Mice*

    PubMed Central

    Sleat, David E.; Sun, Pengling; Wiseman, Jennifer A.; Huang, Ling; El-Banna, Mukarram; Zheng, Haiyan; Moore, Dirk F.; Lobel, Peter

    2013-01-01

    In mammals, most newly synthesized lumenal lysosomal proteins are delivered to the lysosome by the mannose 6-phosphate (Man6P) targeting pathway. Man6P -containing proteins can be affinity-purified and characterized using proteomic approaches, and such studies have led to the discovery of new lysosomal proteins and associated human disease genes. One limitation to this approach is that in most cell types the Man6P modification is rapidly removed by acid phosphatase 5 (ACP5) after proteins are targeted to the lysosome, and thus, some lysosomal proteins may escape detection. In this study, we have extended the analysis of the lysosomal proteome using high resolution/accuracy mass spectrometry to identify and quantify proteins in a combined analysis of control and ACP5-deficient mice. To identify Man6P glycoproteins with limited tissue distribution, we analyzed multiple tissues and used statistical approaches to identify proteins that are purified with high specificity. In addition to 68 known Man6P glycoproteins, 165 other murine proteins were identified that may contain Man6P and may thus represent novel lysosomal residents. For four of these lysosomal candidates, (lactoperoxidase, phospholipase D family member 3, ribonuclease 6, and serum amyloid P component), we demonstrate lysosomal residence based on the colocalization of fluorescent fusion proteins with a lysosomal marker. PMID:23478313

  10. Mannose-6-Phosphate Reductase, a Key Enzyme in Photoassimilate Partitioning, Is Abundant and Located in the Cytosol of Photosynthetically Active Cells of Celery (Apium graveolens L.) Source Leaves.

    PubMed

    Everard, J. D.; Franceschi, V. R.; Loescher, W. H.

    1993-06-01

    Mannitol, a major photosynthetic product and transport carbohydrate in many plants, accounts for approximately 50% of the carbon fixed by celery (Apium graveolens L.) leaves. Previous subfractionation studies of celery leaves indicated that the enzymes for mannitol synthesis were located in the cytosol, but these data are inconsistent with that published for the sites of sugar alcohol synthesis in other families and taxa, including apple (Malus) and a brown alga (Fucus). Using antibodies to a key synthetic enzyme, NADPH-dependent mannose-6-phosphate reductase (M6PR), and immunocytochemical techniques, we have resolved both the inter-cellular and intracellular sites of mannitol synthesis. In leaves, M6PR was found only in cells containing ribulose-1,5-bisphosphate carboxylase/oxygenase. M6PR was almost exclusively cytosolic in these cells, with the nucleus being the only organelle to show labeling. The key step in transport carbohydrate biosynthesis that is catalyzed by M6PR displays no apparent preferential association with vascular tissues or with the bundle sheath. These results show that M6PR and, thus, mannitol synthesis are closely associated with the distribution of photosynthetic carbon metabolism in celery leaves. The principal role of M6PR is, therefore, in the assimilation of carbon being exported from the chloroplast, and it seems unlikely that this enzyme plays even an indirect role in phloem loading of mannitol.

  11. Structural and Functional Characterization of the Clostridium perfringens N-Acetylmannosamine-6-phosphate 2-Epimerase Essential for the Sialic Acid Salvage Pathway*

    PubMed Central

    Pélissier, Marie-Cécile; Sebban-Kreuzer, Corinne; Guerlesquin, Françoise; Brannigan, James A.; Bourne, Yves; Vincent, Florence

    2014-01-01

    Pathogenic bacteria are endowed with an arsenal of specialized enzymes to convert nutrient compounds from their cell hosts. The essential N-acetylmannosamine-6-phosphate 2-epimerase (NanE) belongs to a convergent glycolytic pathway for utilization of the three amino sugars, GlcNAc, ManNAc, and sialic acid. The crystal structure of ligand-free NanE from Clostridium perfringens reveals a modified triose-phosphate isomerase (β/α)8 barrel in which a stable dimer is formed by exchanging the C-terminal helix. By retaining catalytic activity in the crystalline state, the structure of the enzyme bound to the GlcNAc-6P product identifies the topology of the active site pocket and points to invariant residues Lys66 as a putative single catalyst, supported by the structure of the catalytically inactive K66A mutant in complex with substrate ManNAc-6P. 1H NMR-based time course assays of native NanE and mutated variants demonstrate the essential role of Lys66 for the epimerization reaction with participation of neighboring Arg43, Asp126, and Glu180 residues. These findings unveil a one-base catalytic mechanism of C2 deprotonation/reprotonation via an enolate intermediate and provide the structural basis for the development of new antimicrobial agents against this family of bacterial 2-epimerases. PMID:25320079

  12. Assembly of the ligand-binding conformation of Mr 46,000 mannose 6- phosphate-specific receptor takes place before reaching the Golgi complex

    PubMed Central

    1990-01-01

    The early steps in the biosynthesis of Mr 46,000 mannose 6-phosphate- specific receptor (MPR 46) have been studied by in vivo labeling of transfected BHK cells. The acquisition of phosphomannan-binding activity was compared with changes in protein structure and posttranslational modifications of MPR 46. Intramolecular disulfide bonds were formed before MPR 46 acquired a ligand-binding conformation. A conformational change that resulted in increased trypsin resistance, formation of highly immunogenic epitopes and assembly to noncovalently linked homodimers was observed almost simultaneously with the acquisition of ligand-binding activity. MPR 46 was shown to acquire ligand-binding activity before N-linked oligosaccharides were processed to complex-type forms. Maturation of the ligand-binding conformation was observed under conditions where transport to the Golgi was blocked by lowering the temperature to 16 degrees C, or by addition of brefeldin A or dinitrophenol to the medium at 37 degrees C. This suggests that receptor maturation and assembly take place before reaching the Golgi complex. The affinity towards phosphomannan- containing ligands was shown to be similar for the high-mannose and complex-glycosylated forms of MPR 46. PMID:2157722

  13. Non-enzymatic synthesis of the coenzymes, uridine diphosphate glucose and cytidine diphosphate choline, and other phosphorylated metabolic intermediates

    NASA Technical Reports Server (NTRS)

    Mar, A.; Dworkin, J.; Oro, J.

    1987-01-01

    Using urea and cyanamide, the two condensing agents considered to have been present on the primitive earth, uridine diphosphate glucose (UDPG), cytidine diphosphate choline (CDP-choline), glucose-1-phosphate (G1P), and glucose-6-phosphate (G6P) were synthesized under simulated prebiotic conditions. The reaction products were separated and identified using paper chromatography, thin layer chromatography, enzymatic analyses, and ion-pair reverse-phase high performance liquid chromatography. The possibility of nonenzymatic synthesis of metabolic intermediates on the primitive earth from simple precursors was thus demonstrated.

  14. Metabolic engineering of Corynebacterium glutamicum to produce GDP-L-fucose from glucose and mannose.

    PubMed

    Chin, Young-Wook; Park, Jin-Byung; Park, Yong-Cheol; Kim, Kyoung Heon; Seo, Jin-Ho

    2013-06-01

    Wild-type Corynebacterium glutamicum was metabolically engineered to convert glucose and mannose into guanosine 5'-diphosphate (GDP)-L-fucose, a precursor of fucosyl-oligosaccharides, which are involved in various biological and pathological functions. This was done by introducing the gmd and wcaG genes of Escherichia coli encoding GDP-D-mannose-4,6-dehydratase and GDP-4-keto-6-deoxy-D-mannose-3,5-epimerase-4-reductase, respectively, which are known as key enzymes in the production of GDP-L-fucose from GDP-D-mannose. Coexpression of the genes allowed the recombinant C. glutamicum cells to produce GDP-L-fucose in a minimal medium containing glucose and mannose as carbon sources. The specific product formation rate was much higher during growth on mannose than on glucose. In addition, the specific product formation rate was further increased by coexpressing the endogenous phosphomanno-mutase gene (manB) and GTP-mannose-1-phosphate guanylyl-transferase gene (manC), which are involved in the conversion of mannose-6-phosphate into GDP-D-mannose. However, the overexpression of manA encoding mannose-6-phosphate isomerase, catalyzing interconversion of mannose-6-phosphate and fructose-6-phosphate showed a negative effect on formation of the target product. Overall, coexpression of gmd, wcaG, manB and manC in C. glutamicum enabled production of GDP-L-fucose at the specific rate of 0.11 mg g cell(-1) h(-1). The specific GDP-L-fucose content reached 5.5 mg g cell(-1), which is a 2.4-fold higher than that of the recombinant E. coli overexpressing gmd, wcaG, manB and manC under comparable conditions. Well-established metabolic engineering tools may permit optimization of the carbon and cofactor metabolisms of C. glutamicum to further improve their production capacity.

  15. Prooxidative effects of aspartame on antioxidant defense status in erythrocytes of rats.

    PubMed

    Prokic, Marko D; Paunovic, Milica G; Matic, Milos M; Djordjevic, Natasa Z; Ognjanovic, Branka I; Stajn, Andras S; Saicic, Zorica S

    2014-12-01

    Since aspartame (L-aspartyl-L-phenylalanine methyl ester, ASP) is one of the most widely used artificial sweeteners, the aim of the present study was to investigate its effects on serum glucose and lipid levels as well as its effects on oxidative/antioxidative status in erythrocytes of rats. The experiment included two groups of animals: the control group was administered with water only, while the experimental group was orally administered with ASP (40 mg/kg b.w.) daily, for a period of six weeks. When compared with the control group, the group administrated with ASP indicated higher values of serum glucose, cholesterol and triglycerides. Significantly increased concentrations of superoxide anion (O2 .-), hydrogen peroxide (H2O2), peroxynitrite (?N??-) and lipid peroxides (LPO) were recorded in the erythrocytes of ASP treated group in comparison to the control group. In the course of chronic ASP administration, the following was observed: the concentration of reduced glutathione (GSH) and the activity of catalase (CAT) increased. Thus, these findings suggest that long-term consumption of ASP leads to hyperglycemia and hyperlipidemia, as well as to oxidative stress in erythrocytes. PMID:25431414

  16. Prooxidative effects of aspartame on antioxidant defense status in erythrocytes of rats.

    PubMed

    Prokic, Marko D; Paunovic, Milica G; Matic, Milos M; Djordjevic, Natasa Z; Ognjanovic, Branka I; Stajn, Andras S; Saicic, Zorica S

    2014-12-01

    Since aspartame (L-aspartyl-L-phenylalanine methyl ester, ASP) is one of the most widely used artificial sweeteners, the aim of the present study was to investigate its effects on serum glucose and lipid levels as well as its effects on oxidative/antioxidative status in erythrocytes of rats. The experiment included two groups of animals: the control group was administered with water only, while the experimental group was orally administered with ASP (40 mg/kg b.w.) daily, for a period of six weeks. When compared with the control group, the group administrated with ASP indicated higher values of serum glucose, cholesterol and triglycerides. Significantly increased concentrations of superoxide anion (O2 .-), hydrogen peroxide (H2O2), peroxynitrite (?N??-) and lipid peroxides (LPO) were recorded in the erythrocytes of ASP treated group in comparison to the control group. In the course of chronic ASP administration, the following was observed: the concentration of reduced glutathione (GSH) and the activity of catalase (CAT) increased. Thus, these findings suggest that long-term consumption of ASP leads to hyperglycemia and hyperlipidemia, as well as to oxidative stress in erythrocytes.

  17. Effects of muscular exercise on erythrocyte adenosine triphosphate concentration in patients with insulin-dependent diabetes mellitus.

    PubMed

    Donatelli, M; Verga, S; Terrizzi, C; Russo, V; Bucalo, M L; Scarpinato, A; Cerasola, G

    1987-01-01

    Type I diabetes mellitus represents a metabolic disorder in which intracellular glycolytic pathway is inhibited by insulin deficiency, with the subsequent decreased availability of energetic substrates such as ATP. Some aspects of the energetic metabolism in response to an intensive demand (muscular exercise) were investigated, in a group of 10 ketotic diabetic patients, by measuring erythrocyte adenosine triphosphate (ATP) and blood glucose, free fatty acids (FFA) and lactate levels. In the diabetic subjects, in comparison with normal subjects, the decreased levels of erythrocyte ATP at rest did not increase after exercise, while the increased levels of FFA at rest did not diminish after exercise. The results show that the impaired erythrocyte glycolysis may produce reduced levels of ATP not only at rest, but also after exercise, when muscular contraction results in a manifold increase in cellular energy requirements. In addition, other metabolic systems providing energy for the exercising muscle, such as FFA utilization, are impaired in the ketotic diabetic patients.

  18. Regulation of aflatoxin biosynthesis: effect of glucose on activities of various glycolytic enzymes.

    PubMed

    Buchanan, R L; Lewis, D F

    1984-08-01

    Catabolism of carbohydrates has been implicated in the regulation of aflatoxin synthesis. To characterize this effect further, the activities of various enzymes associated with glucose catabolism were determined in Aspergillus parasiticus organisms that were initially cultured in peptone-mineral salts medium and then transferred to glucose-mineral salts and peptone-mineral salts media. After an initial increase in activity, the levels of glucose 6-phosphate dehydrogenase, mannitol dehydrogenase, and malate dehydrogenase were lowered in the presence of glucose. Phosphofructokinase activity was greater in the peptone-grown mycelium, but fructose diphosphatase was largely unaffected by carbon source. Likewise, carbon source had relatively little effect on the activities of pyruvate kinase, malic enzyme, isocitrate-NADP dehydrogenase, and isocitrate-NAD dehydrogenase. The results suggest that glucose may, in part, regulate aflatoxin synthesis via a carbon catabolite repression of NADPH-generating and tricarboxylic acid cycle enzymes.

  19. A rapid method of reconstituting human erythrocyte sugar transport proteins.

    PubMed

    Carruthers, A; Melchior, D L

    1984-06-01

    A rapid reconstitution procedure for human erythrocyte hexose transfer activity is described. The procedure (reverse-phase evaporation) avoids exposure of the isolated proteins to detergent, organic solvent, sonication, or freeze-thaw steps during insertion into synthetic membranes and may be effected within 15 min. The so-formed vesicles are unilamellar structures with a large encapsulated volume, narrow size range, and low passive permeabilities. Contamination by carry-through of endogenous (red cell) lipids is less than 1%. Reconstituted hexose transfer activity was examined by using unfractionated proteins (bands 3, 4.5, and 6) and purified proteins (bands 4.5 and 3). With unfractionated proteins, hexose transport activity is low [0.34 mumol X (mg of protein)-1 X min-1], is inhibited by cytochalasin B, and increases monotonically with protein concentration. Kinetic analysis indicates that Vmax values for both influx and efflux of D-glucose are identical. Reconstitution of the cytochalasin B binding protein (band 4.5) results in hexose transport with high specific activity [5 mumol X (mg of protein)-1 X min-1] and symmetry in transfer kinetics. Band 3 proteins also appear to mediate cytochalasin B sensitive D-glucose transport activity.

  20. The effect of inhibition of hexose-monophosphate shunt on the metabolism of glucose and function in rat brain in vivo

    SciTech Connect

    Gaitonde, M.K.; Evans, G.M.

    1982-09-01

    Rats treated 4 hr previously with 6-aminonicotinamide showed a twenty-four fold increase of (/sup 14/C)phosphogluconate in the adult brain at 30 min after intection of (U-/sup 14/C)glucose indicating a blockade of the hexosemmonophosphate shunt. There was a significant increase in the /sup 14/C-content of glucose and glucose-6-phosphate, and a decrease in that of amino acids. (/sup 14/C)Phosphoglycerate content showed no consistent change after 6-aminonicotinamide treatment. The concentration of glucose and glucose 6-phosphate increased significantly without a significant change in the lactate pool in the brain of 6-aminonicotinamide treated rats. The rate of utilization of glucose in the brain of control rats was 0.73 mumol/min per g of brain. It decreased by 16% in rats treated with 6-aminonicotinamide; the results suggested that both glycolysis and pyruvate oxidation were affected. The amount of glucose utilized in the brain by the hexosemonophosphate shunt was approximately 0.0093 mumol/min per g of brain, i.e. 1.3% of the total rate of utilization of glucose. The observed changes were not due to hypothermia. The rate of glucose utilization was higher in animals exposed to higher ambient temperature and to stress caused by handling. The results were explained by postulating a role for the hexosemonophosphate shunt in providing neurotransmitter amino acids glutamate and gamma-aminobutyrate, and interdependence of brain function and glucose utilization.

  1. Glucose control.

    PubMed

    Preiser, Jean-Charles

    2013-01-01

    Stress-related hyperglycemia is a common finding in acutely ill patients, and is related to the severity and outcome of the critical illness. The pathophysiology of stress hyperglycemia includes hormonal and neural signals, leading to increased production of glucose by the liver and peripheral insulin resistance mediated by the translocation of transmembrane glucose transporters. In one pioneering study, tight glycemic control by intensive insulin therapy in critically ill patients was associated with improved survival. However, this major finding was not confirmed in several other prospective randomized controlled trials. The reasons underlying the discrepancy between the first and the subsequent studies could include nutritional strategy (amount of calories provided, use of parenteral nutrition), case-mix, potential differences in the optimal blood glucose level (BG) in different types of patients, hypoglycemia and its correction, and the magnitude of glucose variability. Therefore, an improved understanding of the physiology and pathophysiology of glycemic regulation during acute illness is needed. Safe and effective glucose control will need improvement in the definition of optimal BG and in the measurement techniques, perhaps including continuous monitoring of insulin algorithms and closed-loop systems. PMID:23075589

  2. Blood viscosity: influence of erythrocyte deformation.

    PubMed

    Chien, S; Usami, S; Dellenback, R J; Gregersen, M I

    1967-08-18

    Suspensions of canine and human erythocytes hardened with acetaldehyde differ from the suspensions of normal erythrocytes with respect to their rheological behavior. Normal erythrocytes can be packed by centrifugation so that the sediment volume is nearly 100 percent cells, but the hardened erythrocytes (RBC's) can be packed only to approximately 60 percent cells. At the same cell percentage the viscosity of the hardened RBC suspension is higher than that of the suspension of normal erythocytes. An increase in shear stress deforms the normal erythocytes and lowers the suspension viscosity, but has no influence on the viscosity of the hardened cell suspension. In blood with high cell percentages, the shear deformation of normal RBC's plays an important role in reducing viscosity and facilitating flow at high shear stresses. PMID:17842793

  3. Purification and Structural and Kinetic Characterization of the Pyrophosphate:Fructose-6-Phosphate 1-Phosphotransferase from the Crassulacean Acid Metabolism Plant, Pineapple.

    PubMed

    Tripodi, KEJ.; Podesta, F. E.

    1997-03-01

    Pyrphosphate-dependent phosphofructokinase (PFP) was purified to electrophoretic homogeneity from illuminated pineapple (Ananas comosus) leaves. The purified enzyme consists of a single subunit of 61.5 kD that is immunologically related to the potato tuber PFP [beta] subunit. The native form of PFP likely consists of a homodimer of 97.2 kD, as determined by gel filtration. PFP's glycolytic activity was strongly dependent on pH, displaying a maximum at pH 7.7 to 7.9. Gluconeogenic activity was relatively constant between pH 6.7 and 8.7. Activation by Fru-2,6-bisphosphate (Fru-2,6-P2) was dependent on assay pH. In the glycolytic direction, it activated about 10-fold at pH 6.7, but only 2-fold at pH 7.7. The gluconeogenic reaction was only weakly affected by Fru-2,6-P2. The true substrates for the PFP forward and reverse reactions were Fru-6-phosphate and Mg-pyrophosphate, and Fru-1,6-P2, orthophosphate, and Mg2+, respectively. The results suggest that pineapple PFP displays regulatory properties consistent with a pH-based regulation of its glycolytic activity, in which a decrease in cytosolic pH caused by nocturnal acidification during Crassulacean acid metabolism, which could curtail its activity, is compensated by a parallel increase in its sensitivity to Fru-2,6-P2. It is also evident that the [beta] subunit alone is sufficient to confer PFP with a high catalytic rate and the regulatory properties associated with activation by Fru-2,6-P2.

  4. Sugar-induced increases in trehalose 6-phosphate are correlated with redox activation of ADPglucose pyrophosphorylase and higher rates of starch synthesis in Arabidopsis thaliana.

    PubMed

    Lunn, John E; Feil, Regina; Hendriks, Janneke H M; Gibon, Yves; Morcuende, Rosa; Osuna, Daniel; Scheible, Wolf-Rüdiger; Carillo, Petronia; Hajirezaei, Mohammad-Reza; Stitt, Mark

    2006-07-01

    Tre6P (trehalose 6-phosphate) is implicated in sugar-signalling pathways in plants, but its exact functions in vivo are uncertain. One of the main obstacles to discovering these functions is the difficulty of measuring the amount of Tre6P in plant tissues. We have developed a highly specific assay, using liquid chromatography coupled to MS-Q3 (triple quadrupole MS), to measure Tre6P in the femto-picomole range. The Tre6P content of sucrose-starved Arabidopsis thaliana seedlings in axenic culture increased from 18 to 482 pmol x g(-1) FW (fresh weight) after adding sucrose. Leaves from soil-grown plants contained 67 pmol x g(-1) FW at the end of the night, which rose to 108 pmol x g(-1)FW after 4 h of illumination. Even greater changes in Tre6P content were seen after a 6 h extension of the dark period, and in the starchless mutant, pgm. The intracellular concentration of Tre6P in wild-type leaves was estimated to range from 1 to 15 microM. It has recently been reported that the addition of Tre6P to isolated chloroplasts leads to redox activation of AGPase (ADPglucose pyrophosphorylase) [Kolbe, Tiessen, Schluepmann, Paul, Ulrich and Geigenberger (2005) Proc. Natl. Acad. Sci. U.S.A. 102, 11118-11123]. Using the new assay for Tre6P, we found that rising sugar levels in plants are accompanied by increases in the level of Tre6P, redox activation of AGPase and the stimulation of starch synthesis in vivo. These results indicate that Tre6P acts as a signalling metabolite of sugar status in plants, and support the proposal that Tre6P mediates sucrose-induced changes in the rate of starch synthesis.

  5. Enzymes Catalyzing the Reversible Conversion of Fructose-6-Phosphate and Fructose-1,6-Bisphosphate in Maize (Zea mays L.) Kernels 1

    PubMed Central

    Tobias, Rowel B.; Boyer, Charles D.; Shannon, Jack C.

    1992-01-01

    The significance of the glycolytic and gluconeogenic conversion of fructose-6-phosphate and fructose-1,6-bisphosphate on sugar metabolism was investigated in maize (Zea mays L.) kernels. Maximum extractable activities of the pyrophosphate (PPi) dependent phosphofructokinase, fructose-1,6-bisphosphatase, and the ATP-dependent phosphofructokinase were measured in normal and four maize genotypes, which accumulate relatively more sugars and less starch, to determine how these enzymes are affected by the genetic lesions. Normal endosperm accumulated more dry matter than the high sugar/low starch genotypes, but protein contents did not differ greatly among the genotypes. Mutation of several starch biosynthetic enzymes had little impact on the activities of PPi-dependent phosphofructokinase, fructose-1,6-bisphosphatase, and ATP-dependent phosphofructokinase, despite the altered capacity of the cell to synthesize starch. The PPi-dependent phosphofructokinase appeared to be more active toward glycolysis in all genotypes studied. Activity of the PPi-dependent phosphofructokinase in shrunken (low sucrose synthase genotype) did not differ from the activity in other genotypes, suggesting that the gluconeogenic production of PPi may not be the primary role of the enzyme. As expected, shrunken kernels contained more sugars and less starch than normal kernels throughout kernel development except at the very early stages. Developmental profiles of normal kernels also showed marked changes in the PPi-dependent phosphofructokinase activity, whereas the level of ATP-dependent phosphofructokinase activity remained relatively steady during kernel development. In addition, the ATP-dependent phosphofructokinase, and not the PPi-dependent phosphofructokinase, appeared to correlate more closely with respiration rate. These findings suggest that glycolysis catalyzed by the ATP-dependent phosphofructokinase may serve primarily to support energy production, and glycolysis catalyzed by the PPi

  6. Sugar-induced increases in trehalose 6-phosphate are correlated with redox activation of ADPglucose pyrophosphorylase and higher rates of starch synthesis in Arabidopsis thaliana

    PubMed Central

    Lunn, John E.; Feil, Regina; Hendriks, Janneke H. M.; Gibon, Yves; Morcuende, Rosa; Osuna, Daniel; Scheible, Wolf-Rüdiger; Carillo, Petronia; Hajirezaei, Mohammad-Reza; Stitt, Mark

    2006-01-01

    Tre6P (trehalose 6-phosphate) is implicated in